WO2021081110A2 - Peptides and use thereof - Google Patents

Peptides and use thereof Download PDF

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Publication number
WO2021081110A2
WO2021081110A2 PCT/US2020/056686 US2020056686W WO2021081110A2 WO 2021081110 A2 WO2021081110 A2 WO 2021081110A2 US 2020056686 W US2020056686 W US 2020056686W WO 2021081110 A2 WO2021081110 A2 WO 2021081110A2
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WO
WIPO (PCT)
Prior art keywords
peptide
acid
amino acid
seq
derivatives
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Application number
PCT/US2020/056686
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French (fr)
Other versions
WO2021081110A3 (en
Inventor
Jason ROLAND
Andrew Goodrich
Sergio Duron
Deborah Nguyen
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Anexigen, Inc.
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Publication of WO2021081110A2 publication Critical patent/WO2021081110A2/en
Publication of WO2021081110A3 publication Critical patent/WO2021081110A3/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • peptides wherein at least three of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , or X 21 are independently a non- canonical amino acid.
  • peptides wherein X 1 and X 2 are independently G, S, A, V, L, I, M, F, W, or P.
  • peptides wherein X 3 is K, R, H, DAB (2,4-diaminobutyric acid), DAP (diaminopimelic acid), or absent.
  • peptides wherein X 4 is K, R, H, DAB (2,4-diaminobutyric acid), or DAP (diaminopimelic acid).
  • peptides wherein X 5 and X 7 are each independently a constrained amino acid.
  • peptides wherein X 5 is P, X 7 is P, and X 6 is V, L, or F
  • peptides wherein X 8 is I or F.
  • peptides wherein X 9 is I, T, N, S, V, E, Y, or F.
  • peptides wherein X 10 is F, W, or Y.
  • peptides wherein X 11 and X 18 are taken together to form an intramolecular linkage.
  • peptides wherein at least one of X 11 and X 18 is C or penicillamine.
  • the intramolecular linkage comprises a disulfide linkage.
  • the intramolecular linkage comprises the structure: substituent, Ar is aryl, and HA is heteroaryl.
  • peptides wherein R is alkyl, aralkyl, or cycloalkyl; Ar is optionally substituted phenyl or naphthyl; and HA is optionally substituted: pyridinyl, imidazolyl, thiophenyl, pyrrolyl, thiazolyl, oxazolyl, or furanyl.
  • peptides wherein X 12 is S, T, C, Y, N, or Q.
  • peptides wherein X 13 is R, Q, L, or F.
  • peptides wherein X 14 , X 17 , and X 20 are independently a positively charged amino acid.
  • peptides wherein X 14 and X 17 , and X 20 are independently a K, R, H, DAB (2,4-diaminobutyric acid), or DAP (diaminopimelic acid).
  • peptides wherein X 16 is G, A, V, L, I, M, F, W, or P.
  • non-canonical amino acid is selected from the group consisting of alanine derivatives; alicyclic amino acids; arginine derivatives; aromatic amino acids; asparagine derivatives; aspartic acid derivatives; beta-amino acids; cysteine derivatives; dab (2,4-diaminobutyric acid); dap (2,3- diaminopropionic acid); glutamic acid derivatives; glutamine derivatives; glycine derivatives; homo-amino acids; isoleucine derivatives; leucine derivatives; linear core amino acids; lysine derivatives; methionine derivatives; n-methyl amino acids; norleucine derivatives; norvaline derivatives; ornithine derivatives; penicillamine derivatives; phenylalanine derivatives; phenylglycine derivatives; proline derivatives; pyroglutamine derivatives; serine derivatives; threonine derivatives; tryptophan derivative
  • peptides wherein at least one of X 4 , X 17 , or X 20 are DAB (2,4- diaminobutryic acid) or DAP (diaminopimelic acid).
  • peptides wherein at least one of X 1 , X 2 , X 3 , and Y 2 are non-canonical amino acids.
  • peptides wherein at least one of X 20 , X 21 , or Y 1 are non-canonical amino acids. Further provided herein are peptides wherein Y 1 is absent or an N-terminal modification; X 1 is absent, or G; X 2 is absent, or S; X 3 is absent, K, DAB, or DAP; X 4 is K, DAB, or DAP; X 5 is P; X 6 is V, I, L, or F; X 7 is P; X 8 is I or F; X 9 is I, T, N, S, V, E, Y, F; X 10 is Y, F, H, or L; X 11 is C or penicillamine; X 12 is N, S, H, or R; X 13 is R, Q, L, F, DAB, or DAP; X 14 is any amino acid; X 15 is T or S; X 16 is G; X 17 is any amino acid; X 18 is C or
  • X 21 is absent, I, L, V, M, F, or a non-canonical amino acid; and Y 2 is absent or a C-terminal modification.
  • the non-canonical amino acid is linked to a half-life extending moiety or a moiety which broadens the antibiotic spectrum.
  • the peptide comprises at least one N-terminal modification or C-terminal modification.
  • the N- terminal modification or C-terminal modification comprises at least one targeting agent.
  • the targeting agent increases the local concentration of the peptide.
  • the targeting agent broadens the antibiotic spectrum.
  • peptides wherein Y 2 comprises an amide. Further provided herein are peptides wherein Y 2 is selected from the group consisting of -NEB, -NH(C 1 -C 6 alkyl), and -N(C 1 -C 6 alkyl) 2 .
  • isolated peptides wherein the peptide has at least 70% identity with SEQ ID NO: 1, wherein in the peptide is not identical to SEQ ID NO: 1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
  • peptides wherein the peptide has at least 75% identity with SEQ ID NO: 1. Further provided herein are peptides wherein the peptide has at least 85% identity with SEQ ID NO: 1. Further provided herein are peptides wherein the peptide has at least 90% identity with SEQ ID NO: 1. Further provided herein are peptides wherein the peptide has at least 95% identity with SEQ ID NO:l.
  • isolated peptides wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 2-313, wherein in the peptide is not identical to SEQ ID NO: 1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
  • peptides wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 2-50.
  • isolated peptides wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 51-100, wherein in the peptide is not identical to SEQ ID NO: 1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. Further provided herein are peptides wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 51-100.
  • isolated peptides wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 101- 154, wherein in the peptide is not identical to SEQ ID NO: 1, and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
  • peptides wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 101-150.
  • isolated peptides wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 151-200, wherein in the peptide is not identical to SEQ ID NO: 1, and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
  • peptides wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 151- 200.
  • isolated peptides wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 201-250, wherein in the peptide is not identical to SEQ ID NO: 1, and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
  • peptides wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 201-250.
  • isolated peptides wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 251- 313, wherein in the peptide is not identical to SEQ ID NO: 1, and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
  • peptides wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 251-313.
  • isolated peptides wherein the peptide has at least 70% identity with SEQ ID NO: 1 over a range of at least 10 amino acids and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R;
  • peptides wherein the peptide has at least 80% identity with SEQ ID NO: 1 over a range of at least 13 amino acids. Further provided herein are peptides wherein the peptide has at least 80% identity with SEQ ID NO: 1 over a range of at least 16 amino acids. Further provided herein are peptides wherein the peptide has at least 80% identity with SEQ ID NO: 1 over a range of at least 9-18 amino acids. Further provided herein are peptides wherein the peptide comprises an N-terminal deletion. Further provided herein are peptides wherein the peptide comprises at least one non-canonical amino acid.
  • peptides wherein the non-canonical amino acid is selected from the group consisting of a D-amino acid, a beta-amino acid, an N-acyl amino acid, or a C- amidyl amino acid.
  • the peptide comprises at least one C-terminal or N-terminal modification.
  • the at least one C-terminal or N-terminal modification comprises a modification which extends half-life or broadens the antibiotic spectrum.
  • peptides wherein the peptide comprises at least one intramolecular linkage.
  • the intramolecular linkage is formed by two amino acid side chains.
  • the peptide comprises at least one substitution relative to SEQ ID NO: 1.
  • compositions comprising a peptide described herein and an excipient, delivery vehicle, second therapeutic agent, or a combination thereof. Further provided herein are pharmaceutical compositions wherein the pharmaceutical composition comprises an excipient. Further provided herein are pharmaceutical compositions wherein the pharmaceutical composition comprises a second therapeutic agent. Further provided herein are pharmaceutical compositions wherein the pharmaceutical composition is formulated for topical administration. Further provided herein are pharmaceutical compositions wherein the pharmaceutical composition is formulated for intravenous administration. Further provided herein are pharmaceutical compositions wherein the pharmaceutical composition is formulated for oral administration. Further provided herein are pharmaceutical compositions wherein the pharmaceutical composition is formulated for intramuscular or subcutaneous administration.
  • kits for treating an infection comprising administering to a subject a therapeutically effective amount of a peptide or pharmaceutical composition described herein, or a peptide comprising SEQ ID NO: 1.
  • the infection comprises a bacterial infection.
  • the bacterial infection is caused by a Gram-negative bacterium.
  • the bacterial infection is caused by an enterobacterium.
  • the bacterium is Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii, or Pseudomonas aeruginosa.
  • bacterium is a drug-resistant or multi-drug resistant bacterium.
  • methods wherein the subject is a mammal.
  • methods wherein the mammal is a human.
  • the peptide is administered orally, enterically, topically, intravenously, intraperitoneally, intramuscularly, endoscopically, percutaneously, subcutaneously, regionally, by inhalation, or by direct injection.
  • methods wherein the orally administered peptide is a capsule or tablet.
  • methods further comprising administering a second therapeutic agent Further provided herein are methods the second therapeutic agent is an antibiotic or a protease inhibitor.
  • the antibiotic is a beta-lactam antibiotic. Further provided herein are methods wherein the antibiotic is amoxicillin, bacitracin, chloramphenicol, clindamycin, capreomycin, colistimethate, ciprofloxacin, doxycycline, erythromycin, fusidic acid, fosfomycin, fusidate sodium, gramicidin, gentamycin, lincomycin, minocycline, macrolides, monobactams, nalidixic acid, novobiocin, ofloxcin, rifamycins, tetracyclines, vancomycin, tobramycin, fluoroquinolones, polymyxins, DNA gyrase inhibitors, bacterial polymerase inhibitors, folate synthesis inhibitors, or trimethoprim.
  • Figure 1A depicts Compound A tested in a time-kill assay against E. coli ATCC 25922.
  • Figure IB depicts Compound A tested in a beta-lactamase release assay.
  • Figure 2A depicts Compound A tested for red blood cell hemolysis.
  • Figure 2B depicts Compound A tested for release of LDH from HEK293 cells.
  • peptides described herein are used to treat, prevent occurrence or prevent recurrence of bacterial infections in living organisms, including those caused by multi-drug resistant bacteria.
  • peptides described herein are used to disinfect surfaces, including medical devices.
  • methods are provided for the manufacturing of antimicrobial peptides.
  • peptides having antimicrobial activity comprise the sequence of Formula (I):
  • Y 1 is absent or an N-terminal modification; X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 ,
  • X 21 are any amino acid
  • X 1 , X 2 , and X 3 are absent or independently any amino acid; and Y 2 is absent or a C-terminal modification.
  • a peptide of Formula (I) is not identical to SEQ ID NO: 1. In some embodiments of a peptide of Formula (I), the peptide has a sequence of any one of SEQ NOs: 2-313. In some embodiments of a peptide of Formula (I), X 1 and X 2 are absent. In some embodiments of a peptide of Formula (I), X 5 is P, X 6 is V, and X 7 is P. In some embodiments of a peptide of Formula (I), X 11 is C or penicillamine. In some embodiments of a peptide of Formula (I), X 13 is R.
  • a peptide of Formula (I) is T, X 16 is G, and X 18 is C or penicillamine. In some embodiments of a peptide of Formula (I), X 20 is R. In some embodiments of a peptide of Formula (I), X 21 is absent. In some embodiments a peptide of Formula (I) is not identical to SEQ ID NO: 1. In some embodiments a peptide of Formula (I), the peptide is not identical to a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. In some embodiments a peptide of Formula (I), C-terminal and/or N-terminal modifications are configured to increase the half-life of the peptide.
  • peptides of Formula (I) comprising one or more non- canonical amino acids.
  • X 4 is a positively charged amino acid.
  • at least one of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , or X 21 is a non-canonical amino acid.
  • At least one of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , or X 21 is a D amino acid.
  • at least one of X 3 , X 4 , X 13 , X 14 , X 20 is a non-canonical amino acid.
  • At least one of X 3 , X 4 , X 13 , X 14 , X 20 is a positively charged non-canonical amino acid. In some embodiments, X 3 , X 4 , X 13 , X 14 , X 20 , are positively charged non-canonical amino acids. In some embodiments, at least one of X 3 , X 4 , X 13 , X 14 , X 20 , is a DAP or DAB.
  • the non-canonical amino acid selected from 2,4-diaminobutryic acid; alpha- methylarginine; homophenylalanine; homoleucine; homoisoleucine; Diaminopropionic acid; N-acetylglycine; 6-aminohexanoic acid; gamma-aminobutyric acid; alpha-methylserine; alpha-methyltyrosine; 4-(trifluoromethyl)-L-phenylalanine; 4-amino-L-phenylalanine; Penicillamine; beta-homophenylalanine; beta-homoisoleucine; t-butyl D-serine, Statine, N- methyl glycine, N-methyl serine, N-methyl lysine, beta-phenylalanine, L-alpha-cyclohexyl glycine, L-cyclopropylglycine, or beta-homoleucine.
  • peptides of Formula (I) comprising an N-terminal half-life extension moiety.
  • Y 1 comprises a half-life extension moiety.
  • Y 1 is absent or an N- terminal modification.
  • Y 1 comprises a water-soluble polymer, a lipid, or a peptide.
  • Y 1 comprises a polyethylene glycol polymer.
  • Y 1 is acyl.
  • Y 1 is C 1 -C 6 acyl. In some embodiments of a peptide of Formula (I), Y 1 is hexenoyl. In some embodiments of a peptide of Formula (I), Y 1 is acetyl. In some embodiments of a peptide of Formula (I), Y 1 comprises an XTEN peptide. In some embodiments of a peptide of Formula (I), Y 1 comprises an XTEN peptide polymer comprising 700-900 repeats. In some embodiments of a peptide of Formula (I), Y 1 comprises a PAS peptide.
  • Y 1 comprises a PAS peptide polymer comprising 500-900 repeats. In some embodiments of a peptide of Formula (I), Y 1 comprises a protein. In some embodiments of a peptide of Formula (I), Y 1 is serum albumin. In some embodiments of a peptide of Formula (I), Y 1 comprises an antibody or antibody fragment. In some embodiments of a peptide of Formula (I), Y 1 comprises an Fc. In some embodiments of a peptide of Formula (I), Y 1 comprises IgG.
  • peptides of Formula (I) comprising amino acids of different charges.
  • X 1 is a non-polar amino acid.
  • X 2 is a polar amino acid.
  • X 3 is absent or any amino acid.
  • X 4 is a positively charged amino acid.
  • At least one of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , or X 21 is a non-canonical amino acid.
  • At least one of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , or X 21 is a D amino acid.
  • at least one of X 3 , X 4 , X 13 , X 14 , X 20 is a non- canonical amino acid.
  • At least one of X 3 , X 4 , X 13 , X 14 , X 20 is a positively charged non-canonical amino acid. In some embodiments, X 3 , X 4 , X 13 , X 14 , X 20 , are positively charged non-canonical amino acids. In some embodiments, at least one of X 3 , X 4 , X 13 , X 14 , X 20 , is a DAP or DAB. In some embodiments of a peptide of Formula (I), X 5 is a constrained amino acid. In some embodiments of a peptide of Formula (I), X 5 and X 7 are each independently a constrained amino acid.
  • X 6 is a non-polar amino acid.
  • X 7 is a constrained amino acid.
  • X 8 is a non-polar amino acid.
  • X 9 is a polar or non-polar amino acid.
  • X 9 is any amino acid.
  • X 10 is any amino acid.
  • X 10 is a phenylalanine derivative.
  • X 10 is an phenyl -substituted phenylalanine. In some embodiments of a peptide of Formula (I), X 10 is p-CF 3 -phenylalanine, p-fluorophenylalanine, p-NFh-phenylalanine, p- MeO-phenylalanine, alpha-methyl tyrosine, or p-methylphenyalanine. In some embodiments of a peptide of Formula (I), X 11 is a bridging amino acid. In some embodiments of a peptide of Formula (I), at least one of X 11 and X 18 is a non-cannonicial amino acid.
  • X 12 is a polar amino acid. In some embodiments of a peptide of Formula (I), X 12 is a positively charged amino acid. In some embodiments of a peptide of Formula (I), X 13 is a non-polar amino acid. In some embodiments of a peptide of Formula (I), X 14 is any amino acid. In some embodiments of a peptide of Formula (I), X 15 is a polar amino acid. In some embodiments of a peptide of Formula (I), X 16 is a non-polar amino acid. In some embodiments of a peptide of Formula (I), X 17 is any amino acid.
  • X 18 is a bridging amino acid.
  • X 19 is a polar amino acid.
  • X 20 is a positively charged amino acid.
  • X 21 is a non-polar amino acid or a homoamino acid analog.
  • X 11 and X 18 are bridging amino acids.
  • X 11 and X 18 form an intermolecular linkage.
  • peptides of Formula (I) comprising substitutions at various positions, hi some embodiments of a peptide of Formula (I), X 6 is V, I, L, or F. In some embodiments of a peptide of Formula (I), X 8 is I or F. In some embodiments of a peptide of Formula (I), X 9 is I, T, N, S, V, E, Y, or F. In some embodiments of a peptide of Formula (I), X 10 is Y, F, H, or L. In some embodiments of a peptide of Formula (I), X 12 is N, S, H, or R.
  • X 13 is R, Q, L, or F.
  • X 15 is T or S.
  • X 19 is Q or T.
  • X 21 is M, L, V, or F.
  • peptides of Formula (I) comprising a C-terminal half-life extension moiety.
  • Y 2 comprises a half-life extension moiety.
  • Y 2 is absent or a C- terminal modification.
  • Y 2 comprises a peptide.
  • Y 2 comprises a water-soluble polymer, a lipid, or a peptide.
  • Y 2 comprises a polyethylene glycol polymer.
  • Y 2 comprises an XTEN peptide. In some embodiments of a peptide of Formula (I), Y 2 comprises an XTEN peptide polymer comprising 700-900 repeats. In some embodiments of a peptide of Formula (I), Y 2 comprises a PAS peptide. In some embodiments of a peptide of Formula (I), Y 2 comprises a PAS peptide polymer comprising 500-900 repeats. In some embodiments of a peptide of Formula (I), Y 2 comprises a protein. In some embodiments of a peptide of Formula (I), Y 2 is serum albumin.
  • Y 2 comprises an antibody or antibody fragment. In some embodiments of a peptide of Formula (I), Y 2 comprises an Fc. In some embodiments of a peptide of Formula (I), Y 2 comprises IgG. In some embodiments of a peptide of Formula (I), Y 2 comprises at least one K. In some embodiments of a peptide of Formula (I), Y 2 comprises at least one Y. In some embodiments of a peptide of Formula (I), Y 2 comprises at least one DAB or DAP. In some embodiments of a peptide of Formula (I), Y 2 comprises ⁇ DAB ⁇ DAB ⁇ or ⁇ DAP ⁇ DAP ⁇ .
  • Y 2 comprises YYKK. In some embodiments of a peptide of Formula (I), Y 2 comprises a peptide comprising at least two positively charged amino acids. In some embodiments of a peptide of Formula (I), Y 2 comprises YY ⁇ DAB ⁇ ⁇ DAB ⁇ or YY ⁇ DAP ⁇ DAP ⁇ .
  • peptides of Formula (I) comprising a hairpin structure.
  • X 1 is absent.
  • X 2 is absent.
  • X 1 and X 2 if present are independently G, S, A, V, L, I, M, F, W, or P.
  • X 3 is K, R, or H.
  • X 4 is K, R, or H.
  • X 4 is a positively charged amino acid. In some embodiments, at least one of X 3 , X 4 , X 13 , X 14 , X 20 , is a DAP or DAB. In some embodiments of a peptide of Formula (I), X 5 is P. In some embodiments of a peptide of Formula (I), X 6 is G, A, V, L, I, M, F, W, or P.
  • X 5 is a constrained amino acid selected from proline, a proline analog, Om(i-PrCO-Hao), 5-hydrazino-2-methoxybenzoic acid (Hao), an N-alkyl amino acid, or an alpha, alpha- disubstituted amino acid.
  • X 7 is a constrained amino acid selected from proline, a proline analog, Orn(i-PrCO-Hao), 5- hydrazino-2-methoxybenzoic acid (Hao), an N-alkyl amino acid, or an alpha, alpha- disubstituted amino acid.
  • X 7 is P.
  • X 8 is G, A, V, L, I, M, F, W, or P.
  • X 9 is G, A, V, L, I, M, F, W, or P.
  • X 9 is S, T, C, Y, N, or Q.
  • X 10 is F, W, or Y.
  • X 11 and X 18 are taken together to form an intramolecular linkage.
  • At least one of X 11 and X 18 is C. In some embodiments of a peptide of Formula (I), at least one of X 11 and X 18 is penicillamine. In some embodiments of a peptide of Formula (I), at least one of X 11 and X 18 is aspartic acid or glutamic acid. In some embodiments of a peptide of Formula (I), at least one of X 11 and X 18 is dehydroalanine. In some embodiments of a peptide of Formula (I), X 11 and X 18 are both C or penicillamine.
  • X 12 is S, T, C, Y, N, or Q.
  • X 13 is G, A, V, L, I, M, F, W, or P.
  • X 14 is a positively charged amino acid.
  • X 14 is K, R, or H.
  • X 15 is S, T, C, Y, N, or Q.
  • X 16 is G, A, V, L, I, M, F, W, or P.
  • X 17 is a positively charged amino acid.
  • X 17 is K, R, or H.
  • X 19 is S, T, C, Y, N, or Q.
  • X 20 is absent, K, R, or H.
  • X 21 is a homoamino acid.
  • X 21 is a beta-amino acid. In some embodiments of a peptide of Formula (I), X 21 is a beta-homoamino acid. In some embodiments of a peptide of Formula (I), ), X 21 is a homoamino acid selected from the group consisting of homophenylalanine, homoleucine, homocysteine, homomethionine, and homoisoleucine. In some embodiments of a peptide of Formula (I), X 21 is G, A, V, L, I, M, F, W, or P.
  • X 3 , X 4 , X 17 , or X 20 are DAB (2,4-diaminobutryic acid). In some embodiments of a peptide of Formula (I), X 3 , X 4 , X 17 , are DAB (2,4- diaminobutryic acid). In some embodiments of a peptide of Formula (I), X 1 is a non- canonical amino acid, and X 2 is absent. In some embodiments of a peptide of Formula (I), X 1 is an N-methyl amino acid.
  • X 1 is N-methyl serine, N-methyl lysine, or N-methyl glycine. In some embodiments of a peptide of Formula (I), X 1 is t-butyl D-serine, Statine, N-methyl glycine, N-methyl serine, N-methyl lysine, beta- phenylalanine, L-alpha-cyclohexyl glycine, L-cyclopropylglycine, or beta-homoleucine. In some embodiments of a peptide of Formula (I), X 1 is Statine, DAB, 6-AHA, GABA, or DAP.
  • X 2 , X 5 , and X 7 are each independently a D- amino acid.
  • X 2 is D-serine.
  • X 5 is D-proline.
  • X 7 is D-proline.
  • X 3 is lysine.
  • X 5 is valine.
  • X 7 is isoleucine.
  • X 8 is threonine.
  • X 12 is arginine.
  • X 17 is cysteine.
  • X 20 is phenylalanine.
  • the peptide comprises at least one of the mutations 17, T8, R12, F20, or Cl 7.
  • the peptide comprises at least two of the mutations 17, T8, R12, F20, or C17.
  • Y 1 is absent or an N-terminal modification
  • X 3 is absent, K or a non-canonical amino acid
  • X 4 is K or a non-canonical amino acid
  • X 6 is V, I, L, orF.
  • X 8 is I or F
  • X 9 is I, T, N, S, V, E, Y, or F;
  • X 10 is Y, F, H, or L
  • X 12 is N, S, H, or R;
  • X 13 is R, Q, L, F, or a non-canonical amino acid
  • X 14 is R, K, or a non-canonical amino acid
  • X 15 is R or a non-canonical amino acid
  • X 16 is T or S
  • X 17 is K or a non-canonical amino acid
  • X 19 is Q or T
  • X 21 is absent, M, L, V, or F;
  • Y 2 is absent or a C-terminal modification.
  • Y 1 is absent or an N-terminal modification
  • X 3 is K, DAB, or DAP
  • X 4 is absent, K, DAB, or DAP;
  • X 9 is I or T
  • X 10 is any amino acid
  • X 12 is N or S
  • X 14 is any amino acid
  • X 17 is any amino acid
  • X 19 is Q or T
  • X 20 is absent, R or DAB;
  • X 21 is absent, I, L, V, M, F, or a beta homoamino acid
  • Y 2 is absent or a C-terminal modification.
  • peptides of Formula (II) or (III) comprising an N-terminal half-life extension moiety.
  • the peptide is not identical to SEQ ID NO: 1.
  • the peptide is not identical to a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
  • Y 1 is absent or an N-terminal modification.
  • Y 1 is a half-life extending moiety or broadens the antibiotic spectrum.
  • Y 1 comprises a water-soluble polymer, a lipid, or a peptide.
  • Y 1 comprises a polyethylene glycol polymer.
  • Y 1 is acyl.
  • Y 1 is C 1 -C 6 acyl.
  • Y 1 is hexenoyl. In some embodiments of a peptide of Formula (II) or Formula (III), Y 1 is acetyl. In some embodiments of a peptide of Formula (II) or Formula (III), Y 1 comprises an XTEN peptide. In some embodiments of a peptide of Formula (II) or Formula (III), Y 1 comprises an XTEN peptide polymer comprising 700-900 repeats.
  • Y 1 comprises an XTEN peptide polymer comprising 20-50, 20-100, 100-300, 200-500, 400-800, 500-1000, or 750-1500 repeats. In some embodiments of a peptide of Formula (II) or Formula (III), Y 1 comprises a PAS peptide. In some embodiments of a peptide of Formula (II) or Formula (III), Y 1 comprises a PAS peptide polymer comprising 500-900 repeats.
  • Y 1 compress an PAS peptide polymer comprising 20-50, 20-100, 100-300, 200-500, 400-800, 500-1000, or 750-1500 repeats.
  • Y 1 comprises a protein.
  • Y 1 is serum albumin.
  • Y 1 comprises an antibody or antibody fragment.
  • Y 1 comprises an Fc.
  • Y 1 comprises IgG.
  • peptides of Formula (II) or Formula (III) comprising a C- terminal half-life extension moiety or a moiety which broadens the antibiotic spectrum.
  • Y 2 comprises a half-life extension moiety.
  • Y 2 is absent or a C-terminal modification.
  • Y 2 comprises a peptide.
  • Y 2 comprises a water-soluble polymer, a lipid, or a peptide. In some embodiments of a peptide of Formula (II) or Formula (III), Y 2 comprises a polyethylene glycol polymer. In some embodiments of a peptide of Formula (II) or Formula (III), Y 2 comprises an XTEN peptide. In some embodiments of a peptide of Formula (II) or Formula (III), Y 2 comprises an XTEN peptide polymer comprising 700-900 repeats.
  • Y 2 comprises an XTEN peptide polymer comprising 20-50, 20-100, 100-300, 200-500, 400-800, 500-1000, or 750-1500 repeats. In some embodiments of a peptide of Formula (II) or Formula (III), Y 2 comprises a PAS peptide. In some embodiments of a peptide of Formula (II) or Formula (III), Y 2 comprises a PAS peptide polymer comprising 500-900 repeats.
  • Y 2 compress an PAS peptide polymer comprising 20-50, 20-100, 100-300, 200-500, 400-800, 500-1000, or 750-1500 repeats.
  • Y 2 comprises a protein.
  • Y 2 is serum albumin.
  • Y 2 comprises an antibody or antibody fragment.
  • Y 2 comprises an Fc.
  • Y 2 comprises IgG. In some embodiments of a peptide of Formula (II) or Formula (III), Y 2 comprises at least one K. In some embodiments of a peptide of Formula (II) or Formula (III), Y 2 comprises at least one Y. In some embodiments of a peptide of Formula (II) or Formula (III), Y 2 comprises YYKK. In some embodiments of a peptide of Formula (II) or Formula
  • Y 2 comprises a peptide comprising at least two positively charged amino acids.
  • a peptide of Formula (II) or Formula (III) one or more both cysteines are replaced with penicillamine or other amino acid capable of forming a disulfide bond.
  • Y 1 is absent or an N-terminal modification
  • X 1 and X 2 are independently present or absent, and when present are any amino acid;
  • X 3 X 4 X 5 c6 X 9 X 11 X 13 c 14 c 15 c 16 c 18 c 19 and c 21 are any aminO acid;
  • Y 2 is absent or a C-terminal modification.
  • peptides of Formula (IV) comprising an N-terminal half-life extension moiety.
  • the peptide is not identical to SEQ ID NO: 1.
  • the peptide is not identical to a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
  • Y 1 is absent or an N-terminal modification.
  • Y 1 is a half-life extending moiety or broadens the antibiotic spectrum.
  • Y 1 comprises a water-soluble polymer, a lipid, or a peptide.
  • Y 1 comprises a polyethylene glycol polymer.
  • Y 1 is acyl.
  • Y 1 is Ci-C 6 acyl.
  • Y 1 is hexenoyl.
  • Y 1 is acetyl.
  • Y 1 comprises an XTEN peptide.
  • Y 1 comprises an XTEN peptide polymer comprising 700-900 repeats. In some embodiments of a peptide of Formula (IV), Y 1 comprises an XTEN peptide polymer comprising 20-50, 20-100, 100-300, 200-500, 400-800, 500-1000, or 750-1500 repeats. In some embodiments of a peptide of Formula (IV), Y 1 comprises a PAS peptide. In some embodiments of a peptide of Formula (IV), Y 1 comprises a PAS peptide polymer comprising 500-900 repeats.
  • Y 1 compress an PAS peptide polymer comprising 20-50, 20-100, 100-300, 200-500, 400-800, 500-1000, or 750- 1500 repeats.
  • Y 1 comprises a protein.
  • Y 1 is serum albumin.
  • Y 1 comprises an antibody or antibody fragment.
  • Y 1 comprises an Fc.
  • Y 1 comprises IgG.
  • a peptide of Formula (IV) at least one of X 3 , X 4 , X 5 , X 6 , X 9 , X 11 , X 13 , X 14 , X 15 , X 16 , X 18 , X 19 , or X 21 is a non-canonical amino acid.
  • a peptide of Formula (IV) at least one of X 3 , X 4 , X 5 , X 6 , X 9 , X 11 , X 13 c 14 c 15 c 16 c 18 c 19 or c 21 K, DAB, or DAP.
  • a peptide of Formula (IV) one or more both cysteines are replaced with penicillamine or other amino acid capable of forming a disulfide bond.
  • Peptides disclosed herein include peptides as well as structurally similar compounds (i.e., small molecules) that are in some embodiments formulated to mimic the key portions of a peptide.
  • the peptide or peptide has at least 90, 91, 92,
  • the peptide or peptide has at least 70, 75, 80, 85, 90, 95, or 98% sequence identity with any one of SEQ ID NOs: 1-313.
  • the peptide comprises an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99% sequence identity with SEQ ID NO: 1.
  • the peptide or peptide has a sequence identity of 70-80%, 75-85%, 80-90%, 85-95%, or 90-100% with any one of SEQ ID NOs: 1-313. In some embodiments, the peptide or peptide has a sequence identity of 70- 80% with any one of SEQ ID NOs: 1-313. In some embodiments, the peptide or peptide has a sequence identity of 80-89% with any one of SEQ ID NOs: 1-313. In some embodiments, the peptide or peptide has a sequence identity of 90-99% with any one of SEQ ID NOs: 1-313. In some embodiments, the peptide or peptide has a sequence identity of 70-79% with any one of SEQ ID NOs: 1-313. In certain embodiments, the peptide or peptide has at least 90, 91, 92,
  • the peptide comprises an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99% sequence similarity with SEQ ID NO: 1.
  • the peptide or peptide has a sequence similarity of 70-80%, 75-85%, 80-90%, 85-95%, or 90-100% with any one of SEQ ID NOs: 1-313.
  • the peptide or peptide has a sequence similarity of 70-80% with any one of SEQ ID NOs: 1-313.
  • the peptide or peptide has a sequence similarity of 80-89% with any one of SEQ ID NOs: 1-313.
  • the peptide or peptide has a sequence similarity of 90-99% with any one of SEQ ID NOs: 1-313. In some embodiments, the peptide or peptide has a sequence similarity of 70-79% with any one of SEQ ID NOs: 1-313.
  • peptides comprising any one of SEQ ID NOs: 1-313, wherein one or more residues susceptible to proteolytic cleavage is replaced with a residue less susceptible to proteolytic cleavage.
  • one or more lysine residues of a peptide comprising any one of SEQ ID NOs: 1-313 is replaced with an non-canonical amino acid.
  • one or more arginine residues of a peptide comprising any one of SEQ ID NOs: 1-313 is replaced with an non-canonical amino acid.
  • all lysine and arginine residues of a peptide comprising any one of SEQ ID NOs: 1-313 is replaced with an non-canonical amino acid.
  • one or more arginine or lysine residues of a peptide comprising any one of SEQ ID NOs: 1-313 is replaced with a conservative non-canonical amino acid.
  • one or more arginine or lysine residues of a peptide comprising any one of SEQ ID NOs: 1-313 is replaced with an non- canonical amino acid, such as DAP or DAB.
  • one or more arginine or lysine residues of a peptide comprising any one of SEQ ID NOs: 1-313 is replaced with a D- amino acid.
  • Peptide lengths described herein are in some embodiments about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
  • peptide lengths are 10-50 residues, 15-50 residues, 20-25 residues 21-25, residues, 20-30 residues, 30-40 residues, and 35-45 residues, or 25-35 residues. In some embodiments, peptide lengths are less than 30, 25, 20, 15, or less than 10 amino residues.
  • peptides described herein comprise L-configuration amino acids, D-configuration amino acids, non- canonical amino acids, or a mixture thereof. In some embodiments, peptides comprise at least 1, 2, 3, 4, 5, 7, 9, 12, 15, 20, or more than 20 D-amino acids.
  • peptides comprise not more than 1, 2, 3, 4, 5, 7, 9, 12, 15, 20, or more than 20 D-amino acids. In some embodiments, peptides comprise 1-5, 2-7, 2-10, 5-10, 7-15, or 4-8 D-amino acids. In some embodiments, all amino acids in a peptide described herein are D-amino acids.
  • amino acids described herein comprise amino acids.
  • amino acids comprise naturally occurring and synthetic amino acids (e.g., non-canonical amino acids such as beta-amino acids, homo-amino acids, or others), as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O- phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.
  • Peptides described herein are often isolated from natural sources of from recombinant organisms.
  • a peptide or peptide has been separated from any natural environment, such as a body fluid, e.g., blood, and separated from the components that naturally accompany the peptide.
  • Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring or a nonconventional/non- canonical amino acid residue. Such substitutions in some embodiments are classified as conservative, in which case an amino acid residue contained in a peptide or peptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size. Such conservative substitutions are well known in the art.
  • substitutions encompassed by the present disclosure may also be non-conservative, in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties, such as naturally-occurring amino acid from a different group (e.g, substituting a charged or hydrophobic amino; acid with alanine), or alternatively, in which a naturally occurring amino acid is substituted with a non- conventional amino acid.
  • amino acid substitutions are conservative.
  • polynucleotide or peptide refers to a polynucleotide or peptide that can vary in primary, secondary, or tertiary structure, as compared to a reference polynucleotide or peptide, respectively (e.g, as compared to a wild- type polynucleotide or peptide).
  • Peptides described herein may comprise insertions, deletions, or substitutions.
  • insertions and deletions are in the range of about 1 to 5 amino acids.
  • the variation allowed in some embodiments is experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.
  • substitution comprises a change in an amino acid for a different entity, for example another amino acid or amino-acid moiety. Substitutions can be conservative or non-conservative substitutions.
  • the peptide is a variant comprising at least one amino acid substitution, deletion, or insertion relative to the amino acid sequence of any one of SEQ ID NOS: 1-313.
  • Variants in some embodiments are synthetic, recombinant, or chemically modified peptides isolated or generated using methods well known in the art. Variants can include conservative or non-conservative amino acid changes, as described below. Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the peptide encoded by the reference sequence. Variants can also include insertions, deletions or substitutions of amino acids, including insertions and substitutions of amino acids and other molecules that do not normally occur in the peptide sequence that is the basis of the variant, for example but not limited to insertion of ornithine which do not normally occur in human proteins.
  • conservative substitution when describing a peptide, refers to a change in the amino acid composition of the peptide that does not substantially alter the peptide's activity.
  • a conservative substitution refers to substituting an amino acid residue for a different amino acid residue that has similar chemical properties.
  • Conservative amino acid substitutions include replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Conservative amino acid substitutions result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • a conservative substitution of a particular amino acid sequence refers to substitution of those amino acids that are not critical for peptide activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or nonpolar) such that the substitution of even critical amino acids does not reduce the activity of the peptide.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Groups of amino acids are categorized in some embodiments based on polarity or charge of their respective side chains.
  • non-polar amino acids include but are not limited to Glycine, Alanine, Valine, Leucine, Isoleucine, Methionine, Phenylalanine, Tryptophan, or Proline.
  • polar amino acids include but are not limited to Serine, Threonine, Cysteine, Tryptophan, Asparagine, or Glutamine.
  • positively charged amino acids include but are not limited to Lysine, Arginine, or Histidine.
  • negatively charged amino acid include but are not limited to Aspartic acid or Glutamic acid.
  • Non- canonical amino acids are also in some embodiments are conservatively substituted for such amino acids.
  • non-canonical amino acids comprising a basic side chain e.g., DAB or DAP
  • an amino acid is a negatively charged amino acid.
  • negatively charged amino acids comprise side-chain functional groups which are negatively charged under aqueous physiological conditions (e.g., pH ⁇ 7).
  • negatively charged amino acids comprise acidic functional group side chains.
  • acidic functional groups include but are not limited to carboxylic acids, sulfonic acids, sulfmic acids, thioacids, dithioacids, sulfamates, sulfimates, phosphates, diketoacids, phosphites, boronic acid, phenols, or other acidic functional group.
  • an amino acid is a positively charged amino acid.
  • positively charged amino acids comprise side-chain functional groups which are positively charged under aqueous physiological conditions (e.g., pH ⁇ 7).
  • positively charged amino acids comprise basic functional group side chains.
  • basic functional groups include but are not limited to heteroaryl rings (e.g., pyridines, pyrimidines, imidazoles, pyrroles), amines (substituted or unsubstituted), guanidines, piperidines, pyrrolidines, morpholines, hydrazines, other basic functional group.
  • amino acids are non-canonical amino acids.
  • individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids can also be considered conservative substitutions if the change does not significantly reduce the activity of the peptide. Insertions or deletions are typically in the range of about 1 to 5 amino acids.
  • the choice of conservative amino acids in some embodiments is selected based on the location of the amino acid to be substituted in the peptide, for example if the amino acid is on the exterior of the peptide and expose to solvents, or on the interior and not exposed to solvents.
  • amino acid is exposed to solvents or is present on the outer surface of the peptide or peptide as compared to internally localized amino acids not exposed to solvents). Selection of such conservative amino acid substitutions are well known in the art. Accordingly, one can select conservative amino acid substitutions suitable for amino acids on the exterior of a protein or peptide (i.e. amino acids exposed to a solvent).
  • substitutions can be used: substitution of Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with Tor A, R with K, G with N or A, K with R, A with S, K or P.
  • one can also select conservative amino acid substitutions suitable for amino acids on the interior of a protein or peptide for example one can use suitable conservative substitutions for amino acids is on the interior of a protein or peptide (i.e. the amino acids are not exposed to a solvent).
  • suitable conservative substitutions for amino acids is on the interior of a protein or peptide (i.e. the amino acids are not exposed to a solvent).
  • nonconservative amino acid substitutions are also encompassed within the term of variants.
  • the peptides or peptides disclosed herein are derivatives of the SEQ ID NOs:1-313.
  • the term derivative in some embodiments comprises peptides which have been chemically modified, for example but not limited to by techniques such as ubiquitination, labeling, pegylation (i.e., derivatization with polyethylene glycol), lipidation, glycosylation, or addition of other molecules.
  • a molecule is also in some embodiments a derivative of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties can improve the molecule's potency, solubility, absorption, biological half-life, etc.
  • a peptide described herein comprises a half-life extending moiety (e.g., water soluble polymer, lipid, protein, or peptide).
  • the moieties can alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, increase antibiotic spectrum, or have other effects.
  • amino acid substitutions can be made in a peptide at one or more positions wherein the substitution is for an amino acid having a similar hydrophilicity.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art.
  • the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • conservative substitution can be made in a peptide and will likely only have minor effects on their activity.
  • hydrophilicity values may be assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine ( -0.4); proline ( -0.5 ⁇ 1); alanine ( 0.5); histidine -0.5); cysteine ( -1.0); methionine ( -1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • any of the peptides or peptides described herein in some embodiments are modified by the substitution of an amino acid, for a different, but homologous amino acid with a similar hydrophilicity value. Amino acids with hydrophilicities within+/- 1.0, or+/- 0.5 points are considered homologous.
  • a modification comprises a co-translational and/or post- translational (C-terminal peptide cleavage) modification.
  • a modification comprises, for example, disulfide bond formation, backbone cyclization, glycosylation, acetylation, phosphorylation, and proteolytic cleavage (e.g., cleavage by furins or metalloproteases). Such modifications in some instances are enabled by bridging amino acids.
  • modification comprise intramolecular cyclizations to form intramolecular linkages.
  • intramolecular linkages comprise amide, ether, disulfide, sulfide, alkyl, ester, or other bond.
  • bridging amino acids include aspartic acid, glutamic acid, serine, cysteine, penicillamine, dehydroalanine, or other amino acid capable of intramolecular reaction to form one or more rings.
  • Bridging amino acids in some instances comprise canonical or non-canonical amino acids described herein.
  • the antimicrobial peptide (or peptide) comprises nonnaturally occurring or non-canonical amino acids.
  • the antimicrobial peptides can comprise a combination of naturally occurring and non-naturally occurring amino acids, or may comprise only nonnaturally occurring amino acids.
  • the non-naturally occurring amino acids optionally include synthetic non-native amino acids, substituted amino acids, or one or more D-amino acids into the peptides (or other components of the composition, with exception for protease recognition sequences) is desirable in certain situations.
  • D-amino acid-containing peptides exhibit increased stability in vitro or in vivo compared to L-amino acid-containing forms.
  • the construction of peptides incorporating D-amino acids can be particularly useful when greater in vivo or intracellular stability is desired or required.
  • D-peptides are resistant to endogenous peptidases and proteases, thereby providing better oral trans-epithelial and transdermal delivery of linked drugs and conjugates, improved bioavailability of membrane -permanent complexes, and prolonged intravascular and interstitial lifetimes when such properties are desirable.
  • the use of D isomer peptides can also enhance trans dermal and oral trans-epithelial delivery of linked drugs and other cargo molecules.
  • D-peptides are often not processed efficiently for major histocompatibility complex class II-restricted presentation to T helper cells, and are therefore less likely to induce humoral immune responses in the whole organism
  • Peptide conjugates can therefore be constructed using, for example, D-isomer forms of cell penetrating peptide sequences, L-isomer forms of cleavage sites, and D-isomer forms of therapeutic peptides.
  • D-amino acids or non-standard (or non- canonical) modified or unusual amino acids which are wel1-defined in the art are also contemplated for use in the present disclosure including phosphorylated amino acids (Ser,
  • Peptides described herein often comprise non-standard (or non-canonical) amino acids.
  • Selenocysteine is incorporated into some proteins at a UGA codon, which is normally a stop codon.
  • Pyrrolysine is used by some methanogenic archaea in enzymes that they use to produce methane. It is coded for with the codon UAG.
  • non-standard amino acids that are not found in proteins include lanthionine, 2-aminoisobutyric acid, dehydroalanine and the neurotransmitter gammaaminobutyric acid.
  • Non-standard amino acids often occur as intermediates in the metabolic pathways for standard amino acids; for example ornithine and citrulline occur in the urea cycle, part of amino acid catabolism.
  • Non-standard amino acids are usually formed through modifications to standard amino acids. For example, homocysteine is formed through the transsulfuration pathway or by the demethylation of methionine via the intermediate metabolite S-adenosyl methionine, while hydroxyproline is made by a posttranslational modification of proline.
  • analogs comprising a molecule similar in function to either the entire molecule or to a fragment thereof.
  • analogs comprise allelic species and induced variants. Analogs typically differ from naturally occurring peptides at one or a few positions, often by virtue of conservative substitutions. Analogs typically exhibit at least 80% or 90% sequence identity with natural peptides. Some analogs also include non-canonical amino acids or modifications of N or C terminal amino acids.
  • non-canonical amino acids are, for example but not limited to, disubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, gamma-carboxyglutamate, epsi lon-N,NN-tri methyl 1 ysi ne, epilson-N-acetyllysine, O-phosphoserine, N-acetylserine, N- formylmethionine, 3-methylhistidine, 5-hydroxylysine, and si gm a-A-m ethyl argi nine.
  • a non-canonical amino acid is selected from 2,4-diaminobutryic acid; alpha- methylarginine; homophenylalanine; homoleucine; homoisoleucine; Diaminopropionic acid; N-acetylglycine; 6-aminohexanoic acid; gamma-aminobutyric acid; alpha-methylserine; alpha-methyltyrosine; 4-(trifluoromethyl)-L-phenylalanine; 4-amino-L-phenylalanine; Penicillamine; beta-homophenylalanine; beta-homoisoleucine; and beta-homoleucine.
  • non-canonical amino acids comprise similar structures to natural amino acids, and/or are derivatives thereof.
  • non-canonical amino acids include but are not limited to alanine derivatives; alicyclic amino acids; arginine derivatives; aromatic amino acids; asparagine derivatives; aspartic acid derivatives; beta- amino acids; cysteine derivatives; dab (2,4-diaminobutyric acid); dap (2,3-diaminopropionic acid); glutamic acid derivatives; glutamine derivatives; glycine derivatives; homo-amino acids; isoleucine derivatives; leucine derivatives; linear core amino acids; lysine derivatives; methionine derivatives; n-methyl amino acids; norleucine derivatives; norvaline derivatives; ornithine derivatives; penicillamine derivatives; phenylalanine derivatives; phenylglycine derivatives; proline derivatives; pyroglut
  • a non-canonical amino acid is an alanine derivative.
  • alanine derivatives include but are not limited to: (-)-3-(3,4-dihydroxyphenyl)- 2-methy1-L-alanine; (R)-(+)- ⁇ -allylalanine; (R)-2-(2-propenyl)Ala-OH; (R)-2-(pentenyl)Ala- OH; (R)-propargy1-Ala-OH; (S)-(-)- ⁇ -allylalanine; (S)-(+)-2-Amino-4-phenylbutyric acid; (S)-2-(2-azidoethane)Ala-OH; (S)-2-(4-azidobutane)Ala-OH; (S)-2-(4-pentenyl)Ala-OH; (S)--(4-pentenyl)Ala-OH; (S)--(4-pentenyl)Ala-
  • a non-canonical amino acid is an alicyclic amino acids.
  • alicyclic amino acids include but are not limited to: (cis)-3- aminobicyclo[2.2.1]heptane-2-carboxylic acid; exo-cis-3-aminobicyclo[2.2.1]hept-5-ene-2- carboxylic acid; 1 -amino- 1-cy cl obutanecarboxylic acid; cis-2-aminocycloheptanecarboxylic acid; 1-aminocyclohexanecarboxylic acid; trans-2-aminocyclohexanecarboxylic acid; cis-6- amino-3 -cyclohexene- 1 -carboxylic acid; 2-(1-aminocyclohexyl)acetic acid; cis-2-amino-1- cyclooctanecarboxylic acid; cis-2-amino-3-
  • a non-canonical amino acid is an arginine derivative.
  • arginine derivatives include but are not limited to L-2-amino-3- guanidinopropionic acid or 4-guanidinobutyric acid.
  • a non-canonical amino acid is an asparagine derivative.
  • arginine derivatives include but are not limited to ⁇ -acety1-L-asparagine, ⁇ -methy1-L-asparagine, or ⁇ -xantheny1-L-asparagine.
  • a non-canonical amino acid is an aspartic acid derivative.
  • aspartic acid derivatives include but are not limited to esters, amides, or anhydrides of the aspartic acid side chain.
  • a non-canonical amino acid is a beta amino acid.
  • beta amino acids include but are not limited to: (lS,3R)-(+)-3- (amino)cyclopentanecarboxylic acid; (2R,3R)-3-(amino)-2-hydroxy-4-phenylbutyric acid; (R)-2-methyl- ⁇ -Phe-OH; (R)-3-(amino)-2-methylpropionic acid; (R)-3-(amino)-4-(2- naphthyl)butyric acid; (R)-3-(amino)-5-phenylpentanoic acid; (R)-3-(trifluoromethyl)- ⁇ -Phe- OH; (R)-3,4-dimethoxy- ⁇ -Phe-OH; (R)-3-methoxy- ⁇ -Phe-OH; (R)-3-methyl- ⁇ -Phe-OH; (R)- 4-(3-pyrid
  • a non-canonical amino acid is an aromatic amino acid.
  • aromatic amino acids include but are not limited to: 4-Acetamidobenzoic acid; 4-Acetamido-2-methylbenzoic acid; N-Acetylanthranilic acid; 3-Aminobenzoic acid; 4- Aminobenzoic acid; 2-Aminobenzophenone-2'-carboxylic acid; 2-Amino-4-bromobenzoic acid; 2-Amino-5-bromobenzoic acid; 3- Amino-2 -bromobenzoic acid; 3-Amino-4- bromobenzoic acid; 3-Amino-5-bromobenzoic acid; 4- Amino-3 -bromobenzoic acid; 5- Amino-2 -bromobenzoic acid; 2-Amino-3-bromo-5-methylbenzoic acid; 2-Amino-3- chlorobenzoic acid; 2-Amino-4-chlorobenzoic acid
  • a non-canonical amino acid is a cysteine derivative.
  • cysteine derivatives include but are not limited to: methylselenocysteine; L- cysteic acid; L-cysteinesulfmic acid; D-ethionine; or seleno-L-cystine.
  • a non-canonical amino acid is a Dab or Dap derivative.
  • Dab or Dap derivatives include but are not limited to: L-2,4- Diaminobutyric acid; Dab(Alloc)-OH; Dab(Boc)-OH; Dap(Alloc)-OH; Dap(Fmoc)-OH; Dap-OH; Dap(Z)-OH; D-2,3-Diaminopropionic acid; L-2,3-Diaminopropionic acid; 2,3- Diaminopropionic acid; Dap(Boc)-OH; or D-Dap-OH.
  • a non-canonical amino acid is a glutamic acid or glutamine derivative.
  • glutamic acid or glutamine derivatives include but are not limited to: g-Carboxy-glutamic acid, (4S)-4-(4-Trifluoromethy1-benzyl)-L-glutamic acid, or D-Citrulline.
  • a non-canonical amino acid is a glycine derivative.
  • glycine derivatives include but are not limited to: D-Allylglycine; N- [Bis(methylthio)methylene]glycine; ally1-Gly-OH; Chg-OH; D-Chg-OH; D- cyclopropylglycine; L-cyclopropylglycine; N-iminodiacetic acid; (2-indanyl)-Gly-OH; ( ⁇ )- ⁇ - phosphonoglycine; D-propargylglycine; propargy1-Gly-OH; (R)-2-thienylglycine; (S)-2- thienylglycine; (R)-3 -thienyl glycine; (2S,3R,4S)- ⁇ -(Carboxycyclopropyl)glycine; D- ⁇ - Cyclohexylglycine; Eth
  • a non-canonical amino acid is a homo-amino acid.
  • homo-amino acids include but are not limited to: (R)-2-(Boc-amino)-5- hexynoic acid; Homophe-OH; Homophe-OH; Homoser(Trt)-OH; piperidine-2-carboxylic acid; L-Homoarginine; Homocysteine; L-Homocystine; L-Homophenylalanine; D- Homophenylalanine; D-Homoserine; or L-homoserine.
  • a non-canonical amino acid is a leucine, isoleucine, or valine derivative.
  • leucine, isoleucine, or valine derivatives include but are not limited to: 3-Fluoro-valine; 4,4,4,4',4',4'-Hexafluoro-valine; (R)-(+)- ⁇ -Methylvaline; (S)- (-)- ⁇ -Methylvaline; N-(2,2,2-trifluoromethyl)-L-Valine; D-allo-Isoleucine; N-[(2S,3R)-3- Amino-2-hydroxy-4-phenylbutyryl]-L-leucine; Cycloleucine; N-(3,5-Dinitrobenzoyl)- leucine; tBu-Gly-OH; N-Formy1-Leu-OH; N-(3-Indolylacetyl)-L-isole
  • a non-canonical amino acid is a linear core amino acid.
  • linear core amino acids include but are not limited to: L-Allysine ethylene acetal; 12-Aminododecanoic acid; 2-Aminoheptanoic acid; 7-Aminoheptanoic acid;
  • a non-canonical amino acid is a lysine derivative.
  • lysine derivatives include but are not limited to: Lys(Me,Boc)-OH; Lys(palmitoyl)-OH; 5-Hydroxylysine; Lys(Me) 2 -OH; Lys(Ac)-OH; Lys(C(O)CF 3 )-0H; or Lys(Me)-OH.
  • a non-canonical amino acid is a methionine derivative.
  • methionine derivatives include but are not limited to: (2S)-2-[[[4-[[(2R)- 2-amino-3 -mercaptopropyl] amino] -2-phenylphenyl] -oxom ethyl] amino] -4- (methylthio)butanoic acid; 2-(1,3-benzothiazo1-2-ylamino)-4-(methylthio)butanoic acid; 2- [(6-bromo-4-quinazolinyl)amino]-4-(methylthio)butanoic acid; 2-[[(4-ethylphenyl)- oxomethyl]amino]-4-(methylthio)butanoic acid; 2-amino-4-(methylsulfanyl)butanoic acid; N- acetylmethionine; methionine S
  • a non-canonical amino acid is an N-methyl amino acid.
  • N-methyl amino acids include but are not limited to: L-Abrine; N-Me- Aib-OH; N-Me-Ala-OH; N-Me-D-Ala-OH; N-Me-Leu-OH; N-Me-D-Leu-OH; N-Me-Phe- OH; N-Me-Thr(Bzl)-OH; N-Me-Thr-OH; N-Me-Tyr(Bzl)-OH; N-Me-Tyr-OH; N-Me-Va1- OH; N,N-Dimethylglycine; N-methy1-L-phenylalanine; N-Me-Aib-OH; N-Me-Ala-OH; N- Me-Asp(OtBu)-OH;; N-Me-Asp-OH; N-Me-Me-
  • a non-canonical amino acid is a norleucine, norvaline, ornithine, or penicillamine derivative.
  • norleucine, norvaline, ornithine, or penicillamine derivatives include but are not limited to: 6-azido-L-norleucine; Nle-OH; D- Norleucine; L-Norleucine; Norvaline; (S)-5-Azido-2-(amino)pentanoic acid; Orn(Alloc)-OH; Om(Z)-OH; D-Ornithine; L-Omithine; Pen(Mob)-OH; Pen(Trt)-OH; D-Penicillamine; L- Penicillamine; or D-Penicillamine disulfide.
  • a non-canonical amino acid is a phenylalanine derivative.
  • phenylalanine derivatives include but are not limited to: 2-fluoro- phenylalanine; 4-fluoro-phenylalanine; 4-Amino-L-phenylalanine; 3 -[3, 4- bis(trifluoromethyl)phenyl]-L-alanine; Bpa-OH; D-Bpa-OH; 4-tert-buty1-Phe-OH; 4-tert- buty1-D-Phe-OH; 4-(Fmoc-amino)-L-phenylalanine; ⁇ 2-homophenyl alanine; 2-methoxy-L- phenylalanine; 2-nitro-L-phenylalanine; pentafluoro-D-phenylalanine; pentafluoro-L- phenylalanine; Phe(4-Br)-OH; D-P
  • a non-canonical amino acid is a phenylglycine derivative.
  • phenylglycine derivatives include but are not limited to: 2-(piperazino)- 2-(2-fluorophenyl)acetic acid; 2-(piperazino)-2-(3-pyridyl)acetic acid; 2-(piperazino)-2-[4- (trifluorom ethyl )phenyl] acetic acid; 2-Chlorophenyl glycine; 4-Chlorophenyl glycine; (R)-(-)- 2-(2,5-Dihydrophenyl)glycine; (R)-(-)-N-(3,5-Dinitrobenzoyl)- ⁇ -phenylglycine; (S)-(+)-N- (3,5-Dinitrobenzoyl)- ⁇ -phenylglycine; 2,2-Diphenylglycine; 2-Fl
  • a non-canonical amino acid is a pyroglutamine derivative.
  • pyroglutamine derivatives include but are not limited to: (4R)-4- benzy1-Pyr-OH (4R)-4-(4-bromobenzyl)-Pyr-OH; (4R)-4-(4-methylbenzyl)-Pyr-OH; (R)-5- oxopyrrolidine-2-carboxylic acid; or L-Pyroglutamic acid.
  • a non-canonical amino acid is a serine or threonine derivative.
  • serine or threonine derivatives include but are not limited to: (2R,3S)-3-phenylisoserine; Ser[GalNAc(Ac)3- ⁇ -D]-OH; Isoserine; 3-Phenylserine; Thr[GalNAc(Ac)3- ⁇ -D]-OH; or L-allo-Threonine.
  • a non-canonical amino acid is a tryptophan derivative.
  • tryptophan derivatives include but are not limited to: 5-Fluoro-L- tryptophan; 5-Hydroxy-L-tryptophan; 5-Methoxy-tryptophan; or 5 -Methyl -tryptophan.
  • a non-canonical amino acid is a tyrosine derivative.
  • tyrosine derivatives include but are not limited to: 3-Amino-L-tyrosine; Tyr(3,5-l 2 )-OH; 3-Chloro-L-tyrosine; [CpRu(tyrosin)]CF 3 CO 2 ; ⁇ -Methy1-tyrosine; 3-Nitro-L- tyrosine; or o-tyrosine.
  • exemplary non-canonical amino acids include but are not limited to: (S)- ⁇ -Amino- ⁇ -butyrolactone; 2-Aminocaprylic acid; 7-Aminocephalosporanic acid; (S)-(+)- ⁇ -Aminocyclohexanepropionic acid; (R)-Amino-(4-hydroxyphenyl)acetic acid; 5-Aminolevulinic acid; 4-Amino-nicotinic acid; 3-Aminophenylacetic acid; 4- Aminophenylacetic acid; 2-Amino-2-phenylbutyric acid; 4-(4-Aminophenyl)butyric acid; 2- (4-Aminophenylthio)acetic acid; 5-Aminovaleric acid; 8-Benzyl (S)-2-aminooctanedioate; (lR,3S,4S)-2-azabicyclo[2.2.1]heptane-3-car
  • peptides described herein comprise amino acids which promote secondary structure formation, such as hairpin formation (constrained amino acids).
  • constrained amino acids include but are not limited to analogs of proline.
  • analogs of proline include but are not limited to proline, proline analogs, Om(i-PrCO-Hao), 5-hydrazino-2-methoxybenzoic acid (Hao), N-alkyl amino acid (e.g., N-( C 1 -C 6 alkyl)), or an alpha, alpha-disubstituted amino acid (e.g., alpha, alpha-(C 1 -C 6 dialkyl)).
  • two or more amino acids are replaced with a constrained amino acid or analog or isostere thereof.
  • Analogs of amino acids may comprise non-canonical amino acids with similar shape, charge, or properties.
  • peptides described herein comprise proline analogs.
  • Proline include but are not limited to: (R)- ⁇ -ally1-proline; (R)-(+)4- oxazolidinecarboxylic acid; (S)-(-)-4-oxazolidinecarboxylic acid; (S)- ⁇ -ally1-proline; L- azetidine-2-carboxylic acid; ⁇ -(2-bromobenzyl)-Proline; ⁇ -(4-bromobenzyl)-Proline; ⁇ -(2- chlorobenzyl)-Proline; ⁇ -(3-chlorobenzyl)-Proline; ⁇ -(diphenylmethyl)-proline; (R)- ⁇ -(4- fluorobenzyl)-Proline; (S)- ⁇ -(4-fluorobenzyl)-Pro; ⁇
  • amino acids of the antimicrobial peptides of the present disclosure may also be modified.
  • amino groups in some embodiments are acylated, alkylated, or arylated.
  • Benzyl groups in some embodiments are halogenated, nitrosylated, alkylated, sulfonated or acylated.
  • Carboxy terminal modifications include acylation with carboxylic acids: formic, acetic, propionic, fatty acids (myristic, palmitic, stearic), succinic, benzoic, carbobenzoxy (Cbz); and biotinylation.
  • Amino terminal modifications include: (i) acylation with carboxylic acids: formic, acetic, propionic, fatty acids (myristic, palmitic, stearic, etc.) succinic, benzoic, carbobenzoxy (Cbz); (ii) biotinylation; (iii) attachment of dyes such as fluorescein (FITC, FAM, etc.), 7-hydroxy-4-methylcoumarin-3-acetic acid, 7-hydroxycoumarin-3-acetic acid, 7- methoxycoumarin-3 -acetic acid and other coumarins; rhodamines (5-carboxyrhodamine 110 or 6G, 5(6)-TAMRA, ROX); N-4-(4-dimethylamino)phenylazo]benzoic acid (Dabcyl), 2,4- dinitrobenzene (Dnp), 5-dimethylaminonaphthalene-1-sulfonic acid (Dansyl) and other dyes; and (i
  • beta-alanine beta-Ala
  • other omega-amino acids such as 3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4- aminobutyric acid and so forth
  • alpha-aminoisobutyric acid Alpha-aminoisobutyric acid (Aib); epsilon-aminohexanoic acid (Aha); sigma-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Om); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); norleucine (Nle); 4-chlorophenylalanine (Phe(4-Cl)); 2- fluorophenylalanine (Phe(2-F)); 3-fluoropheny
  • the peptide in some embodiments is modified at its N and/or C termini (“capping” groups) with an acyl (abbreviated “Ac”) and/or an amido (abbreviated “Am”) group, respectively, for example acetyl (CH 3 CO-) at the N terminus and amido (-NH 2 ) at the C terminus.
  • Ac acyl
  • Am amido
  • N-terminal capping functions preferably in a linkage to the terminal amino group, is contemplated, for example: formyl; alkanoyl, having from 1 to 10 carbon atoms (such as acetyl, propionyl, or butyryl); cycloalkanoyl, having from 1 to 10 carbon atoms; alkenoyl, having from 1 to 10 carbon atoms (such as hex-3 -enoyl); alkynoyl, having from 1 to 10 carbon atoms, such as (hex-5 -ynoyl); aroyl, (such as benzoyl or 1- naphthoyl); heteroaroyl (such as 3-pyrroyl or 4-quinoloyl); alkylsulfonyl (such as methanesulfonyl); arylsulfonyl (such as benzenesulfonyl or sulfanilyl); heteroaryl sulfonyl (such as
  • a peptide described herein has a C-terminal modification.
  • the C-terminal modification is an amide or ester.
  • C-terminal modifications that provide for an amide bond are designated as -NR'R 2 wherein R 1 and R 2 are independently hydrogen; alkyl, preferably having from 1 to 10 carbon atoms, such as methyl, ethyl, or isopropyl; cycloalkyl, preferably having from 1 to 10 carbon atoms, such as cyclopropyl, cyclobutyl, or cyclopentyl; alkenyl, preferably having from 1 to 10 carbon atoms, such as prop-2-enyl; alkynyl, preferably having from 1 to 10 carbon atoms, such as prop-2-ynyl; substituted alkyl having from 1 to 10 carbon atoms, such as hydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthi
  • C-terminal modifications providing an ester bond are designated as OR, wherein R may comprise an alkoxy; aryloxy; heteroaryloxy; aralkoxy; heteroaralkoxy; substituted alkoxy; substituted aryloxy; substituted heteroaryloxy; substituted aralkoxy; or substituted heteroaralkoxy group.
  • N-terminal or the C-terminal modifications, or both are in some embodiments of such a structure that the peptide functions as a prodrug (a pharmacologically inactive derivative of the parent drug molecule) that undergoes spontaneous or enzymatic transformation within the body in order to release the active drug and that has improved delivery properties over the parent drug molecule.
  • a prodrug a pharmacologically inactive derivative of the parent drug molecule
  • N or C terminal modification results in attachment of additional chemical groups.
  • the presence of a sulfhydryl group linked to the N- or C-modification permits conjugation of the derivatized peptide to other molecules.
  • the peptides or variants or derivatives thereof can be "retro- inverso peptides.”
  • a “retro-inverso peptide” refers to a peptide with a reversal of the direction of the peptide bond on at least one position, i.e., a reversal of the amino- and carboxy- termini with respect to the side chain of the amino acid.
  • a retro-inverso analogue has reversed termini and reversed direction of peptide bonds while approximately maintaining the topology of the side chains as in the native peptide sequence.
  • the retro-inverso peptide can contain L-amino acids or D-amino acids, or a mixture of L-amino acids and D-amino acids, up to all of the amino acids being the D-isomer.
  • Partial retro-inverso peptide analogues are peptides in which only part of the sequence is reversed and replaced with enantiomeric amino acid residues. Since the retro- inverted portion of such an analogue has reversed amino and carboxyl termini, the amino acid residues flanking the retro-inverted portion are replaced by side-chain-analogous alpha-substituted geminal-diaminomethanes and malonates, respectively. Retro-inverso forms of cell penetrating peptides have been found to work as efficiently in translocating across a membrane as the natural forms.
  • Embodiments of the present disclosure also include longer peptides built from repeating units of an antimicrobial peptide.
  • a peptide multimer may comprise different combinations of peptide.
  • Such multimeric peptides can be made by chemical synthesis or by recombinant DNA techniques as discussed herein.
  • the oligomers When produced by chemical synthesis, the oligomers preferably have from 2-5 repeats of a core peptide sequence, and the total number of amino acids in the multimer should not exceed about 160 residues, preferably not more than 100 residues (or their equivalents, when including linkers or spacers).
  • the present disclosure also contemplates that structurally similar compounds in some embodiments are formulated to mimic the key portions of peptide or peptides of the present disclosure.
  • Such compounds which may be termed peptidomimetics, in some embodiments are used in the same manner as the peptides of the present disclosure and, hence, also are functional equivalents.
  • peptides and proteins describe a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics.
  • the subunits in some embodiments are linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g. ester, ether, etc.
  • peptides comprise a peptidomimetic or peptide mimic wherein the peptide is modified in such a way that it includes at least one non-peptidic bond such as, for example, urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond.
  • a peptidomimetic agent in some embodiments is an unnatural peptide or a non-peptide agent that recreates the stereospatial properties of the binding elements of the antimicrobial peptide such that it has the binding activity and biological activity of the unmodified peptide.
  • the present disclosure also includes compounds that retain partial peptide characteristics. For example, any proteolytically unstable bond within a peptide of the present disclosure could be selectively replaced by a non-peptidic element such as an isostere (N-methylation; D-amino acid) or a reduced peptide bond while the rest of the molecule retains its peptidic nature.
  • one, two, three, four, or five peptide bonds are reduced in the antimicrobial peptides described herein. These reduced peptide bonds result in the conversion of an amide into a amine.
  • azapeptide analogs wherein the alpha-carbon atom is replaced with an isoelectronic nitrogen atom within the azapeptide, the side chains remain unchanged but the hydrogen atom on the alpha-carbon atom is missing. It is contemplated the entire peptide in some embodiments is constructed of azapeptide linkages or only one, two, three, four, five, six, seven, eight, nine, ten, fifteen, or all alpha-carbon atoms are replaced with azapeptide linkages. [091] In yet another aspect, the antimicrobial peptides described herein in some embodiments are chemically prepared as a comparable peptoid.
  • the peptoids in some embodiments are prepared using a glycine backbone in which the respective side chain of the peptide has been attached to the nitrogen atom rather than the alpha-carbon atom.
  • the conversion of the peptides into isomerically similar peptoids results in the production of compound which is more resistant to the activity of proteases or peptidases. It is contemplated one or more side chain from one or more amino acid has been converted into the corresponding peptoid molecule.
  • the number of amino acid residues converted into their peptoid counterpart is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or all of the amino acid residues.
  • Peptidomimetic compounds either agonists, substrates or inhibitors, have been described for a number of bioactive peptides/peptides such as opioid peptides, VIP, thrombin, HIV protease, etc. These methods are used to make peptidomimetics that possess at least the binding capacity and specificity of the desired antimicrobial peptide and also possess the biological activity. Knowledge of peptide chemistry and general organic chemistry available to those skilled in the art are sufficient, in view of the present disclosure, for designing and synthesizing such compounds.
  • such peptidomimetics in some embodiments are identified by inspection of the three-dimensional structure of a peptide or peptide of the present disclosure either free or bound in complex with a ligand.
  • the structure of a peptide of the present bound to its ligand can be gained by the techniques of nuclear magnetic resonance spectroscopy. Greater knowledge of the stereochemistry of the interaction of the peptide with its ligand or receptor will permit the rational design of such peptidomimetic agents.
  • the structure of a peptide or peptide of the invention in the absence of ligand could also provide a scaffold for the design of mimetic molecules.
  • Peptides described herein may be stabilized by one or more bonds, such as covalent bonds.
  • stabilization of peptides comprises use of "Stapled Peptide” technology.
  • the general approach for "stapling" a peptide is that two key residues within the peptide are modified by attachment of linkers through the amino acid side chains. Once synthesized, the linkers are connected, optionally through a catalyst, thereby creating a bridge (or linkage) that physically constrains the peptide into its native alpha-helical shape. In addition to helping retain the native structure needed to interact with a target molecule, this conformation also provides stability against peptidases as well as promotes cell permeating properties.
  • peptide stapling in some embodiments encompasses the joining of two double bond-containing sidechains, two triple bond-containing sidechains, or one double bond-containing and one triple bond-containing side chain, which may be present in a peptide chain, using any number of reaction conditions and/or catalysts to facilitate such a reaction, to provide a singly "stapled" peptide.
  • the introduction of a staple entails a modification of standard peptide synthesis, with alpha-methyl, alpha-alkenyl amino acids being introduced at two positions along the peptide chain, separated by either three or six intervening residues (i + 4 or i + 7).
  • a peptide described herein is attached to a water-soluble polymer.
  • the water-soluble polymer is a nonpeptidic, nontoxic, and biocompatible.
  • a substance is considered biocompatible if the beneficial effects associated with use of the substance alone or with another substance (e.g., an antimicrobial peptide) in connection with living tissues (e.g., administration to a patient) outweighs any deleterious effects as evaluated by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist.
  • a water-soluble polymer is further non-immunogenic.
  • a substance is considered non-immunogenic if the intended use of the substance in vivo does not produce an undesired immune response (e.g., the formation of antibodies) or, if an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist.
  • an undesired immune response e.g., the formation of antibodies
  • an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist.
  • the antimicrobial peptides in some embodiments are conjugated with heterologous peptide segments or polymers, such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the peptides in some embodiments are linked to PEG to increase the hydrodynamic radius and hence increase the serum persistence.
  • the peptides in some embodiments are conjugated to any targeting agent, such as a ligand having the ability to specifically and stably bind to an external receptor.
  • methods and compositions of the embodiments relate to PEGylation of disclosed peptides. PEGylation is the process of covalent attachment of poly(ethylene glycol) polymer chains to another molecule, normally a drug or therapeutic protein.
  • PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target macromolecule.
  • the covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent from the host's immune system (reduced immunogenicity and antigenicity) or increase the hydrodynamic size (size in solution) of the agent, which prolongs its circulatory time by reducing renal clearance.
  • PEGylation can also provide water solubility to hydrophobic drugs and proteins.
  • the first step of the PEGylation is the suitable functionalization of the PEG polymer at one or both terminals.
  • PEGs that are activated at each terminus with the same reactive moiety are known as "homobifunctional,” whereas if the functional groups present are different, then the PEG derivative is referred as “heterobifunctional” or “heterofunctional.”
  • the chemically active or activated derivatives of the PEG polymer are prepared to attach the PEG to the desired molecule.
  • the choice of the suitable functional group for the PEG derivative is based on the type of available reactive group on the molecule that will be coupled to the PEG.
  • typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine.
  • the N-terminal amino group and the C- terminal carboxylic acid in some embodiments are sites of PEGylation.
  • first generation PEG derivatives are generally reacting the PEG polymer with a group that is reactive with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates, and carbonates.
  • hydroxyl groups typically anhydrides, acid chlorides, chloroformates, and carbonates.
  • more efficient functional groups such as aldehyde, esters, amides, etc., are made available for conjugation.
  • heterobifunctional PEGs are very useful in linking two entities, where a hydrophilic, flexible, and biocompatible spacer is needed.
  • Preferred end groups for heterobifunctional PEGs are maleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids, and NHS esters.
  • the most common modification agents, or linkers are based on methoxy PEG (mPEG) molecules. Their activity depends on adding a protein-modifying group to the alcohol end.
  • PEG diol polyethylene glycol
  • the diol is subsequently modified at both ends in order to make a hetero- or homo- dimeric PEGlinked molecule.
  • Proteins are generally PEGylated at nucleophilic sites, such as unprotonated thiols (cysteinyl residues) or amino groups.
  • cysteinyl -specific modification reagents include PEG maleimide, PEG iodoacetate, PEG thiols, and PEG vinylsulfone. All four are strongly cysteiny1-specific under mild conditions and neutral to slightly alkaline pH.
  • the water-soluble polymer is characterized as having from about 2 to about 300 termini.
  • Exemplary water soluble polymers include, but are not limited to, poly(alkylene glycols) such as polyethylene glycol (“PEG”), polypropylene glycol) (“PPG”), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefmic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polydimethylacrylamide (PDAAm), polyphosphazene, polyoxazolines (“POZ”), poly(N-acryloylmorpholine), and combinations thereof.
  • PEG polyethylene glycol
  • PPG polypropylene
  • the water-soluble polymer is not limited to a particular structure.
  • the water-soluble polymer is linear (e.g., an end capped, e.g., alkoxy PEG or a bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), a dendritic (or star) architecture, each with or without one or more degradable linkages.
  • the internal structure of the water-soluble polymer can be organized in any number of different repeat patterns and can be selected from the group consisting of homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer.
  • the weight-average molecular weight of the water-soluble polymer attached to a peptide described herein is from about 100 Daltons to about 150,000 Daltons.
  • Exemplary ranges include, for example, weight-average molecular weights in the range of greater than 5,000 Daltons to about 100,000 Daltons, in the range of from about 6,000 Daltons to about 90,000 Daltons, in the range of from about 10,000 Daltons to about 85,000 Daltons, in the range of greater than 10,000 Daltons to about 85,000 Daltons, in the range of from about 20,000 Daltons to about 85,000 Daltons, in the range of from about 53,000 Daltons to about 85,000 Daltons, in the range of from about 25,000 Daltons to about 120,000 Daltons, in the range of from about 29,000 Daltons to about 120,000 Daltons, in the range of from about 35,000 Daltons to about 120,000 Daltons, and in the range of from about 40,000 Daltons to about 120,000 Daltons.
  • PEGs will typically comprise a number of (OCH 2 CH 2 ) monomers [or (CH 2 CH 2 O) monomers, depending on how the PEG is defined]. As used herein, the number of repeating units is identified by the subscript “n” in “(OCH 2 CH 2 ) n.
  • the value of (n) typically falls within one or more of the following ranges: from 2 to about 3400, from about 100 to about 2300, from about 100 to about 2270, from about 136 to about 2050, from about 225 to about 1930, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1,200 to about 1,900.
  • n the number of repeating units
  • the water-soluble polymer is an end-capped polymer, that is, a polymer having at least one terminus capped with a relatively inert group, such as a lower C 1-6 alkoxy group, or a hydroxyl group.
  • a relatively inert group such as a lower C 1-6 alkoxy group, or a hydroxyl group.
  • a methoxy-PEG commonly referred to as mPEG
  • mPEG is a linear form of PEG wherein one terminus of the polymer is a methoxy ( — OCH 3 ) group, while the other terminus is a hydroxyl or other functional group that can be optionally chemically modified.
  • exemplary water-soluble polymers include, but are not limited to, linear or branched discrete PEG (dPEG); linear, branched, or forked PEGs; and Y-shaped PEG derivatives.
  • a water-soluble polymer comprises a polyglycerol (PG).
  • the polyglycerol is a hyperbranched PG (HPG).
  • the polyglycerol is a linear PG (LPG).
  • the polyglycerol is a midfunctional PG, a linear-block-hyperbranched PG or a side-chain functional PG.
  • a water-soluble polymer is a degradable synthetic PEG alternative.
  • degradable synthetic PEG alternatives include, but are not limited to, poly[oligo(ethylene glycol)methyl methacrylate] (POEGMA); backbone modified PEG derivatives generated by polymerization of telechelic, or di-end-functionalized PEG-based macromonomers; PEG derivatives comprising comonomers comprising degradable linkage such as poly [(ethylene oxide)-co-(methylene ethylene oxide)] [P(EO-co-MEO)], cyclic ketene acetals such as 5,6-benzo-2-methylene-1,3-dioxepane (BMDO), 2-methylene- 1,3- dioxepane (MDO), and 2-methyl ene-4-pheny1-1,3-dioxolane (MPDL) copolymerized with OEGMA; or poly-( ⁇ -caprolactone)-graft-poly(ethylene oxide) (POEGMA); backbone
  • a peptide described herein is conjugated to a degradable synthetic PEG alternative, such as for example, POEGM; backbone modified PEG derivatives generated by polymerization of telechelic, or di-end-functionalized PEG-based macromonomers; P(EO-co-MEO); cyclic ketene acetals such as BMDO, MDO, and MPDL copolymerized with OEGMA; or PCL-g-PEO.
  • a degradable synthetic PEG alternative such as for example, POEGM; backbone modified PEG derivatives generated by polymerization of telechelic, or di-end-functionalized PEG-based macromonomers; P(EO-co-MEO); cyclic ketene acetals such as BMDO, MDO, and MPDL copolymerized with OEGMA; or PCL-g-PEO.
  • a water-soluble polymer comprises a poly(zwitterions).
  • Exemplary poly(zwitterions) include, but are not limited to, poly(sulfobetaine methacrylate) (PSBMA), poly(carboxybetaine methacrylate) (PCBMA), and poly(2-methyacryloyloxyethyl phosphorylcholine) (PMPC).
  • a water-soluble polymer comprises a polycarbonate.
  • Exemplary polycarbones include, but are not limited to, pentafluorophenyl 5- methy1-2-oxo-1,3-dioxane-5-carboxylate (MTC-OC6F5).
  • a water- soluble polymer comprises a polymer hybrid, such as for example, a polycarbonate/PEG polymer hybrid, a peptide/protein-polymer conjugate, or a hydroxyl containing and/or zwitterionic derivatized polymer (e.g., a hydroxyl containing and/or zwitterionic derivatized PEG polymer).
  • a water-soluble polymer comprises a polysaccharide.
  • exemplary polysaccharides include, but are not limited to, dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethy1- starch (HES).
  • a water-soluble polymer comprises a glycan.
  • Exemplary classes of glycans include A-linked glycans, O-linked glycans, glycolipids, O-GlcNAc, and glycosainoglycans.
  • a water-soluble polymer comprises a polyoxazoline polymer.
  • a polyoxazoline polymer is a linear synthetic polymer, and similar to PEG, comprises a low polydispersity.
  • a polyoxazoline polymer is a polydispersed polyoxazoline polymer, characterized with an average molecule weight.
  • the average molecule weight of a polyoxazoline polymer includes, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, 100,000, 200,000, 300,000, 400,000, or 500,000 Da.
  • a polyoxazoline polymer comprises poly(2 -methyl 2- oxazoline) (PMOZ), poly(2-ethyl 2-oxazoline) (PEOZ), or poly(2 -propyl 2-oxazoline) (PPOZ).
  • a water-soluble polymer comprises a polyacrylic acid polymer.
  • a water-soluble polymer comprises polyamine.
  • Polyamine is an organic polymer comprising two or more primary amino groups.
  • a polyamine includes a branched polyamine, a linear polyamine, or cyclic polyamine.
  • a polyamine is a low-molecular-weight linear polyamine.
  • Exemplary polyamines include putrescine, cadaverine, spermidine, spermine, ethylene diamine, 1,3- diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, and piperazine.
  • a peptide described herein is conjugated to a moiety described herein is a lipid.
  • the lipid is a fatty acid.
  • the fatty acid is a saturated fatty acid. In other cases, the fatty acid is an unsaturated fatty acid.
  • Exemplary fatty acids include, but are not limited to, fatty acids comprising from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms.
  • the lipid binds to one or more serum proteins, thereby increasing serum stability and/or serum half-life.
  • the lipid is conjugated to a peptide described herein.
  • the lipid is a fatty acid, e.g., a saturated fatty acid or an unsaturated fatty acid.
  • the fatty acid is from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms.
  • the fatty acid comprises about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbon atoms in length.
  • the fatty acid comprises caproic acid (hexanoic acid), enanthic acid (heptanoic acid), caprylic acid (octanoic acid), pelargonic acid (nonanoic acid), capric acid (decanoic acid), undecylic acid (undecanoic acid), lauric acid (dodecanoic acid), tridecylic acid (tridecanoic acid), myristic acid (tetradecanoic acid), pentadecylic acid (pentadecanoic acid), palmitic acid (hexadecanoic acid), margaric acid (heptadecanoic acid), stearic acid (octadecanoic acid), nonadecylic acid (nonadecanoic acid), arachidic acid (eicosanoic
  • Certain embodiments of the present disclosure concern fusion proteins of the antimicrobial peptides or peptides. These molecules may have the peptides of the embodiments linked at the N- or C-terminus to a heterologous domain.
  • fusions may also employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Fusion proteins can comprise a half-life extender.
  • Another useful fusion includes the addition of a protein affinity tag, such as a serum albumin affinity tag or six histidine residues, or an immunologically active domain, such as an antibody epitope, preferably cleavable, to facilitate purification of the fusion protein.
  • Non-limiting affinity tags include polyhistidine, chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST).
  • the peptides or peptides of the embodiments in some embodiments are linked to a peptide that increases the in vivo half-life, such as an XTEN peptide, IgG Fe domain, albumin, or albumin binding peptide.
  • fusion proteins are well known to those of skill in the art. Such proteins can be produced, for example, by de novo synthesis of the complete fusion protein, or by attachment of the DNA sequence encoding the heterologous domain, followed by expression of the intact fusion protein.
  • fusion proteins that recover the functional activities of the parent proteins in some embodiments are facilitated by connecting genes with a bridging DNA segment encoding a peptide linker that is spliced between the peptides connected in tandem.
  • the linker would be of sufficient length to allow proper folding of the resulting fusion protein.
  • a peptide described herein is attached to a protein or a binding fragment thereof.
  • exemplary proteins include albumin, transferrin, or transthyretin.
  • the protein or a binding fragment thereof comprises an antibody, or its binding fragments thereof.
  • a peptide comprises a protein or a binding fragment thereof.
  • a peptide described herein comprising a protein or a binding fragment thereof has an increased serum half-life, and/or stability.
  • a peptide described herein comprising a protein or a binding fragment thereof has an increased antibiotic spectrum.
  • the protein is albumin.
  • Albumin is a family of water-soluble globular proteins. It is commonly found in blood plasma, comprising about 55-60% of all plasma proteins.
  • Human serum albumin (HSA) is a 585 amino acid polypeptide in which the tertiary structure is divided into three domains, domain I (amino acid residues 1-195), domain II (amino acid residues 196-383), and domain III (amino acid residues 384-585). Each domain further comprises a binding site, which can interact either reversibly or irreversibly with endogenous ligands such as long- and medium-chain fatty acids, bilirubin, or hemin, or exogenous compounds such as heterocyclic or aromatic compounds.
  • the protein is albumin is transferrin.
  • Transferrin is a 679 amino acid polypeptide that is about 80 kDa in size and comprises two Fe 3+ binding sites with one at the N-terminal domain and the other at the C-terminal domain.
  • human transferrin has a half-life of about 7-12 days.
  • the protein is transthyretin (TTR).
  • TTR transthyretin
  • Transthyretin is a transport protein located in the serum and cerebrospinal fluid which transports the thyroid hormone thyroxine (T4) and retino1-binding protein bound to retinol.
  • the protein is an antibody, or its binding fragments thereof.
  • an antibody or its binding fragments thereof comprise a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab’, divalent Fab2, F(ab)' 3 fragments, single-chain variable fragment (scFv), bis- scFv, (SCFV)2, diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof.
  • the protein comprises a scFv, bis-scFv, (scFv)2, dsFv, or sdAb. In some embodiments, the protein comprises a scFv. In some embodiments, the protein comprises a bis-scFv. In some embodiments, the protein comprises a (scFv)2. In some embodiments, the protein comprises a dsFv. In some embodiments, the protein comprises a sdAb. [0132] In some embodiments, the protein comprises an Fc portion of an antibody, e.g., of IgG, IgA, IgM, IgE, or IgD. In some embodiments, the moiety comprises an Fc portion of IgG (e.g, IgGi, IgG 3 , or IgG 4 ).
  • the antimicrobial peptides or peptides of the embodiments in some embodiments are chemically conjugated using bifunctional cross-linking reagents or fused at the protein level with peptide linkers.
  • Bifunctional cross-linking reagents have been extensively used for a variety of purposes, including preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies.
  • Suitable peptide linkers may also be used to link the peptide or peptide of the embodiments, such as Gly-Ser linkers.
  • Homobifunctional reagents that carry two identical functional groups induce cross- linking between identical and different macromolecules or subunits of a macromolecule, and linking of peptide ligands to their specific binding sites.
  • Heterobifunctional reagents contain two different functional groups. By taking advantage of the differential reactivities of the two different functional groups, cross-linking can be controlled both selectively and sequentially.
  • the bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g., amino-, sulfhydryl' guanidine-, indole-, carboxy1-specific groups.
  • reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis, and the mild reaction conditions under which they can be applied.
  • a majority of heterobifunctional cross-linking reagents contain a primary amine-reactive group and a thio1- reactive group.
  • heterobifunctional cross-linking reagents are used to generate peptides described herein.
  • the cross-linking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing coupling, in one example, of aldehydes to free thiols.
  • the cross-linking reagent can be modified to crosslink various functional groups.
  • any other linking/coupling agents and/or mechanisms known to those of skill in the art are in some embodiments used to combine peptides of the embodiments, such as, for example, antibody-antigen interaction, avidin biotin linkages, amide linkages, ester linkages, thioester linkages, ether linkages, thioether linkages, phosphoester linkages, phosphoramide linkages, anhydride linkages, disulfide linkages, ionic and hydrophobic interactions, bispecific antibodies and antibody fragments, or combinations thereof.
  • a cross-linker having reasonable stability in blood or other relevant tissue site of infection will be employed.
  • linkers Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo. These linkers are thus one group of linking agents.
  • non-hindered linkers also can be employed in accordance herewith.
  • Other useful cross-linkers include SATA, SPDP, and 2-iminothiolane. The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.
  • the peptide generally will be purified to separate the conjugate from unconjugated agents and from other contaminants.
  • a large number of purification techniques are available for use in providing conjugates of a sufficient degree of purity to render them clinically useful.
  • Purification methods based upon size separation such as gel filtration, gel permeation, or high performance liquid chromatography, will generally be of most use. Other chromatographic techniques, such as Blue-Sepharose separation, may also be used. Conventional methods to purify the fusion proteins from inclusion bodies in some embodiments are useful, such as using weak detergents, such as sodium N-lauroy1-sarcosine (SLS).
  • weak detergents such as sodium N-lauroy1-sarcosine (SLS).
  • the present disclosure contemplates fusing or conjugating a cel1-penetrating domain (also called a cell delivery domain, or cell transduction domain) to an antimicrobial peptide.
  • a cel1-penetrating domain also called a cell delivery domain, or cell transduction domain
  • Such domains are well known in the art and are generally characterized as short amphipathic or cationic peptides and peptide derivatives, often containing multiple lysine and arginine resides.
  • TAT sequence from HIV1 YGRKKRRQRRR
  • poly-D-Arg and poly-D-Lys sequences e.g, dextrorotary residues, eight residues in length.
  • cell penetrating peptide and “membrane translocation domain” are used interchangeably and refer to segments of peptide sequence that allow a peptide to cross the cell membrane (e.g, the plasma membrane in the case a eukaryotic cell).
  • CPP segments include, but are not limited to, segments derived from HIV Tat (e.g, GRKKRRQRRRPPQ), herpes virus VP22, the Drosophila Antennapedia homeobox gene product, protegrin I, Penetratin (RQIKIWFQNRRMKWKK) or melittin (GIGAVLKVLTTGLPALISWIKRKRQQ).
  • the CPP comprises the T1 (TKIESLKEHG), T2 (TQIENLKEKG), 26 (AALEALAEALEALAEALEALAEAAAA) or INF7 (GLFEAIEGFIENGWEGMIEGW Y GCG) CPP sequence.
  • Peptides in some embodiments are modified for in vivo use by the addition, at the amino- and/or carboxy1-terminal ends, of a blocking agent to facilitate survival of the peptide in vivo are contemplated.
  • a blocking agent to facilitate survival of the peptide in vivo are contemplated.
  • Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. These agents can be added either chemically during the synthesis of the peptide, or by recombinant DNA technology by methods familiar in the art.
  • blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues.
  • nanoparticles could be used for the packaging and delivery of the peptide.
  • a nucleic acid encoding a peptide of the present disclosure may be made by any technique known to one of ordinary skill in the art.
  • Non-limiting examples of a synthetic nucleic acid, particularly a synthetic oligonucleotide include a nucleic acid made by in vitro chemical synthesis using phosphotriester, phosphite or phosphoramidite chemistry and solid phase techniques, or via deoxynucleoside H-phosphonate intermediates.
  • a non-limiting example of enzymatically produced nucleic acids includes one produced by enzymes in amplification reactions such as PCRTM.
  • a non-limiting example of a biologically produced nucleic acid includes recombinant nucleic acid production in living cells, such as recombinant DNA vector production in bacteria.
  • nucleic acid(s) regardless of the length of the sequence itself, is in some embodiments combined with other nucleic acid sequences, including but not limited to, promoters, enhancers, polyadenylation signals, restriction enzyme sites, multiple cloning sites, coding segments, and the like, to create one or more nucleic acid construct(s).
  • the overall length may vary considerably between nucleic acid constructs.
  • a nucleic acid segment of almost any length may be employed, with the total length preferably being limited by the ease of preparation or use in the intended recombinant nucleic acid protocol.
  • Peptides described herein may comprise other peptides, small molecules, antibodies, nucleic acids, or other targeting agent.
  • the N-terminal modification or C-terminal modification comprises at least one targeting agent.
  • the targeting agent increases the local concentration of the peptide.
  • the targeting agent broadens or broadens the antibiotic spectrum.
  • compositions comprising a peptide disclosed herein and a pharmaceutically acceptable excipient, vehicle, or second therapeutic agent.
  • the compositions may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides described herein.
  • the peptides in some embodiments are different.
  • the peptides in some embodiments are the same or similar.
  • the compositions may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides.
  • the peptides in some embodiments are different.
  • the peptides may comprise different therapeutic agents, different peptides, or a combination thereof.
  • the peptides in some embodiments are the same or similar.
  • a pharmaceutical composition comprises a peptide having any one of SEQ ID NOS: 1-313.
  • a pharmaceutical composition comprises a peptide having any one of SEQ ID NOS: 2-313.
  • compositions comprise an anti-microbial peptide described herein are a pharmaceutically acceptable vehicle such as a diluent, adjuvant, excipient, or carrier with which at least one antibody is administered.
  • a pharmaceutically acceptable vehicle such as a diluent, adjuvant, excipient, or carrier with which at least one antibody is administered.
  • the compositions disclosed herein may further comprise one or more pharmaceutically acceptable salts, excipients or vehicles.
  • Pharmaceutically acceptable salts, excipients, or vehicles in some embodiments include carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, aerosolizing agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.
  • compositions described herein comprise a pharmaceutically acceptable excipient, carrier or adjuvant, such as an excipient, carrier or adjuvant that may be administered to a subject, together with at least one antibody of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.
  • a pharmaceutically acceptable excipient, carrier or adjuvant such as an excipient, carrier or adjuvant that may be administered to a subject, together with at least one antibody of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.
  • Neutral buffered saline or saline mixed with serum albumin are exemplary appropriate carriers.
  • the pharmaceutical compositions may include antioxidants such as ascorbic acid; low molecular weight peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, pluronics, or polyethylene glycol (PEG).
  • antioxidants such as ascorbic acid
  • low molecular weight peptides such as serum albumin, gelatin, or immunoglobulins
  • hydrophilic polymers such as polyviny
  • suitable tonicity enhancing agents include alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol, and the like.
  • Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and the like. Hydrogen peroxide in some embodiments is used as a preservative.
  • Suitable cosolvents include glycerin, propylene glycol, and PEG.
  • Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxy- propy1-beta-cyclodextrin.
  • Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal, and the like.
  • the buffers in some embodiments are conventional buffers such as acetate, borate, citrate, phosphate, bicarbonate, or Tris-HCl.
  • Acetate buffer in some embodiments is about pH 4-5.5, and Tris buffer can be about pH 7-8.5.
  • composition comprising a peptide described herein often is in liquid form or in a lyophilized or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and/or bulking agents.
  • a lyoprotectant is included, which is a non-reducing sugar such as sucrose, lactose or trehalose.
  • the amount of lyoprotectant generally included is such that, upon reconstitution, the resulting formulation will be isotonic, although hypertonic or slightly hypotonic formulations also are suitable in some embodiments.
  • lyoprotectant concentrations for sugars e.g., sucrose, lactose, trehalose
  • sugars e.g., sucrose, lactose, trehalose
  • concentrations for sugars in the pre-lyophilized formulation are from about 10 mM to about 400 mM.
  • a surfactant is included, such as for example, nonionic surfactants and ionic surfactants such as polysorbates (e.g., polysorbate 20, polysorbate 80); poloxamers (e.g., poloxamer 188); poly(ethylene glycol) phenyl ethers (e.g., Triton); sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; laury1-, myristy1-, linoley1-, or steary1-sulfobetaine; laury1-, myristy1-, linoley1-or steary1-sarcosine; linoleyl, myristy1-, or cety1-betaine; lauroamidopropy1-, cocamidopropy1-, linoleamidopropy1- , myristamidopropy
  • surfactant present in the pre-lyophilized formulation are from about 0.001-0.5%.
  • High molecular weight structural additives may include for example, acacia, albumin, alginic acid, calcium phosphate (dibasic), cellulose, carboxymethylcellulose, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, dextran, dextrin, dextrates, sucrose, tylose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylates, povidone, sodium alginate, tragacanth microcrystalline cellulose, starch, and
  • compositions in some embodiments are suitable for parenteral administration.
  • Exemplary compositions are suitable for injection or infusion into an animal by any route available to the skilled worker, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, or intralesional routes.
  • a parenteral formulation in some embodiments is a sterile, pyrogen-free, isotonic aqueous solution, optionally containing pharmaceutically acceptable preservatives.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringers' dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like.
  • compositions described herein may be formulated for controlled or sustained delivery in a manner that provides local concentration of the product (e.g., bolus, depot effect) and/or increased stability or half-life in a particular local environment.
  • compositions can include the formulation peptides, nucleic acids, or vectors disclosed herein with particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., as well as agents such as a biodegradable matrix, injectable microspheres, microcapsular particles, microcapsules, bioerodible particles beads, liposomes, and implantable delivery devices that provide for the controlled or sustained release of the active agent which then can be delivered as a depot injection.
  • agents such as a biodegradable matrix, injectable microspheres, microcapsular particles, microcapsules, bioerodible particles beads, liposomes, and implantable delivery devices that provide for the controlled or sustained release of the active agent which then can be delivered as a depot injection.
  • Techniques for formulating such sustained-or controlled-delivery means are known and a variety of polymers have been developed and used for the controlled release and delivery of drugs. Such polymers are typically biodegradable and biocompatible.
  • Polymer hydrogels including those formed by complexation of enantiomeric polymer or peptide segments, and hydrogels with temperature or pH sensitive properties, may be desirable for providing drug depot effect because of the mild and aqueous conditions involved in trapping bioactive protein agents (e.g., antibodies comprising an ultralong CDR3).
  • bioactive protein agents e.g., antibodies comprising an ultralong CDR3
  • pharmaceutical compositions comprise controlled release porous polymeric microparticles.
  • Suitable materials for this purpose include polylactides, polymers of poly-(a- hydroxycarboxylic acids), such as poly-D-(-)-3-hydroxybutyric acid, copolymers of L- glutamic acid and gamma ethy1-L-glutamate, poly(2-hydroxyethy1-methacrylate), and ethylene vinyl acetate, or poly-D-(-)-3-hydroxybutyric acid.
  • Other biodegradable polymers include poly(lactones), poly(acetals), poly(orthoesters), and poly(orthocarbonates).
  • Sustained-release compositions also may include liposomes, which can be prepared by any of several methods known in the art.
  • the carrier itself, or its degradation products, should be nontoxic in the target tissue and should not further aggravate the condition. This can be determined by routine screening in animal models of the target disorder or, if such models are unavailable, in normal animals.
  • Microencapsulation of recombinant proteins for sustained release has been performed successfully with human growth hormone (rhGH), interferon-(rhlFN-), interleukin-2, and MN rgpl20.
  • the sustained-release formulations of these proteins were developed using poly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties.
  • PLGA poly-lactic-coglycolic acid
  • the degradation products of PLGA, lactic and glycolic acids can be cleared quickly within the human body.
  • the degradability of this polymer can be depending on its molecular weight and composition.
  • Bioadhesive polymers are also contemplated for use in or with compositions of the present disclosure.
  • Bioadhesives are synthetic and naturally occurring materials able to adhere to biological substrates for extended time periods.
  • Carbopol and polycarbophil are both synthetic cross-linked derivatives of poly(acrylic acid).
  • Bioadhesive delivery systems based on naturally occurring substances include for example hyaluronic acid, also known as hyaluronan.
  • Hyaluronic acid is a naturally occurring mucopolysaccharide consisting of residues of D-glucuronic and N-acety1-D-glucosamine.
  • Hyaluronic acid is found in the extracellular tissue matrix of vertebrates, including in connective tissues, as well as in synovial fluid and in the vitreous and aqueous humor of the eye. Esterified derivatives of hyaluronic acid have been used to produce microspheres for use in delivery that are biocompatible and biodegradable.
  • Both biodegradable and non-biodegradable polymeric matrices in some embodiments are used to deliver compositions of the present disclosure, and such polymeric matrices may comprise natural or synthetic polymers. Biodegradable matrices are preferred. The period of time over which release occurs is based on selection of the polymer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable.
  • Exemplary synthetic polymers used to form the biodegradable delivery system include: polymers of lactic acid and glycolic acid, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkyl ene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyanhydrides, polyurethanes and copolymers thereof, poly(butyric acid), poly(valeric acid), alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose
  • Exemplary natural polymers include alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
  • the polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.
  • Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and triglycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and triglycerides
  • hydrogel release systems silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • Specific examples include, but are not limited to: (a) erosional systems in which the product is contained in a form within a matrix and (b) diffusional systems in which a product permeates at a controlled rate. Liposomes containing the product are often prepared by methods known by those skilled in the art.
  • Peptides described herein may be administered to a subject to treat a disease or condition, such as an infection.
  • the compositions described herein are administered enterically to the small intestine.
  • Routes of delivery may include non-invasive peroral (through the mouth), topical (skin), transmucosal (nasal, buccal/sublingual, vaginal, ocular and rectal) and inhalation routes, as well as parenteral routes, and other methods known in the art.
  • Parenteral refers to a route of delivery that is generally associated with injection, including intraorbital, infusion, intraarterial, intracarotid, intracap sular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrastemal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.
  • the compositions in some embodiments are in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.
  • administration of peptides described herein comprises inhalation (e.g., vaporization).
  • compositions in some embodiments are administered locally via implantation into the affected area of a membrane, sponge, or other appropriate material on to which a peptide disclosed herein has been absorbed or encapsulated.
  • the device in some embodiments is implanted into any suitable tissue or organ, and delivery of an antibody comprising an ultralong CDR3 antibody fragment, nucleic acid, or vector disclosed herein can be directly through the device via bolus, or via continuous administration, or via catheter using continuous infusion.
  • a pharmaceutical composition comprising a peptide disclosed herein may be formulated for inhalation, such as for example, as a dry powder.
  • Inhalation solutions in some embodiments are formulated in a liquefied propellant for aerosol delivery.
  • solutions in some embodiments are nebulized.
  • Additional pharmaceutical compositions for pulmonary administration include pulmonary delivery of chemically modified proteins.
  • the particle size should be suitable for delivery to the distal lung. For example, the particle size is 1 pm to 5 pm; however, larger particles in some embodiments are used, for example, if each particle is fairly porous.
  • formulations containing antibodies comprising a peptide disclosed herein may be administered orally.
  • Formulations administered in this fashion in some embodiments are formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
  • a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.
  • Additional agents in some embodiments are included to facilitate absorption of a selective binding agent. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders also can be employed.
  • Another preparation may involve an effective quantity of an antibody comprising a peptide disclosed herein in a mixture with non-toxic excipients which are suitable for the manufacture of tablets.
  • excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • Suitable and/or preferred pharmaceutical formulations in some embodiments are determined in view of the present disclosure and general knowledge of formulation technology, depending upon the intended route of administration, delivery format, and desired dosage. Regardless of the manner of administration, an effective dose is often calculated according to patient body weight, body surface area, or organ size. Further refinement of the calculations for determining the appropriate dosage for treatment involving each of the formulations described herein are routinely made in the art and is within the ambit of tasks routinely performed in the art. Appropriate dosages in some embodiments are ascertained through use of appropriate dose-response data.
  • the antimicrobial peptides of the present disclosure are useful in a variety of environments including industrial, clinical, the household, and personal care.
  • the peptide compositions of the present disclosure for industrial, pharmaceutical, household and personal care use may comprise at least one active ingredient, of which the peptide of the present disclosure is an active ingredient acting alone, additively, or synergistically against the target microbe.
  • the antimicrobial compositions of the present disclosure may be used to form contact-killing coatings or layers on a variety of substrates including personal care products (e.g., toothbrushes, contact lens cases and dental equipment), healthcare products, household products, food preparation surfaces and packaging, and laboratory and scientific equipment.
  • substrates include medical devices such as catheters, urological devices, blood collection and transfer devices, tracheotomy devices, intraocular lenses, wound dressings, sutures, surgical staples, membranes, shunts, gloves, tissue patches, prosthetic devices (e.g., heart valves) and wound drainage tubes.
  • other substrates include textile products such as carpets and fabrics, paints and joint cement. A further use is as an antimicrobial soil fumigant.
  • the substrate is a shunt, a catheter, artificial protheses, screw, plate, or pin.
  • a device configured for external fixation of a surgical device is coated with a composition comprising a peptide described herein.
  • peptides comprising antimicrobial activity, wherein the peptide is capable of killing a microbial organism or inhibiting its growth.
  • the antimicrobial activities of the antimicrobial peptides can include, without limitation, antibacterial, antiviral, or antifungal activities.
  • the present disclosure provides antimicrobial peptides with activity against gram-negative bacteria.
  • the present disclosure also provides methods of using the antimicrobial peptides and antimicrobial compositions of the present disclosure to prevent, inhibit or terminate the growth of at least one microbe which may include, for example, bacteria, archaea, fungi, algae, protozoa, multicellular parasites, and viruses.
  • therapeutic agents which may comprise a protein or peptide that modulates the activity of another protein, peptide, cell or tissue. Modulating the activity in some embodiments comprises increasing, decreasing, stimulating, or preventing the activity or expression of the protein, peptide, cell or tissue.
  • Therapeutic agents may modulate the activity of proteins or peptides involved in the etiology of a disease or disorder. Exemplary proteins may include, but are not limited to, at least a portion of a hormone, kinase, receptor, ligand, growth factor, regulatory protein, metabolic protein, cytokine, chemokine, interferon, phosphatase, antibody or any combination thereof.
  • Peptides and compositions of the present disclosure may result in antimicrobial effects on a target microbial organism.
  • peptides and compositions disclosed herein are used to treat a disease or infection associated with the target microbial organism.
  • An antimicrobial effect includes preventing or inhibiting the growth of or killing the target microbial organisms, or interfering with any biological functions of the target microbial organisms.
  • the compositions of the present disclosure can be used to treat or prevent a disease or infection at any place in a host, e.g., at any tissue including surfaces of any tissue or implant.
  • the compositions are used to specifically kill or inhibit bacterial target microbial organisms in body fluid (e.g, blood, sputum).
  • peptides described herein are bacteriostatic.
  • Peptides described herein may be used for the treatment or alleviation of diseases, including but not limited to: 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder; and/or 2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder.
  • treatment comprises clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology.
  • Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • those in need of treatment may include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.
  • treatment of a disease or condition comprises executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance.
  • the administration of one or more antimicrobial peptides provided herein is used to prevent, treat or relieve the symptoms of a microbial infection.
  • peptides described herein comprise antimicrobial activity, including prevention, inhibition or termination of a microbe.
  • prevention is considered to be the obstruction or hindrance of any potential microbial growth
  • inhibition is considered to be a reduction in microbial growth. This may occur via, but is not limited to, a microbiostatic mechanism such as interference in the synthesis of the cell wall or binding to ribosomal subunits to prevent production of microbial proteins.
  • termination is considered to be the actual killing of the microbes by the presence of the composition.
  • peptides described herein treat organisms comprising the phylogenetic domains bacteria and archaea, as well as unicellular and filamentous fungi (e.g., yeasts and molds), unicellular and filamentous algae, unicellular and multicellular parasites, and viruses.
  • compositions of the present disclosure are effective against bacteria including Gram-positive and Gram-negative cocci, Gram-positive and Gram negative straight, curved and helical/vibroid and branched rods, sheathed bacteria, sulfur- oxidizing bacteria, sulfur or sulfate-reducing bacteria, spirochetes, actinomycetes and related genera, myxobacteria, mycoplasmas, rickettsias and chlamydias, cyanobacteria, archea, fungi, parasites, viruses and algae.
  • the target microbial organisms of the present disclosure include, without limitation, Enter obacteriaceae, including Escherichia and Klebsiella spp., Enterococcus spp., Acinetobacter spp., Candida spp., Salmonella spp., Staphylococcus spp., Streptococcus spp.
  • compositions are effective against drug-resistant and multi-drug resistant bacterial species.
  • compositions of the present disclosure are effective against Gram-negative bacteria.
  • Gram-positive and Gram-negative cocci include, but are not limited to, Aerococcus, Enterococcus, Halococcus, Leuconostoc, Micrococcus, Mobiluncus, Moraxella catarrhalis, Neisseria (including N gonorrheae and N. meningitidis), Pediococcus, Peptostreptococcus, Staphylococcus species (including S. aureus, methicillin resistant S. aureus, coagulase-negative S. aureus, and S. saprophyticus), Streptococcus species (including S. pyogenes, S.
  • the Gram -positive and Gram-negative straight, curved, helical/vibrioid and branched rods include, but are not limited to, Acetobacter, Acinetobacter species (including A. baumannii), Actinobacillus equuli, Aeromonas, Agrobacterium, Alcaligenes, Aquaspirillum, Arcanobacterium haemolyticum, Bacillus species (including B. cereus and B. anthracis), Bacteroides species (including B. fragilis), Bartonella, Bordetella species (including B.
  • Flavobacterium species including E. meninosepticum, Francisella species (including E. tularensis), Fusobacterium (including E. nucleatum), Gardnerella species (including G. vaginalis), Gluconobacter, Haemophilus species (including//, influenzae and H. ducreyi), Haftiia, Helicobacter (including//. pylori), Herpetosiphon, Klebsiella species (including K. pneumoniae), Kluyvera, Lactobacillus, Legionella species (including E. pneumophila), Leptotrichia, Listeria species (including E.
  • compositions are effective against drug-resistant and multi-drug resistant bacterial species.
  • the clinical diseases or infections caused by Gram-positive and/or Gram negative bacteria, treatable with the peptides described herein include abscesses, bacteremia, blood stream infection, contamination of peritoneal dialysis fluid, endocarditis, pneumonia, meningitis, osteomyelitis, cellulitis, pharyngitis, otitis media, sinusitis, scarlet fever, arthritis, urinary tract infection, laryngotracheitis, erysipeloid, gas gangrene, tetanus, typhoid fever, acute gastroenteritis, bronchitis, bronchiolitis, epiglottitis, plague, sepsis, chancroid, wound and bum infection, cholera, glanders, periodontitis, genital infections, empyema, granuloma inguinale, Legionnaire's disease, paratyphoid, bacillary dysentery, brucellosis, diphtheria, pertussi
  • swine erysipelas peritonitis, intraabdominal infection, abortion, encephalitis, anthrax, nocardiosis, pericarditis, mycetoma, peptic ulcer, melioidosis, Haverhill fever, tularemia, Moko disease, galls (e.g., crown, cane and leaf), hairy root, bacterial rot, bacterial blight, bacterial brown spot, bacterial wilt, bacterial fin rot, dropsy, columnaris disease, pasteurellosis, furunculosis, enteric redmouth disease, vibriosis, tuberculosis, offish, and fouling of medical devices.
  • the infection may be acute.
  • the infection may be chronic.
  • infections may be community-acquired or hospita1-acquired.
  • peptides are used to treat chronic skin ulcers, infected acute wounds or bum wounds, infected skin eczema, impetigo, atopic dermatitis, acne, external otitis, vaginal infections, seborrheic dermatitis, oral infections, parodontitis, conjunctivitis or pneumonia.
  • Another embodiment of the present disclosure relates to administering an antimicrobial peptide provided herein in combination with an antibiotic.
  • Antibiotics suitable for co-administration with the antimicrobial peptides disclosed herein include substances, produced synthetically or naturally, which can inhibit the growth of or kill microbial organisms.
  • antibiotics include, without any limitation, beta-lactam antibiotics (e.g, ampicillin, aziocillin, aztreonam, carbenicillin, cefoperazone, ceftriaxone, cephaloridine, cephalothin, cloxacillin, moxalactam, penicillin, piperacillin, and ticarcillin), amoxicillin, bacitracin, chloramphenicol, clindamycin, capreomycin, colistimethate, ciprofloxacin, doxycycline, erythromycin, fusidic acid, fosfomycin, fusidate sodium, gramicidin, gentamycin, lincomycin, minocycline, macrolides, monobactams, nalidixic acid, novobiocin, ofloxcin, rifamycins, tetracyclines, vancomycin, tobramycin, fluoroquinolones, polymyxins, DNA gyrase inhibitor
  • compositions comprising an antimicrobial peptide and an agent which can enhance, maintain, or facilitate the function or activity of the peptide.
  • the chemical is a protease inhibitor.
  • the peptide is exposed to a protease-present environment where the presence of the protease may reduce the antimicrobial activity of the peptide via, for example, enzymatic degradation.
  • the combination of a protease inhibitor and the peptide stabilizes the peptide from the protease degradation and thus enhances the activity of the antimicrobial peptide.
  • the protease-present environment includes, for example, body fluid (e.g., urine, blood, serum, salvia, sputum, and mucosal fluid).
  • the protease includes, for example, neutrophil elastase, proteinase-3, cysteine protease, metalloprotease, serine-protease, or other proteases derived from bacteria and/or hosts.
  • the protease inhibitor includes, for example, BMF, EDTA, PMSF, benzamidine, and/or recombinant alpha-1 antitrypsin (rAAT).
  • a peptide, as disclosed herein, may be expressed by recombinant methods.
  • a nucleic acid encoding a peptide is often isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
  • DNA encoding the peptide may be prepared by PCR amplification and sequenced using conventional procedures (e.g, by using oligonucleotide probes that are capable of binding specifically to nucleotides encoding a peptide).
  • nucleic acid encoding a peptide is PCR amplified, restriction enzyme digested and gel purified. The digested nucleic acid in some embodiments is inserted into a replicable vector.
  • the replicable vector containing the nucleic acid insertion in some embodiments is transformed or transduced into a host cell for further cloning (amplification of the DNA) or for expression of the peptide.
  • Host cells in some embodiments are prokaryotic or eukaryotic cells.
  • a vector comprises an expression cassette for a peptide described herein.
  • an expression cassette comprises at least one promoter and a polynucleotide sequence encoding for the peptide.
  • an expression cassette comprises at least one terminator.
  • Polynucleotide sequences encoding peptide components disclosed herein may be obtained by PCR amplification with overlapping oligonucleotide primers.
  • Polynucleotide sequences in some embodiments are isolated and sequenced from peptide-producing cells. Altematively, polynucleotides in some embodiments are synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the peptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic and/or eukaryotic hosts.
  • phage vectors containing replicon and control sequences that are compatible with the host microorganism may be used as transforming vectors in connection with these hosts.
  • bacteriophage such as XGEMTM- 11 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.
  • Peptides in some embodiments are expressed in intracellularly (e.g., cytoplasm) or extracellularly (e.g., secretion).
  • the vector may comprise a secretion signal which enables translocation of the peptide to the outside of the cell.
  • Suitable host cells for cloning or expression of peptide-encoding vectors include prokaryotic or eukaryotic cells.
  • the host cell in some embodiments is a eukaryotic cell.
  • eukaryotic cells include, but are not limited to, Human Embryonic Kidney (HEK) cell, Chinese Hamster Ovary (CHO) cell, fungi, yeasts, invertebrate cells (e.g., plant cells and insect cells), lymphoid cell (e.g., YO, NSO, Sp20 cell).
  • HEK Human Embryonic Kidney
  • CHO Chinese Hamster Ovary
  • fungi fungi
  • yeasts invertebrate cells
  • lymphoid cell e.g., YO, NSO, Sp20 cell
  • the host cell is Pichia pastoris or S. cerevisiae.
  • suitable mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); baby hamster kidney cells (BHK); mouse Sertoli cells; monkey kidney cells (CV1); African green monkey kidney cells (VERO- 76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TR1 cells; MRC 5 cells; and FS4 cells.
  • the host cell in some embodiments is a prokaryotic cell (e.g., E. coli).
  • Host cells in some embodiments are transformed with vectors containing nucleotides encoding a peptide.
  • Transformed host cells in some embodiments are cultured in media.
  • the media may be supplemented with one or more agents for inducing promoters, selecting transformants, or amplifying or expressing the genes encoding the desired sequences.
  • Methods for transforming host cells are known in the art and may include electroporation, calcium chloride, or polyethylene glycol/DMSO.
  • host cells in some embodiments are transfected or transduced with vectors containing nucleotides encoding a peptide.
  • Transfected or transduced host cells in some embodiments are cultured in media.
  • the media is supplemented with one or more agents for inducing promoters, selecting transfected or transduced cells, or expressing genes encoding the desired sequences.
  • the expressed peptides in some embodiments are secreted into and recovered from the periplasm of the host cells or transported into the culture media. Protein recovery from the periplasm may involve disrupting the host cell. Disruption of the host cell may comprise osmotic shock, sonication or lysis. Centrifugation or filtration is often used to remove cell debris or whole cells.
  • the peptides in some embodiments are further purified, for example, by affinity resin chromatography.
  • peptides that are secreted into the culture media may be isolated therein.
  • Cells in some embodiments are removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced.
  • the expressed peptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.
  • PAGE polyacrylamide gel electrophoresis
  • Peptide production may be conducted in large quantity by a fermentation process.
  • Various large-scale fed-batch fermentation procedures are available for production of recombinant proteins.
  • Large-scale fermentations have at least 1000 liters of capacity, preferably about 1,000 to 100,000 liters of capacity. These fermenters use agitator impellers to distribute oxygen and nutrients, especially glucose (a preferred carbon/energy source).
  • Small scale fermentation refers generally to fermentation in a fermenter that is no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters.
  • induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD550 of about 180-220, at which stage the cells are in the early stationary phase.
  • a desired density e.g., an OD550 of about 180-220
  • inducers in some embodiments are used, according to the vector construct employed, as is known in the art and described above.
  • Cells in some embodiments are grown for shorter periods prior to induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction time may be used.
  • chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis,trans-isom erase with chaperone activity) may be used to co-transform the host prokaryotic cells.
  • the chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells.
  • host strains deficient for proteolytic enzymes can be used for the present disclosure.
  • host cell strains in some embodiments are modified to effect genetic mutation(s) in the genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof.
  • known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof.
  • Standard protein purification methods known in the art can be employed.
  • the following procedures are exemplary of suitable purification procedures: fractionation on immunoaffmity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, hydroxyl apatite chromatography, gel electrophoresis, dialysis, and affinity chromatography and gel filtration using, for example, Sephadex G-75.
  • Peptides in some embodiments are concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ® ultrafiltration unit.
  • protease inhibitors or protease inhibitor cocktails in some embodiments are included in any of the foregoing steps to inhibit proteolysis of the peptide.
  • a peptide or fragment thereof may not be biologically active upon isolation.
  • Various methods for "refolding" or converting a peptide to its tertiary structure and generating disulfide linkages can be used to restore biological activity. Such methods include exposing the solubilized peptide to a pH usually above 7 and in the presence of a particular concentration of a chaotrope. The selection of chaotrope is very similar to the choices used for inclusion body solubilization, but usually the chaotrope is used at a lower concentration and is not necessarily the same as chaotropes used for the solubilization.
  • the refolding/oxidation solution will also contain a reducing agent or the reducing agent plus its oxidized form in a specific ratio to generate a particular redox potential allowing for disulfide shuffling to occur in the formation of the protein's cysteine bridge(s).
  • Some of the commonly used redox couples include cystein/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride, dithiothreitol(DTT)/dithiane DTT, and 2-mercaptoethanol(bME)/di-thio-b(ME).
  • a cosolvent may be used to increase the efficiency of the refolding, and common reagents used for this purpose include glycerol, polyethylene glycol of various molecular weights, arginine and the like.
  • peptides described herein comprise disulfide linkages, or their equivalent. Such linkages in some instances are enabled by bridging amino acids.
  • intramolecular linkages comprise amide, ether, disulfide, sulfide, alkyl, ester, or other bond.
  • Non-limiting examples of bridging amino acids include aspartic acid, glutamic acid, serine, cysteine, penicillamine, dehydroalanine, or other amino acid capable of intramolecular reaction to form one or more rings. Bridging amino acids in some instances comprise canonical or non-canonical amino acids described herein.
  • linkages include but are not limited to alkenyl linkages (formed by olefin metathesis), alkyl linkages, lactam formation (amide linkages), lanthionine linkages, thio linkages (formed through displacement of electrophiles or conjugate addition), ether linkages, sulfate linkages, sulfmyl linkages, sulfamide linkages, ester linkages, amine linkages, or other linkage.
  • the linkage comprises the structure of ,or ; wherein R is a substituent, Ar is aryl, and HA is heteroaryl.
  • R is alkyl, aralkyl, or cycloalkyl.
  • R is C 1 -C 6 alkyl.
  • Ar is optionally substituted phenyl or naphthyl.
  • HA is optionally substituted: pyridinyl, imidazolyl, thiophenyl, pyrrol yl, thiazolyl, oxazolyl, or furanyl.
  • two amino acids from a peptide described herein are cyclized to form an intramolecular linkage.
  • Amino refers to the -NH 2 radical.
  • Niro refers to the -NO 2 radical.
  • Alkyl refers to a straight or branched hydrocarbon chain radical, has from one to thirty carbon atoms, and is attached to the rest of the molecule by a single bond. Alkyls comprising any number of carbon atoms from 1 to 30 are included. An alkyl comprising up to 30 carbon atoms is referred to as a C 1 -C 30 alkyl, likewise, for example, an alkyl comprising up to 12 carbon atoms is a C 1 -C 12 alkyl. Alkyls (and other moieties defined herein) comprising other numbers of carbon atoms are represented similarly.
  • Alkyl groups include, but are not limited to, C 1 -C 30 alkyl, C 1 -C 20 alkyl, C 1 -C 15 alkyl, C 1 -C 10 alkyl, C 1 -C 8 alkyl, C 1 -C 6 alkyl, C 1 -C 4 alkyl, C 1 -C 3 alkyl, C 1 -C 2 alkyl, C 2 -C 8 alkyl, C 3 -C 8 alkyl and C 4 -C 8 alkyl.
  • Representative alkyl groups include, but are not limited to, methyl, ethyl, «-propyl,
  • Alkyl comprising unsaturations include alkenyl and alkynyl groups. Unless stated otherwise specifically in the specification, an alkyl group may be optionally substituted as described below.
  • Alkylene or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain, as described for alkyl above. Unless stated otherwise specifically in the specification, an alkylene group may be optionally substituted as described below.
  • Alkoxy refers to a radical of the formula -OR a where R a is an alkyl radical as defined. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted as described below.
  • Aryl refers to a radical derived from a hydrocarbon ring system comprising hydrogen, 6 to 30 carbon atoms and at least one aromatic ring.
  • the aryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems.
  • Aryl radicals include, but are not limited to, aryl radicals derived from the hydrocarbon ring systems of aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, .v-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene.
  • aryl or the prefix “ar-“ (such as in “aralkyl”) is meant to include aryl radicals that are optionally substituted.
  • Cycloalkyl or “carbocycle” refers to a stable, non-aromatic, monocyclic or polycyclic carbocyclic ring, which may include fused or bridged ring systems, which is saturated or unsaturated.
  • Representative cycloalkyls or carbocycles include, but are not limited to, cycloalkyls having from three to fifteen carbon atoms, from three to ten carbon atoms, from three to eight carbon atoms, from three to six carbon atoms, from three to five carbon atoms, or three to four carbon atoms.
  • Monocyclic cycloalkyls or carbocycles include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Polycyclic cycloalkyls or carbocycles include, for example, adamantyl, norbornyl, decalinyl, bicyclo[3.3.0]octane, bicyclo[4.3.0]nonane, cis-decalin, trans-decalin, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, and bicyclo[3.3.2]decane, and 7,7-dimethy1-bicyclo[2.2.1]heptanyl.
  • a cycloalkyl or carbocycle group may be optionally substituted.
  • Illustrative examples of cycloalkyl groups include, but are not limited to, the following moieties:
  • fused refers to any ring structure described herein which is fused to an existing ring structure.
  • the fused ring is a heterocyclyl ring or a heteroaryl ring
  • any carbon atom on the existing ring structure which becomes part of the fused heterocyclyl ring or the fused heteroaryl ring may be replaced with a nitrogen atom.
  • Halo or “halogen” refers to bromo, chloro, fluoro or iodo.
  • Haloalkyl refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g ., trifluoromethyl, difluoromethyl, fluoromethyl, tri chi orom ethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like. Unless stated otherwise specifically in the specification, a haloalkyl group may be optionally substituted.
  • Haloalkoxy similarly refers to a radical of the formula -OR a where R a is a haloalkyl radical as defined. Unless stated otherwise specifically in the specification, a haloalkoxy group may be optionally substituted as described below.
  • Heterocycloalkyl or “heterocyclyl” or “heterocyclic ring” or “heterocycle” refers to a stable 3- to 24-membered non-aromatic ring radical comprising 2 to 23 carbon atoms and from one to 8 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorous and sulfur.
  • the heterocyclyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quatemized; and the heterocyclyl radical may be partially or fully saturated.
  • heterocyclyl radicals include, but are not limited to, azetidinyl, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl
  • heterocyclyl group may be optionally substituted.
  • heterocycloalkyl groups also referred to as non-aromatic heterocycles, include: and the like.
  • heterocycloalkyl also includes all ring forms of the carbohydrates, including but not limited to the monosaccharides, the disaccharides and the oligosaccharides. Unless otherwise noted, heterocycloalkyls have from 2 to 10 carbons in the ring.
  • the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including the heteroatoms) that make up the heterocycloalkyl (i.e. skeletal atoms of the heterocycloalkyl ring). Unless stated otherwise specifically in the specification, a heterocycloalkyl group may be optionally substituted.
  • heteroaryl refers to optionally substituted aromatic monoradicals containing from about five to about twenty skeletal ring atoms, where one or more of the ring atoms is a heteroatom independently selected from among oxygen, nitrogen, sulfur, phosphorous, silicon, selenium and tin but not limited to these atoms and with the proviso that the ring of said group does not contain two adjacent O or S atoms.
  • the two or more heteroatoms can be the same as each another, or some or all of the two or more heteroatoms can each be different from the others.
  • heteroaryl includes optionally substituted fused and non-fused heteroaryl radicals having at least one heteroatom.
  • heteroaryl also includes fused and non-fused heteroaryls having from five to about twelve skeletal ring atoms, as well as those having from five to about ten skeletal ring atoms. Bonding to a heteroaryl group can be via a carbon atom or a heteroatom.
  • an imidiazole group may be attached to a parent molecule via any of its carbon atoms (imidazo1-2-yl, imidazo1-4-yl or imidazo1-5-yl), or its nitrogen atoms (imidazo1- 1-yl or imidazo1-3-yl).
  • a heteroaryl group may be further substituted via any or all of its carbon atoms, and/or any or all of its heteroatoms.
  • a fused heteroaryl radical may contain from two to four fused rings where the ring of attachment is a heteroaromatic ring and the other individual rings in some embodiments are alicyclic, heterocyclic, aromatic, heteroaromatic or any combination thereof.
  • a non-limiting example of a single ring heteroaryl group includes pyridyl; fused ring heteroaryl groups include benzimidazolyl, quinolinyl, acridinyl; and a non-fused bi-heteroaryl group includes bipyridinyl.
  • heteroaryls include, without limitation, furanyl, thienyl, oxazolyl, acridinyl, azepinyl, phenazinyl, benzimidazolyl, benzindolyl, benzofuranyl, benzofuranonyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzothiophenyl, benzoxadiazolyl, benzodioxolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzotriazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzothienyl (benzothiophenyl), benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cinn
  • substituted means any of the above groups (e.g ., alkyl, alkylene, alkoxy, aryl, cycloalkyl, haloalkyl, heterocyclyl and/or heteroaryl) may be further functionalized wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atom substituent.
  • a substituted group may include one or more substituents selected from: oxo, amino, -CO 2 H, nitrile, nitro, hydroxyl, thiooxy, alkyl, alkylene, alkoxy, aryl, cycloalkyl, heterocyclyl, heteroaryl, dialkylamines, arylamines, alkylarylamines, diarylamines, trialkylammonium (-N + R.
  • N-oxides, imides, and enamines a silicon atom in groups such as trialkyl silyl groups, di al ky 1 aryl si 1 y 1 groups, al ky 1 di aryl si 1 y 1 groups, triarylsilyl groups, perfluoroalkyl or perfluoroalkoxy, for example, trifluoromethyl or trifluoromethoxy.
  • “Substituted” also means any of the above groups in which one or more hydrogen atoms are replaced by a higher-order bond (e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles.
  • a higher-order bond e.g., a double- or triple-bond
  • nitrogen in groups such as imines, oximes, hydrazones, and nitriles.
  • R g and R h are the same or different and independently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, /V-heterocyclyl, heterocyclylalkyl, heteroaryl, A'-heteroaryl and/or heteroaryl alkyl.
  • each of the foregoing substituents may also be optionally substituted with one or more of the above substituents.
  • any of the above groups in some embodiments are substituted to include one or more internal oxygen, sulfur, or nitrogen atoms.
  • an alkyl group may be substituted with one or more internal oxygen atoms to form an ether or polyether group.
  • an alkyl group may be substituted with one or more internal sulfur atoms to form a thioether, disulfide, etc.
  • optionally substituted alkyl means either "alkyl” or "substituted alkyl” as defined above.
  • an optionally substituted group may be un-sub stituted (e.g., -CH 2 CH 3 ), fully substituted (e.g., -CF 2 CF 3 ), mono- sub stituted (e.g., -CH 2 CH 2 F) or substituted at a level anywhere in-between fully substituted and mono-substituted
  • any substituents described should generally be understood as having a maximum molecular weight of about 1,000 daltons, and more typically, up to about 500 daltons.
  • a “tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule.
  • the compounds presented herein may exist as tautomers. Tautomers are compounds that are interconvertible by migration of a hydrogen atom, accompanied by a switch of a single bond and adjacent double bond. In bonding arrangements where tautomerization is possible, a chemical equilibrium of the tautomers will exist. All tautomeric forms of the compounds disclosed herein are contemplated. The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH. Some examples of tautomeric interconversions include:
  • a “metabolite” of a compound disclosed herein is a derivative of that compound that is formed when the compound is metabolized.
  • active metabolite refers to a biologically active derivative of a compound that is formed when the compound is metabolized.
  • metabolized refers to the sum of the processes (including, but not limited to, hydrolysis reactions and reactions catalyzed by enzymes, such as, oxidation reactions) by which a particular substance is changed by an organism. Thus, enzymes may produce specific structural alterations to a compound.
  • Metabolites of the compounds disclosed herein can be identified either by administration of compounds to a host and analysis of tissue samples from the host, or by incubation of compounds with hepatic cells in vitro and analysis of the resulting compounds. Both methods are well known in the art. Metabolites of a compound may be formed by oxidative processes and correspond to the corresponding hydroxy-containing compound. A compound may be metabolized to one or more pharmacologically active metabolites.
  • a derivative of a peptide refers to a fragment, analog, homolog, complex and/or aggregate of the peptide.
  • identity or “homology” shall be construed to mean the percentage of amino acid residues in the candidate sequence that are identical with the residue of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity.
  • Conservative substitutions in some embodiments involve substitution of one amino acid of similar shape or charge for another, including but not limited to substitution of a natural amino acid for a different natural amino acid, substitution of a non-canonical amino acid for a different non- canonical amino acid, substitution of a non-canonical amino acid for a natural amino acid, or substitution of a natural amino acid for a non-canonical amino acid.
  • a polynucleotide or polynucleotide region has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" or "homology" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. Neither N- or C-terminal extensions nor insertions shall be construed as reducing identity or homology. Alignment and the percent homology or sequence identity can be determined using software programs known in the art. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters.
  • substantially pure a peptide or peptide that has been separated and purified to at least some degree from the components that naturally accompany it.
  • a peptide or peptide is substantially pure when it is at least about 60%, or at least about 70%, at least about 80%, at least about 90%, at least about 95%, or even at least about 99%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • a substantially pure peptide or peptide may be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in a cell that does not normally express that protein, or by chemical synthesis.
  • essentially free in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts.
  • the total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%.
  • Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
  • Disorder or “disease” refers to a condition that would benefit from treatment with a substance/molecule (e.g., a peptide as disclosed herein) or method disclosed herein. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
  • a substance/molecule e.g., a peptide as disclosed herein
  • This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, rodents (e.g., mice and rats), and monkeys; domestic and farm animals; and zoo, sports, laboratory, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.
  • the mammal is selected from a human, rodent, or monkey.
  • subject and “individual” and “patient” are used interchangeably herein, and refer to an animal, for example a human or non-human animal (e.g., a mammal), to whom treatment, including prophylactic treatment, with a pharmaceutical composition as disclosed herein, is provided.
  • subject refers to human and nonhuman animals.
  • non-human animals includes all vertebrates, e.g, mammals, such as nonhuman primates (particularly higher primates), sheep, dogs, rodents (e.g. mouse or rat), guinea pigs, goats, pigs, cats, rabbits, cows, and non-mammals such as chickens, amphibians, reptiles etc.
  • the subject is human.
  • the subject is an experimental animal or animal substitute as a disease model.
  • Non-human mammals include mammals such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g. mouse or rat), guinea pigs, goats, pigs, cats, rabbits and cows.
  • Embodiment 1 An isolated peptide, wherein the peptide has a sequence comprising Formula (I): Y1-X1-X2- X3 -X4-X5 -X6-X7 -X8-X9-X 10-X 11 -X 12-X 13 -X 14-X 15 -X 16-X 17-X 18-X 19-X20-X21 - Y2 Formula (I); wherein Y1 is absent or an N-terminal modification; X1 and X2 are independently absent, or any amino acid; X3 is absent or any amino acid; X4 is positively charged amino acid; X5 is any amino acid; X6 is a non-polar amino acid; X7 is any amino acid; X8 is a non-polar amino acid; X9 is any amino acid; X10 is any amino acid; X11 is a bridging amino acid; X
  • Embodiment 3. The peptide of embodiment 1 or 2, wherein X2 is absent.
  • Embodiment 4. The peptide of any one of embodiments 1-3, wherein X1 and X2 if present are independently a non-polar amino acid.
  • Embodiment 5. The peptide of any one of embodiments 1-3, wherein X1 and X2 if present are independently G, A, V, L, I, M, F, W, or P.
  • Embodiment 6. The peptide of any one of embodiments 1-5, wherein X3 is absent.
  • Embodiment 7. The peptide of any one of embodiments 1-6, wherein X4 is K, R, or H.
  • Embodiment 8. The peptide of any one of embodiments 1-7, wherein X5 is a constrained amino acid.
  • Embodiment 9. The peptide of any one of embodiments 1-8, wherein X7 is
  • Embodiment 10 The peptide of any one of embodiments 1-7, wherein X5 and X7 are each independently a constrained amino acid.
  • Embodiment 11 The peptide of any one of embodiments 1-7, wherein X5 is P.
  • Embodiment 12 The peptide of any one of embodiments 1-11, wherein X6 is G, A, V, L, I, M, F, W, or P.
  • Embodiment 13 The peptide of any one of embodiments 1-11, wherein X6 is V, L, I, F.
  • the peptide of embodiment 8 or 9, wherein the constrained amino acid is proline, a proline analog, Orn(i-PrCO-Hao), 5- hydrazino-2-methoxybenzoic acid (Hao), an N-alkyl amino acid, or an alpha, alpha- disubstituted amino acid.
  • Embodiment 15. The peptide of any one of embodiments 1-14, wherein X7 is P.
  • Embodiment 16 The peptide of any one of embodiments 1-15, wherein X8 is G, A, V, L, I, M, F, W, or P.
  • Embodiment 17 The peptide of any one of embodiments 1- 15, wherein X8 is I or F.
  • Embodiment 23 The peptide of any one of embodiments 1-20, wherein X10 is F, H, L, or Y.
  • Embodiment 23 The peptide of any one of embodiments 1-22, wherein X11 and X18 are taken together to form an intramolecular linkage.
  • Embodiment 24 The peptide of embodiment 23, wherein at least one of X11 and X18 is C.
  • Embodiment 25 The peptide of embodiment 23 or 24, wherein at least one of X11 and X18 is dehydroalanine.
  • Embodiment 26 The peptide of embodiment 23, wherein X11 and X18 are both C.
  • Embodiment 27 The peptide of embodiment 23, wherein the intramolecular linkage comprises the structure: , or ; wherein R is a substituent, Ar is aryl, and HA is heteroaryl.
  • Embodiment 28 The peptide of embodiment 27, wherein R is alkyl, aralkyl, or cycloalkyl.
  • Embodiment 29 The peptide of embodiment 28, wherein R is C 1 -C 6 alkyl.
  • Embodiment 30 The peptide of embodiment 27, wherein Ar is optionally substituted phenyl or naphthyl.
  • Embodiment 31 The peptide of embodiment 27, wherein HA is optionally substituted: pyridinyl, imidazolyl, thiophenyl, pyrrolyl, thiazolyl, oxazolyl, or furanyl.
  • Embodiment 32 The peptide of embodiment 27, wherein HA is optionally substituted: pyridinyl, imidazolyl, thiophenyl, pyrrolyl, thiazolyl, oxazolyl, or furanyl.
  • X12 is S, T, C, Y, N, or Q.
  • Embodiment 33 The peptide of any one of embodiments 1-31, wherein X12 is S, N, H, or R.
  • Embodiment 34 The peptide of any one of embodiments 1-33, wherein X13 is G, A, V, L, I, M, F, W, or P.
  • Embodiment 35 The peptide of any one of embodiments 1-33, wherein X13 is R, Q, L, or F.
  • Embodiment 36 The peptide of any one of embodiments 1-35, wherein X14 is a positively charged amino acid.
  • Embodiment 37 The peptide of any one of embodiments 1-35, wherein X14 is a positively charged amino acid.
  • Embodiment 38 The peptide of any one of embodiments 1-37, wherein X15 is S, T, C, Y, N, or Q.
  • Embodiment 39 The peptide of any one of embodiments 1-37, wherein X15 is S or T.
  • Embodiment 40 The peptide of any one of embodiments 1-39, wherein X16 is G, A, V, L, I, M, F, W, or P.
  • Embodiment 41 The peptide of any one of embodiments 1-40, wherein X17 is a positively charged amino acid.
  • Embodiment 42 The peptide of embodiment 41, wherein X17 is K, R, or H.
  • Embodiment 43 The peptide of embodiment 41, wherein X17 is Q or T.
  • Embodiment 44 The peptide of any one of embodiments 1-43, wherein X19 is S, T, C, Y, N, or Q.
  • Embodiment 45 The peptide of any one of embodiments 1-44, wherein X20 is K, R, or H.
  • Embodiment 46 The peptide of any one of embodiments 1-45, wherein X21 is M, L, V, or F.
  • Embodiment 47 The peptide of any one of embodiments 1-45, wherein X21 is absent.
  • Embodiment 48 The peptide of embodiment 41, wherein X17 is Q or T.
  • Embodiment 44 The peptide of any one of embodiments 1-43, wherein X19 is S, T, C, Y, N, or Q.
  • Embodiment 45 The peptide of any one of embodiments 1-44, wherein X20 is K, R,
  • X21 is a beta-homoamino acid analog.
  • Embodiment 49 The peptide of embodiment 48, wherein the beta-homoamino acid analog is selected from the group consisting of (lS,3R)-(+)-3-(amino)cyclopentanecarboxylic acid; (2R,3R)-3-(amino)-
  • Embodiment 50 The peptide of embodiment 48, wherein the beta-homo amino acid analog is selected from the group consisting of beta-homophenylalanine, beta-homoleucine, beta-homocysteine, beta- homomethionine, and beta-homoisoleucine.
  • Embodiment 51 The peptide of any one of embodiments 1-50, wherein X21 is G, A, V, L, I, M, F, W, or P.
  • Embodiment 52 The peptide of any one of embodiments 1-51, wherein at least one of X1, X2, X3, X4, X5, X6,
  • X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, or X21 is an non- canonical amino acid.
  • Embodiment 53 The peptide of any one of embodiments 1-51, wherein at least two of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, or X21 are independently a no-canonical amino acid.
  • Embodiment 54 Embodiment 54.
  • X20, or X21 are independently a no-canonical amino acid.
  • Embodiment 55 The peptide of any one of embodiments 1-51, wherein at least four of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, or X21 are independently a no- canonical amino acid.
  • Embodiment 56 Embodiment 56.
  • non-canonical amino acid is selected from the group consisting of alanine derivatives; alicyclic amino acids; arginine derivatives; aromatic amino acids; asparagine derivatives; aspartic acid derivatives; beta-amino acids; cysteine derivatives; dab (2,4-diaminobutyric acid); dap (2,3- diaminopropionic acid); glutamic acid derivatives; glutamine derivatives; glycine derivatives; homo-amino acids; isoleucine derivatives; leucine derivatives; linear core amino acids; lysine derivatives; methionine derivatives; n-methyl amino acids; norleucine derivatives; norvaline derivatives; ornithine derivatives; penicillamine derivatives; phenylalanine derivatives; phenylglycine derivatives; proline derivatives; pyroglutamine derivatives; serine derivatives; threonine derivatives;
  • Embodiment 58 The peptide of any one of embodiments 52-56, wherein the non-canonical amino acid is selected from 2,4-diaminobutryic acid; alpha-methylarginine; homophenylalanine; homoleucine; homoisoleucine; diaminopropionic acid; N-acetylglycine; 6-aminohexanoic acid; gamma-aminobutyric acid; alpha-methyl serine; alpha-methyltyrosine; 4-(trifluoromethyl)-L-phenylalanine; 4-amino-L-phenylalanine; Penicillamine, t-butyl D- serine, Statine, N-methyl glycine, N-methyl serine, N-methyl lysine, beta-phenylalanine, L- alpha-cyclohexyl glycine, L-cyclopropylglycine, or beta-homoleucine.
  • Embodiment 59 The peptide of embodiment 1, wherein at least one of X3, X4, X17, or X20 are DAB (2,4- diaminobutryic acid) or DAP (diaminopimelic acid).
  • Embodiment 60 The peptide of embodiment 1, wherein at least one of X3, X4, X17, are DAB (2,4-diaminobutryic acid) or DAP (diaminopimelic acid).
  • Embodiment 61 The peptide of embodiment 1, wherein at least one of X1, X2, X3, and Y2 are non-canonical amino acids.
  • Embodiment 62 The peptide of embodiment 1, wherein at least one of X1, X2, X3, and Y2 are non-canonical amino acids.
  • the peptide of embodiment 66, wherein the half-life extending moiety comprises PEG (polyethylene glycol).
  • the peptide of embodiment 65, wherein the half-life extending moiety comprises a peptide or protein.
  • the peptide of embodiment 69, wherein the half-life extending moiety comprises XTEN or PAS.
  • the peptide of embodiment 70 comprises a polymer having 100-1000 XTEN or PAS repeats.
  • the peptide of embodiment 65, wherein the half-life extending moiety comprises an antibody or antibody fragment.
  • the peptide of embodiment 72, wherein the half-life extending moiety comprises an Fc.
  • the peptide of embodiment 72, wherein the half-life extending moiety comprises an IgG.
  • Y1 is absent or an N-terminal modification
  • X1 is absent, or G
  • X2 is absent, or S
  • X3 is absent, K, DAB, or DAP
  • X4 is K, DAB, or DAP
  • X5 is P
  • X6 is V, I, L, or F
  • X7 is P
  • X8 is I or F
  • X9 is I, T, N, S, V, E, Y, F
  • X10 is Y, F, H, or L
  • X11 is C
  • X12 is N, S, H, or R
  • X13 is R, Q, L, F, DAB, or DAP
  • X14 is any amino acid
  • X15 is T or S
  • X16 is G
  • X17 is any amino acid
  • X18 is C
  • X19 is Q or T
  • X20 is R, DAB or DAP
  • X21 is absent, I, L, V,
  • Embodiment 76 The peptide of any one of embodiments 1-75, wherein the N-terminal modification or C-terminal modification comprises at least one targeting agent. 77. The peptide of embodiment 76, wherein the targeting agent increases the local concentration of the peptide. 78. The peptide of embodiment 76, wherein the targeting agent broadens the antibiotic spectrum. 79. The peptide of any one of embodiments 1-75, wherein Y2 comprises an amide.
  • Embodiment 80 The peptide of embodiment 76, wherein Y2 is selected from the group consisting of -NH2, -NH(C 1 -C 6 alkyl), and -N(C 1 -C 6 alkyl)2.
  • Y2 comprises a water-soluble polymer.
  • Embodiment 82 The peptide of embodiment 81, wherein Y2 comprises PEG (polyethylene glycol).
  • Embodiment 83 The peptide of embodiment 82, wherein PEG has an average molecular weight of 10,000-85,000 daltons.
  • 84 The peptide of any one of embodiments 1-75, wherein Y2 comprises a peptide or protein.
  • Embodiment 85 The peptide of embodiment 84, wherein Y2 comprises XTEN or PAS.
  • the peptide of embodiment 85 comprises a polymer having 100-1000 XTEN or PAS repeats.
  • the peptide of embodiment 84, wherein Y2 comprises Y or K.
  • the peptide of embodiment 84, wherein Y2 comprises Y, K, DAB, or DAP.
  • the peptide of embodiment 84, wherein Y2 comprises at least one DAP or DAB amino acids.
  • the peptide of embodiment 84, wherein Y2 comprises at least one homoamino acid.
  • the peptide of embodiment 84, wherein Y2 comprises YY ⁇ DAB ⁇ DAB ⁇ or YY ⁇ DAP ⁇ DAP ⁇ .
  • Embodiment 93 The peptide of any one of embodiments 1-75, wherein Y2 comprises an antibody or antibody fragment.
  • Embodiment 94 The peptide of embodiment 93, wherein Y2 comprises an Fc.
  • Embodiment 95 The peptide of embodiment 93, wherein Y2 comprises an IgG.
  • Embodiment 96 The peptide of embodiment 93, wherein Y2 comprises a lipid.
  • Embodiment 97 The peptide of any one of embodiments 1-96, wherein Y1 comprises an acyl group.
  • Y1 is selected from the group consisting of formyl, acetyl, propionyl, butyryl, and isovaleryl.
  • Embodiment 100 The peptide of any one of embodiments 1-96, wherein Y1 comprises a water-soluble polymer.
  • 101. The peptide of embodiment 97, wherein Y1 comprises PEG (polyethylene glycol).
  • Embodiment 102. The peptide of embodiment 101, wherein PEG has an average molecular weight of 10,000-85,000 daltons.
  • Embodiment 103 The peptide of any one of embodiments 1-96, wherein Y1 comprises a peptide or protein.
  • Embodiment 104 The peptide of any one of embodiments 1-96, wherein Y1 comprises a peptide or protein.
  • the peptide of embodiment 104 comprises a polymer having 100-1000 XTEN or PAS repeats.
  • Embodiment 106. The peptide of any one of embodiments 1-96, wherein Y1 comprises an antibody or antibody fragment.
  • Embodiment 107. The peptide of embodiment 106, wherein Y1 comprises an Fc.
  • Embodiment 112 The peptide of embodiment 110, wherein the peptide has at least 85% identity with SEQ ID NO : 1.
  • Embodiment 115 An isolated peptide, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 2-154, wherein in the peptide is not identical to SEQ ID NO: 1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
  • Embodiment 116 An isolated peptide, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 2-154, wherein in the peptide is not identical to SEQ ID NO: 1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
  • an isolated peptide wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 2-50, wherein in the peptide is not identical to SEQ ID NO: 1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
  • Embodiment 117. The peptide of embodiment 116, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 2-50.
  • the peptide of embodiment 116, wherein the peptide has at least 90% identity with any one of SEQ ID NOs: 2-50.
  • Embodiment 120 An isolated peptide, wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 51-100, wherein in the peptide is not identical to SEQ ID NO:
  • Embodiment 121 The peptide of embodiment 120, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 51-100.
  • Embodiment 122. The peptide of embodiment 120, wherein the peptide has at least 90% identity with any one of SEQ ID NOs: 51-100.
  • Embodiment 123. The peptide of embodiment 120, wherein the peptide has at least 95% identity with any one of SEQ ID NOs: 51-100.
  • Embodiment 124 The peptide of embodiment 120, wherein the peptide has at least 95% identity with any one of SEQ ID NOs: 51-100.
  • an isolated peptide wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 101-154, wherein in the peptide is not identical to SEQ ID NO: 1, and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
  • Embodiment 125 The peptide of embodiment 123, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 101-154.
  • the peptide of embodiment 123, wherein the peptide has at least 90% identity with any one of SEQ ID NOs: 101-154.
  • Embodiment 128 An isolated peptide, wherein the peptide has at least 70% identity with SEQ ID NO: 1 over a range of at least 10 amino acids and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
  • Embodiment 129 The peptide of embodiment 128, wherein the peptide has at least 80% identity with SEQ ID NO: 1 over a range of at least 13 amino acids.
  • Embodiment 130 Embodiment 130.
  • Embodiment 132. The peptide of any one of embodiments 110-131, wherein the peptide comprises an N- terminal deletion.
  • the peptide of embodiment 132, wherein the N-terminal deletion consists of one amino acid.
  • the peptide of embodiment 132, wherein the N-terminal deletion consists of two amino acids.
  • the peptide of embodiment 132, wherein the N-terminal deletion consists of three amino acids.
  • Embodiment 136. The peptide of embodiment 132, wherein the N-terminal deletion consists of one to five amino acids.
  • Embodiment 137. The peptide of any one of embodiments 110- 136, wherein the peptide comprises at least one non-canonical amino acid.
  • Embodiment 138. The peptide of embodiment 137, wherein the non-canonical amino acid is selected from the group consisting of a D-amino acid, a beta-amino acid, an N-acyl amino acid, or a C-amidyl amino acid.
  • the peptide of embodiment 141, wherein the at least one C-terminal or N-terminal modification comprises a PEG group.
  • the peptide of embodiment any one of embodiments 110-138, wherein the at least one C-terminal or N-terminal modification comprises a lipid.
  • the peptide of embodiment any one of embodiments 110-138, wherein the at least one C-terminal or N-terminal modification comprises a protein or half-life extending peptide.
  • the peptide of embodiment any one of embodiments 110-139, wherein the peptide comprises at least one intramolecular linkage.
  • Embodiment 149 A pharmaceutical composition comprising a peptide of any one of embodiments 1-148 and an excipient, delivery vehicle, second therapeutic agent, or a combination thereof.
  • Embodiment 150 The pharmaceutical composition of embodiment 149, wherein the pharmaceutical composition comprises an excipient.
  • Embodiment 151 The pharmaceutical composition of embodiment 149, wherein the pharmaceutical composition comprises a second therapeutic agent.
  • Embodiment 152 The pharmaceutical composition of embodiment 149, wherein the pharmaceutical composition is formulated for topical administration.
  • Embodiment 153 The pharmaceutical composition of embodiment 149, wherein the pharmaceutical composition is formulated for intravenous administration.
  • Embodiment 154. The pharmaceutical composition of embodiment 149, wherein the pharmaceutical composition is formulated for oral administration.
  • Embodiment 155 The pharmaceutical composition of embodiment 149, wherein the pharmaceutical composition is formulated for intramuscular or subcutaneous administration.
  • Embodiment 156. A method of treating an infection comprising administering to a subject a therapeutically effective amount of a peptide or pharmaceutical composition of any one of embodiments 1-155 or SEQ ID NO: 1.
  • Embodiment 157. The method of embodiment 156, wherein the infection comprises a bacterial infection.
  • Embodiment 157 wherein the bacterial infection is caused by a Gram-negative bacterium.
  • Embodiment 159 The method of embodiment 157, wherein the bacterial infection is caused by an enterobacterium.
  • Embodiment 160 The method of embodiment 158, wherein the bacterium is Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, or Pseudomonas aeruginosa.
  • Embodiment 161. The method of any one of embodiments 156-159, wherein the bacterium is a drug- resistant or multi-drug resistant bacterium.
  • Embodiment 162. The method of any one of embodiments 156-161, wherein the subject is a mammal.
  • Embodiment 162 wherein the mammal is a human.
  • Embodiment 164 The method of any one of embodiments 156-163, wherein the peptide is administered orally, enterically, topically, intravenously, intraperitoneally, intramuscularly, endoscopically, percutaneously, subcutaneously, regionally, by inhalation, or by direct injection.
  • Embodiment 165 The method of embodiment 164, wherein the orally administered peptide is a capsule or tablet.
  • Embodiment 166 The method of any one of embodiments 156-165, further comprising administering a second therapeutic agent.
  • Embodiment 167 The method of embodiment 166, wherein the second therapeutic agent is an antibiotic or a protease inhibitor.
  • Embodiment 168 The method of embodiment 162, wherein the mammal is a human.
  • Embodiment 164 The method of any one of embodiments 156-163, wherein the peptide is administered orally, enterically, topically, intravenously
  • the method of embodiment 167, wherein the antibiotic is a beta-lactam antibiotic.
  • Embodiment 169. The method of embodiment 167, wherein the antibiotic is amoxicillin, bacitracin, chloramphenicol, clindamycin, capreomycin, colistimethate, ciprofloxacin, doxycycline, erythromycin, fusidic acid, fosfomycin, fusidate sodium, gramicidin, gentamycin, lincomycin, minocycline, macrolides, monobactams, nalidixic acid, novobiocin, ofloxcin, rifamycins, tetracyclines, vancomycin, tobramycin, fluoroquinolones, polymyxins, DNA gyrase inhibitors, bacterial polymerase inhibitors, folate synthesis inhibitors, or trimethoprim.
  • Example 1 Measurement of MICs of Peptides
  • Minimum inhibitory concentration (MIC) was determined by broth microdilution using Mueller-Hinton broth (MHB) as the test medium. Bacterial strains were cultured at 37°C in a shaking incubator until log phase was reached between 0.5-0.8 at O.D. 600nm. Cell concentration was adjusted to approximately 5 x 10 5 cells/ml. Peptides were prepared as 10X stock solutions and serially diluted 2-fold in bovine serum albumin (BSA) acetic acid media to prevent binding to the plastic. Using 96 well plates 90 microliters of cells and 10 ul of 10X peptide dilutions were added to each well. Growth control and media control lanes were included in each plate. Each plate was then parafilmed and incubated at 37°C for 16-18 hours. Plates were read with a spectrophotometer at O.D. 600 nm. The MIC was defined as the lowest concentration of peptide with an O.D. of 0.
  • Table 2 shows MIC99 data for Compound A (SEQ ID NO: 1) against several drug resistant isolates, including multi-drug resistant E. coli , colistin resistant E. coli , and E. coli with resistant mutations in New Delhi metallo-beta-lactamase 1.
  • Compound A also shows activity against Klebsiella pneumoniae and its New Delhi mutations. In addition, activity was measured against the New Delhi mutant of enterbacter cloacae.
  • Table 3 shows sequences and data associated with Compound A (SEQ ID NO: 1) and analogs (SEQ ID NOS: 1-8).
  • Table 4 shows MIC data for various bacterial strains.
  • Compound A demonstrated increased potency compared to thanatin against a panel of MDR E. coli strains including multi-drug (MDR), carbapenem (NDM) and colistin resistant (Col R ) isolates.
  • MDR multi-drug
  • NDM carbapenem
  • Col R colistin resistant
  • KPC K. pneumoniae carbapenemase
  • MDR multi-drug resistant
  • CoP colistin resistant
  • NDM New Delhi metallo-fi-lactamase-l .
  • Compound A was tested in both a time-kill assay (Fig. 1A), and beta-lactamase release assay (Fig. IB, 4 hours). Compound A showed rapid bactericidal activity consistent with other bactericidal antibiotics and distinct from membrane lytic agents such as melittin. The D-form of Compound A was not active (Fig. 1A). Compound A did not demonstrate measurable release of periplasmic bacterial ⁇ -lactamase from E. coli even well above its MIC (Fig. IB). Colistin was used as a positive control for the beta-lactamase release assay.
  • Example 4 In-vitro toxicity study with Compound A in mammalian cells
  • Compound A, thanatin, and melittin were tested for toxicity in red blood cells and HEK293 cells using standard protocols.
  • Traditional cationic AMPS anti-microbial peptides, such as cAMPS, e.g., melittin
  • cAMPS anti-microbial peptides
  • melittin e.g., melittin
  • Compound A showed greater than 2 log reduction in peripheral organ bacterial load, similar to positive control gentamicin.
  • the resin was incubated with a mixture of Fmoc-AA-OH: HBTU: DIEA (3 eq: 2.85 eq: 6 eq, in DMF) for 45 min with N2 bubbling. The coupling process was monitored by ninhydrin test, where colorless beads indicated no free amine thus the reaction was complete. Between coupling of amino acids and de-protection of Fmoc groups, the resin was washed exhaustively with DMF.
  • the crude peptide was purified by prep-HPLC (TFA condition; 30 °C, A:0.075% TFA in H 2 O, B:CH 3 CN) to give the peptide, then the peptide was purified by prep-HPLC (AcOH condition; 50 °C, A:0.5% AcOH in H 2 O, B:CH 3 CN) to give Compound B (41.6 mg, 1.66% yield, 99.2% purity, AcOH).
  • Example 8 In-vitro antimicrobial activity assay
  • Example 6 Following the general procedure of Example 6, peptides were synthesized. These peptides were then tested for antimicrobial activity following the general procedures of Example 1 (Tables 10 and Table 11).

Abstract

Described herein are peptides comprising anti-microbial activity. Such peptides in some embodiments are used as pharmaceutical compositions to treat infectious diseases, such as bacterial infections. In some embodiments, the bacterial infections are caused by multi-drug resistant bacteria.

Description

PEPTIDES AND USE THEREOF
CROSS-REFERENCE
[001] This application claims the benefit of U.S. provisional patent application number 62/924,629 filed on October 22, 2019, which is incorporated by reference in its entirety.
BACKGROUND
[002] Pathogenic bacteria which are resistant to conventional antibiotic treatments are regarded as a serious threat to human health and agriculture. There is a need for the development of medicines to prevent or treat such infections. The present disclosure provides peptides having antimicrobial activity which are in some embodiments used to treat or prevent infections.
BRIEF SUMMARY
[003] Provided herein are isolated peptides, wherein the peptide has a sequence comprising Formula (I):
Y1-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-Y2
Formula (I); wherein Y1 is absent or an N-terminal modification; X1 and X2 are independently absent, or any amino acid; X3 is absent or any amino acid; X4 is positively charged amino acid; X5 is any amino acid; X6 is a non-polar amino acid; X7 is any amino acid; X8 is a non-polar amino acid; X9 is any amino acid; X10 is any amino acid; X11 is a bridging amino acid; X12 is a polar amino acid or a positively charged amino acid; X13 is any amino acid; X14 is any amino acid; X15 is a polar amino acid; X16 is a non-polar amino acid; X17 is any amino acid; X18 is a bridging amino acid; X19 is a polar amino acid; X20 is a positively charged amino acid; X21 is absent or any amino acid; Y2 is absent or a C-terminal modification, wherein if: X1 and X16 are G; X2 is S, X3, X4, X17 are K; X5 and X7 are P; X6 is V; X8 and X9 are I; X10 is Y; X11 is C; X12 is N; X13, X14, and X20 are R; X15 is T, X16 is G, X18 is C, X19 is Q; then X21 is not M; and the peptide is not identical to SEQ ID NO: 1. Further provided herein are peptides wherein at least three of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, or X21 are independently a non- canonical amino acid. Further provided herein are peptides wherein X1 and X2 are independently G, S, A, V, L, I, M, F, W, or P. Further provided herein are peptides wherein X3 is K, R, H, DAB (2,4-diaminobutyric acid), DAP (diaminopimelic acid), or absent. Further provided herein are peptides wherein X4 is K, R, H, DAB (2,4-diaminobutyric acid), or DAP (diaminopimelic acid). Further provided herein are peptides wherein X5 and X7 are each independently a constrained amino acid. Further provided herein are peptides wherein X5 is P, X7 is P, and X6 is V, L, or F Further provided herein are peptides wherein X8 is I or F. Further provided herein are peptides wherein X9 is I, T, N, S, V, E, Y, or F. Further provided herein are peptides wherein X10 is F, W, or Y. Further provided herein are peptides wherein X11 and X18 are taken together to form an intramolecular linkage. Further provided herein are peptides wherein at least one of X11 and X18 is C or penicillamine. Further provided herein are peptides wherein the intramolecular linkage comprises a disulfide linkage. Further provided
Figure imgf000003_0001
herein are peptides wherein the intramolecular linkage comprises the structure:
Figure imgf000003_0002
substituent, Ar is aryl, and HA is heteroaryl. Further provided herein are peptides wherein R is alkyl, aralkyl, or cycloalkyl; Ar is optionally substituted phenyl or naphthyl; and HA is optionally substituted: pyridinyl, imidazolyl, thiophenyl, pyrrolyl, thiazolyl, oxazolyl, or furanyl. Further provided herein are peptides wherein X12 is S, T, C, Y, N, or Q. Further provided herein are peptides wherein X13 is R, Q, L, or F. Further provided herein are peptides wherein X14, X17, and X20 are independently a positively charged amino acid. Further provided herein are peptides wherein X14 and X17, and X20 are independently a K, R, H, DAB (2,4-diaminobutyric acid), or DAP (diaminopimelic acid). Further provided herein are peptides wherein X16 is G, A, V, L, I, M, F, W, or P. Further provided herein are peptides wherein X21 is M, L, V, or F. Further provided herein are peptides wherein the non-canonical amino acid is selected from the group consisting of alanine derivatives; alicyclic amino acids; arginine derivatives; aromatic amino acids; asparagine derivatives; aspartic acid derivatives; beta-amino acids; cysteine derivatives; dab (2,4-diaminobutyric acid); dap (2,3- diaminopropionic acid); glutamic acid derivatives; glutamine derivatives; glycine derivatives; homo-amino acids; isoleucine derivatives; leucine derivatives; linear core amino acids; lysine derivatives; methionine derivatives; n-methyl amino acids; norleucine derivatives; norvaline derivatives; ornithine derivatives; penicillamine derivatives; phenylalanine derivatives; phenylglycine derivatives; proline derivatives; pyroglutamine derivatives; serine derivatives; threonine derivatives; tryptophan derivatives; tyrosine derivatives; and valine derivatives. Further provided herein are peptides wherein at least one of X4, X17, or X20 are DAB (2,4- diaminobutryic acid) or DAP (diaminopimelic acid). Further provided herein are peptides wherein at least one of X3, X4, X17, are DAB (2,4-diaminobutryic acid) or DAP (diaminopimelic acid). Further provided herein are peptides wherein at least one of X1, X2, X3, and Y2 are non-canonical amino acids. Further provided herein are peptides wherein at least one of X20, X21, or Y1 are non-canonical amino acids. Further provided herein are peptides wherein Y1 is absent or an N-terminal modification; X1 is absent, or G; X2 is absent, or S; X3 is absent, K, DAB, or DAP; X4 is K, DAB, or DAP; X5 is P; X6 is V, I, L, or F; X7 is P; X8 is I or F; X9 is I, T, N, S, V, E, Y, F; X10 is Y, F, H, or L; X11 is C or penicillamine; X12 is N, S, H, or R; X13 is R, Q, L, F, DAB, or DAP; X14 is any amino acid; X15 is T or S; X16 is G; X17 is any amino acid; X18 is C or penicillamine; X19 is Q or T; X20 is R, DAB or DAP;
X21 is absent, I, L, V, M, F, or a non-canonical amino acid; and Y2 is absent or a C-terminal modification. Further provided herein are peptides wherein the non-canonical amino acid is linked to a half-life extending moiety or a moiety which broadens the antibiotic spectrum. Further provided herein are peptides wherein the peptide comprises at least one N-terminal modification or C-terminal modification. Further provided herein are peptides wherein the N- terminal modification or C-terminal modification comprises at least one targeting agent. Further provided herein are peptides wherein the targeting agent increases the local concentration of the peptide. Further provided herein are peptides wherein the targeting agent broadens the antibiotic spectrum. Further provided herein are peptides wherein Y2 comprises an amide. Further provided herein are peptides wherein Y2 is selected from the group consisting of -NEB, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2. Provided herein are isolated peptides, wherein the peptide has at least 70% identity with SEQ ID NO: 1, wherein in the peptide is not identical to SEQ ID NO: 1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. Further provided herein are peptides wherein the peptide has at least 75% identity with SEQ ID NO: 1. Further provided herein are peptides wherein the peptide has at least 85% identity with SEQ ID NO: 1. Further provided herein are peptides wherein the peptide has at least 90% identity with SEQ ID NO: 1. Further provided herein are peptides wherein the peptide has at least 95% identity with SEQ ID NO:l. Provided herein are isolated peptides, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 2-313, wherein in the peptide is not identical to SEQ ID NO: 1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. Further provided herein are peptides wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 2-50. Provided herein are isolated peptides, wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 51-100, wherein in the peptide is not identical to SEQ ID NO: 1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. Further provided herein are peptides wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 51-100. Provided herein are isolated peptides, wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 101- 154, wherein in the peptide is not identical to SEQ ID NO: 1, and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
Further provided herein are peptides wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 101-150. Provided herein are isolated peptides, wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 151-200, wherein in the peptide is not identical to SEQ ID NO: 1, and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. Further provided herein are peptides wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 151- 200. Provided herein are isolated peptides, wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 201-250, wherein in the peptide is not identical to SEQ ID NO: 1, and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. Further provided herein are peptides wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 201-250. Provided herein are isolated peptides, wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 251- 313, wherein in the peptide is not identical to SEQ ID NO: 1, and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
Further provided herein are peptides wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 251-313. Provided herein are isolated peptides, wherein the peptide has at least 70% identity with SEQ ID NO: 1 over a range of at least 10 amino acids and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R;
T18Q; and F20M. Further provided herein are peptides wherein the peptide has at least 80% identity with SEQ ID NO: 1 over a range of at least 13 amino acids. Further provided herein are peptides wherein the peptide has at least 80% identity with SEQ ID NO: 1 over a range of at least 16 amino acids. Further provided herein are peptides wherein the peptide has at least 80% identity with SEQ ID NO: 1 over a range of at least 9-18 amino acids. Further provided herein are peptides wherein the peptide comprises an N-terminal deletion. Further provided herein are peptides wherein the peptide comprises at least one non-canonical amino acid. Further provided herein are peptides wherein the non-canonical amino acid is selected from the group consisting of a D-amino acid, a beta-amino acid, an N-acyl amino acid, or a C- amidyl amino acid. Further provided herein are peptides wherein the peptide comprises at least one C-terminal or N-terminal modification. Further provided herein are peptides wherein the at least one C-terminal or N-terminal modification comprises a modification which extends half-life or broadens the antibiotic spectrum. Further provided herein are peptides wherein the peptide comprises at least one intramolecular linkage. Further provided herein are peptides wherein the intramolecular linkage is formed by two amino acid side chains. Further provided herein are peptides wherein the peptide comprises at least one disulfide linkage. Further provided herein are peptides wherein the peptide comprises at least one substitution relative to SEQ ID NO: 1.
[004] Provided herein are pharmaceutical compositions comprising a peptide described herein and an excipient, delivery vehicle, second therapeutic agent, or a combination thereof. Further provided herein are pharmaceutical compositions wherein the pharmaceutical composition comprises an excipient. Further provided herein are pharmaceutical compositions wherein the pharmaceutical composition comprises a second therapeutic agent. Further provided herein are pharmaceutical compositions wherein the pharmaceutical composition is formulated for topical administration. Further provided herein are pharmaceutical compositions wherein the pharmaceutical composition is formulated for intravenous administration. Further provided herein are pharmaceutical compositions wherein the pharmaceutical composition is formulated for oral administration. Further provided herein are pharmaceutical compositions wherein the pharmaceutical composition is formulated for intramuscular or subcutaneous administration.
[005] Provided herein are methods of treating an infection comprising administering to a subject a therapeutically effective amount of a peptide or pharmaceutical composition described herein, or a peptide comprising SEQ ID NO: 1. Further provided herein are methods wherein the infection comprises a bacterial infection. Further provided herein are methods wherein the bacterial infection is caused by a Gram-negative bacterium. Further provided herein are methods wherein the bacterial infection is caused by an enterobacterium. Further provided herein are methods wherein the bacterium is Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii, or Pseudomonas aeruginosa. Further provided herein are methods wherein the bacterium is a drug-resistant or multi-drug resistant bacterium. Further provided herein are methods wherein the subject is a mammal. Further provided herein are methods wherein the mammal is a human. Further provided herein are methods wherein the peptide is administered orally, enterically, topically, intravenously, intraperitoneally, intramuscularly, endoscopically, percutaneously, subcutaneously, regionally, by inhalation, or by direct injection. Further provided herein are methods wherein the orally administered peptide is a capsule or tablet. Further provided herein are methods further comprising administering a second therapeutic agent. Further provided herein are methods the second therapeutic agent is an antibiotic or a protease inhibitor. Further provided herein are methods wherein the antibiotic is a beta-lactam antibiotic. Further provided herein are methods wherein the antibiotic is amoxicillin, bacitracin, chloramphenicol, clindamycin, capreomycin, colistimethate, ciprofloxacin, doxycycline, erythromycin, fusidic acid, fosfomycin, fusidate sodium, gramicidin, gentamycin, lincomycin, minocycline, macrolides, monobactams, nalidixic acid, novobiocin, ofloxcin, rifamycins, tetracyclines, vancomycin, tobramycin, fluoroquinolones, polymyxins, DNA gyrase inhibitors, bacterial polymerase inhibitors, folate synthesis inhibitors, or trimethoprim.
INCORPORATION BY REFERENCE
[0006] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS
[0007] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0008] Figure 1A depicts Compound A tested in a time-kill assay against E. coli ATCC 25922.
[0009] Figure IB depicts Compound A tested in a beta-lactamase release assay.
[0010] Figure 2A depicts Compound A tested for red blood cell hemolysis.
[0011] Figure 2B depicts Compound A tested for release of LDH from HEK293 cells.
DETAILED DESCRIPTION OF THE INVENTION
[012] Described herein are isolated peptides, pharmaceutical compositions, disinfectant compositions, and methods of treating bacterial infections or disinfecting surfaces. In some embodiments, peptides described herein are used to treat, prevent occurrence or prevent recurrence of bacterial infections in living organisms, including those caused by multi-drug resistant bacteria. In other embodiments, peptides described herein are used to disinfect surfaces, including medical devices. In others embodiments, methods are provided for the manufacturing of antimicrobial peptides.
[013] Antimicrobial Peptides
[014] Described herein are peptides having antimicrobial activity. In some embodiments, peptides comprise the sequence of Formula (I):
Y1-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-Y2
Formula (I); wherein
Y1 is absent or an N-terminal modification; X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20,
X21 are any amino acid;
X1, X2, and X3 are absent or independently any amino acid; and Y2 is absent or a C-terminal modification.
[015] In some embodiments of a peptide of Formula (I), the peptide is not identical to SEQ ID NO: 1. In some embodiments of a peptide of Formula (I), the peptide has a sequence of any one of SEQ NOs: 2-313. In some embodiments of a peptide of Formula (I), X1 and X2 are absent. In some embodiments of a peptide of Formula (I), X5 is P, X6 is V, and X7 is P. In some embodiments of a peptide of Formula (I), X11 is C or penicillamine. In some embodiments of a peptide of Formula (I), X13 is R. In some embodiments of a peptide of Formula (I), X15 is T, X16 is G, and X18 is C or penicillamine. In some embodiments of a peptide of Formula (I), X20 is R. In some embodiments of a peptide of Formula (I), X21 is absent. In some embodiments a peptide of Formula (I) is not identical to SEQ ID NO: 1. In some embodiments a peptide of Formula (I), the peptide is not identical to a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. In some embodiments a peptide of Formula (I), C-terminal and/or N-terminal modifications are configured to increase the half-life of the peptide.
[016] Described herein are peptides of Formula (I) comprising one or more non- canonical amino acids. In some embodiments of a peptide of Formula (I), X4 is a positively charged amino acid. In some embodiments, at least one of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, or X21 is a non-canonical amino acid. In some embodiments, at least one of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, or X21 is a D amino acid. In some embodiments, at least one of X3, X4, X13, X14, X20, is a non-canonical amino acid. In some embodiments, at least one of X3, X4, X13, X14, X20, is a positively charged non-canonical amino acid. In some embodiments, X3, X4, X13, X14, X20, are positively charged non-canonical amino acids. In some embodiments, at least one of X3, X4, X13, X14, X20, is a DAP or DAB. In some embodiments, the non-canonical amino acid selected from 2,4-diaminobutryic acid; alpha- methylarginine; homophenylalanine; homoleucine; homoisoleucine; Diaminopropionic acid; N-acetylglycine; 6-aminohexanoic acid; gamma-aminobutyric acid; alpha-methylserine; alpha-methyltyrosine; 4-(trifluoromethyl)-L-phenylalanine; 4-amino-L-phenylalanine; Penicillamine; beta-homophenylalanine; beta-homoisoleucine; t-butyl D-serine, Statine, N- methyl glycine, N-methyl serine, N-methyl lysine, beta-phenylalanine, L-alpha-cyclohexyl glycine, L-cyclopropylglycine, or beta-homoleucine.
[017] Described herein are peptides of Formula (I) comprising an N-terminal half-life extension moiety. In some embodiments of a peptide of Formula (I), Y1 comprises a half-life extension moiety. In some embodiments of a peptide of Formula (I), Y1 is absent or an N- terminal modification. In some embodiments of a peptide of Formula (I), Y1 comprises a water-soluble polymer, a lipid, or a peptide. In some embodiments of a peptide of Formula (I), Y1 comprises a polyethylene glycol polymer. In some embodiments of a peptide of Formula (I), Y1 is acyl. In some embodiments of a peptide of Formula (I), Y1 is C1-C6 acyl. In some embodiments of a peptide of Formula (I), Y1 is hexenoyl. In some embodiments of a peptide of Formula (I), Y1 is acetyl. In some embodiments of a peptide of Formula (I), Y1 comprises an XTEN peptide. In some embodiments of a peptide of Formula (I), Y1 comprises an XTEN peptide polymer comprising 700-900 repeats. In some embodiments of a peptide of Formula (I), Y1 comprises a PAS peptide. In some embodiments of a peptide of Formula (I), Y1 comprises a PAS peptide polymer comprising 500-900 repeats. In some embodiments of a peptide of Formula (I), Y1 comprises a protein. In some embodiments of a peptide of Formula (I), Y1 is serum albumin. In some embodiments of a peptide of Formula (I), Y1 comprises an antibody or antibody fragment. In some embodiments of a peptide of Formula (I), Y1 comprises an Fc. In some embodiments of a peptide of Formula (I), Y1 comprises IgG.
[018] Described herein are peptides of Formula (I) comprising amino acids of different charges. In some embodiments of a peptide of Formula (I), X1 is a non-polar amino acid. In some embodiments of a peptide of Formula (I), X2 is a polar amino acid. In some embodiments of a peptide of Formula (I), X3 is absent or any amino acid. In some embodiments of a peptide of Formula (I), X4 is a positively charged amino acid. In some embodiments, at least one of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, or X21 is a non-canonical amino acid. In some embodiments, at least one of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, or X21 is a D amino acid. In some embodiments, at least one of X3, X4, X13, X14, X20, is a non- canonical amino acid. In some embodiments, at least one of X3, X4, X13, X14, X20, is a positively charged non-canonical amino acid. In some embodiments, X3, X4, X13, X14, X20, are positively charged non-canonical amino acids. In some embodiments, at least one of X3, X4, X13, X14, X20, is a DAP or DAB. In some embodiments of a peptide of Formula (I), X5 is a constrained amino acid. In some embodiments of a peptide of Formula (I), X5 and X7 are each independently a constrained amino acid. In some embodiments of a peptide of Formula (I), X6 is a non-polar amino acid. In some embodiments of a peptide of Formula (I), X7 is a constrained amino acid. In some embodiments of a peptide of Formula (I), X8 is a non-polar amino acid. In some embodiments of a peptide of Formula (I), X9 is a polar or non-polar amino acid. In some embodiments of a peptide of Formula (I), X9 is any amino acid. In some embodiments of a peptide of Formula (I), X10 is any amino acid. In some embodiments of a peptide of Formula (I), X10 is a phenylalanine derivative. In some embodiments of a peptide of Formula (I), X10 is an phenyl -substituted phenylalanine. In some embodiments of a peptide of Formula (I), X10 is p-CF3-phenylalanine, p-fluorophenylalanine, p-NFh-phenylalanine, p- MeO-phenylalanine, alpha-methyl tyrosine, or p-methylphenyalanine. In some embodiments of a peptide of Formula (I), X11 is a bridging amino acid. In some embodiments of a peptide of Formula (I), at least one of X11 and X18 is a non-cannonicial amino acid. In some embodiments of a peptide of Formula (I), X12 is a polar amino acid. In some embodiments of a peptide of Formula (I), X12 is a positively charged amino acid. In some embodiments of a peptide of Formula (I), X13 is a non-polar amino acid. In some embodiments of a peptide of Formula (I), X14 is any amino acid. In some embodiments of a peptide of Formula (I), X15 is a polar amino acid. In some embodiments of a peptide of Formula (I), X16 is a non-polar amino acid. In some embodiments of a peptide of Formula (I), X17 is any amino acid. In some embodiments of a peptide of Formula (I), X18 is a bridging amino acid. In some embodiments of a peptide of Formula (I), X19 is a polar amino acid. In some embodiments of a peptide of Formula (I), X20 is a positively charged amino acid. In some embodiments of a peptide of Formula (I), X21 is a non-polar amino acid or a homoamino acid analog. In some embodiments of a peptide of Formula (I), X11 and X18 are bridging amino acids. In some embodiments of a peptide of Formula (I), X11 and X18 form an intermolecular linkage.
[019] Described herein are peptides of Formula (I) comprising substitutions at various positions, hi some embodiments of a peptide of Formula (I), X6 is V, I, L, or F. In some embodiments of a peptide of Formula (I), X8 is I or F. In some embodiments of a peptide of Formula (I), X9 is I, T, N, S, V, E, Y, or F. In some embodiments of a peptide of Formula (I), X10 is Y, F, H, or L. In some embodiments of a peptide of Formula (I), X12 is N, S, H, or R.
In some embodiments of a peptide of Formula (I), X13 is R, Q, L, or F. In some embodiments of a peptide of Formula (I), X15 is T or S. In some embodiments of a peptide of Formula (I), X19 is Q or T. In some embodiments of a peptide of Formula (I), X21 is M, L, V, or F.
[020] Described herein are peptides of Formula (I) comprising a C-terminal half-life extension moiety. In some embodiments of a peptide of Formula (I), Y2 comprises a half-life extension moiety. In some embodiments of a peptide of Formula (I), Y2 is absent or a C- terminal modification. In some embodiments of a peptide of Formula (I), Y2 comprises a peptide. In some embodiments of a peptide of Formula (I), Y2 comprises a water-soluble polymer, a lipid, or a peptide. In some embodiments of a peptide of Formula (I), Y2 comprises a polyethylene glycol polymer. In some embodiments of a peptide of Formula (I), Y2 comprises an XTEN peptide. In some embodiments of a peptide of Formula (I), Y2 comprises an XTEN peptide polymer comprising 700-900 repeats. In some embodiments of a peptide of Formula (I), Y2 comprises a PAS peptide. In some embodiments of a peptide of Formula (I), Y2 comprises a PAS peptide polymer comprising 500-900 repeats. In some embodiments of a peptide of Formula (I), Y2 comprises a protein. In some embodiments of a peptide of Formula (I), Y2 is serum albumin. In some embodiments of a peptide of Formula (I), Y2 comprises an antibody or antibody fragment. In some embodiments of a peptide of Formula (I), Y2 comprises an Fc. In some embodiments of a peptide of Formula (I), Y2 comprises IgG. In some embodiments of a peptide of Formula (I), Y2 comprises at least one K. In some embodiments of a peptide of Formula (I), Y2 comprises at least one Y. In some embodiments of a peptide of Formula (I), Y2 comprises at least one DAB or DAP. In some embodiments of a peptide of Formula (I), Y2 comprises {DAB}{DAB} or {DAP}{DAP}. In some embodiments of a peptide of Formula (I), Y2 comprises YYKK. In some embodiments of a peptide of Formula (I), Y2 comprises a peptide comprising at least two positively charged amino acids. In some embodiments of a peptide of Formula (I), Y2 comprises YY {DAB } {DAB } or YY{DAP}{DAP}.
[021] Described herein are peptides of Formula (I) comprising a hairpin structure. In some embodiments of a peptide of Formula (I), X1 is absent. In some embodiments of a peptide of Formula (I), X2 is absent. In some embodiments of a peptide of Formula (I) X1 and X2 if present are independently G, S, A, V, L, I, M, F, W, or P. In some embodiments of a peptide of Formula (I), X3 is K, R, or H. In some embodiments of a peptide of Formula (I), X4 is K, R, or H. In some embodiments of a peptide of Formula (I), X4 is a positively charged amino acid. In some embodiments, at least one of X3, X4, X13, X14, X20, is a DAP or DAB. In some embodiments of a peptide of Formula (I), X5 is P. In some embodiments of a peptide of Formula (I), X6 is G, A, V, L, I, M, F, W, or P. In some embodiments of a peptide of Formula (I), X5 is a constrained amino acid selected from proline, a proline analog, Om(i-PrCO-Hao), 5-hydrazino-2-methoxybenzoic acid (Hao), an N-alkyl amino acid, or an alpha, alpha- disubstituted amino acid. In some embodiments of a peptide of Formula (I), X7 is a constrained amino acid selected from proline, a proline analog, Orn(i-PrCO-Hao), 5- hydrazino-2-methoxybenzoic acid (Hao), an N-alkyl amino acid, or an alpha, alpha- disubstituted amino acid. In some embodiments of a peptide of Formula (I), X7 is P. In some embodiments of a peptide of Formula (I), X8 is G, A, V, L, I, M, F, W, or P. In some embodiments of a peptide of Formula (I), X9 is G, A, V, L, I, M, F, W, or P. In some embodiments of a peptide of Formula (I), X9is S, T, C, Y, N, or Q. In some embodiments of a peptide of Formula (I), X10 is F, W, or Y. In some embodiments of a peptide of Formula (I), X11 and X18 are taken together to form an intramolecular linkage. In some embodiments of a peptide of Formula (I), at least one of X11 and X18 is C. In some embodiments of a peptide of Formula (I), at least one of X11 and X18 is penicillamine. In some embodiments of a peptide of Formula (I), at least one of X11 and X18 is aspartic acid or glutamic acid. In some embodiments of a peptide of Formula (I), at least one of X11 and X18 is dehydroalanine. In some embodiments of a peptide of Formula (I), X11 and X18 are both C or penicillamine. In some embodiments of a peptide of Formula (I), X12is S, T, C, Y, N, or Q. In some embodiments of a peptide of Formula (I), X13 is G, A, V, L, I, M, F, W, or P. In some embodiments of a peptide of Formula (I), X14 is a positively charged amino acid. In some embodiments of a peptide of Formula (I), X14 is K, R, or H. In some embodiments of a peptide of Formula (I), X15 is S, T, C, Y, N, or Q. In some embodiments of a peptide of Formula (I), X16 is G, A, V, L, I, M, F, W, or P. In some embodiments of a peptide of Formula (I), X17 is a positively charged amino acid. In some embodiments of a peptide of Formula (I), X17 is K, R, or H. In some embodiments of a peptide of Formula (I), X19 is S, T, C, Y, N, or Q. In some embodiments of a peptide of Formula (I), X20 is absent, K, R, or H. In some embodiments of a peptide of Formula (I), X21 is a homoamino acid. In some embodiments of a peptide of Formula (I), X21 is a beta-amino acid. In some embodiments of a peptide of Formula (I), X21 is a beta-homoamino acid. In some embodiments of a peptide of Formula (I), ), X21 is a homoamino acid selected from the group consisting of homophenylalanine, homoleucine, homocysteine, homomethionine, and homoisoleucine. In some embodiments of a peptide of Formula (I), X21 is G, A, V, L, I, M, F, W, or P. In some embodiments of a peptide of Formula (I), X3, X4, X17, or X20 are DAB (2,4-diaminobutryic acid). In some embodiments of a peptide of Formula (I), X3, X4, X17, are DAB (2,4- diaminobutryic acid). In some embodiments of a peptide of Formula (I), X1 is a non- canonical amino acid, and X2 is absent. In some embodiments of a peptide of Formula (I), X1 is an N-methyl amino acid. In some embodiments of a peptide of Formula (I), X1 is N-methyl serine, N-methyl lysine, or N-methyl glycine. In some embodiments of a peptide of Formula (I), X1 is t-butyl D-serine, Statine, N-methyl glycine, N-methyl serine, N-methyl lysine, beta- phenylalanine, L-alpha-cyclohexyl glycine, L-cyclopropylglycine, or beta-homoleucine. In some embodiments of a peptide of Formula (I), X1 is Statine, DAB, 6-AHA, GABA, or DAP. In some embodiments of a peptide of Formula (I), X2, X5, and X7 are each independently a D- amino acid. In some embodiments of a peptide of Formula (I), X2 is D-serine. In some embodiments of a peptide of Formula (I), X5 is D-proline. In some embodiments of a peptide of Formula (I), X7 is D-proline. In some embodiments of a peptide of Formula (I), X3 is lysine. In some embodiments of a peptide of Formula (I), X5 is valine. In some embodiments of a peptide of Formula (I), X7 is isoleucine. In some embodiments of a peptide of Formula (I), X8 is threonine. In some embodiments of a peptide of Formula (I), X12 is arginine. In some embodiments of a peptide of Formula (I), X17is cysteine. In some embodiments of a peptide of Formula (I), X20is phenylalanine. In some embodiments of a peptide of Formula (I), the peptide comprises at least one of the mutations 17, T8, R12, F20, or Cl 7. In some embodiments of a peptide of Formula (I), the peptide comprises at least two of the mutations 17, T8, R12, F20, or C17.
[022] Described herein are peptides comprising the sequence of Formula (II):
Y1-GS-X3-X4-P-X6-P-X8-X9-X10-C-X12-X13-X14-X15-G-X17-C-X19-R-X21-Y2
Formula (II); wherein:
Y1 is absent or an N-terminal modification;
X3 is absent, K or a non-canonical amino acid;
X4 is K or a non-canonical amino acid;
X6 is V, I, L, orF.
X8 is I or F;
X9 is I, T, N, S, V, E, Y, or F;
X10 is Y, F, H, or L;
X12 is N, S, H, or R;
X13 is R, Q, L, F, or a non-canonical amino acid;
X14 is R, K, or a non-canonical amino acid;
X15 is R or a non-canonical amino acid;
X16 is T or S;
X17 is K or a non-canonical amino acid;
X19 is Q or T;
X21 is absent, M, L, V, or F;
Y2 is absent or a C-terminal modification.
[023] Described herein are peptides comprising the sequence of Formula (III): Formula (III); wherein:
Y1 is absent or an N-terminal modification;
X3 is K, DAB, or DAP;
X4 is absent, K, DAB, or DAP;
X9 is I or T;
X10 is any amino acid;
X12 is N or S;
X14 is any amino acid;
X17 is any amino acid;
X19 is Q or T;
X20 is absent, R or DAB;
X21 is absent, I, L, V, M, F, or a beta homoamino acid; and
Y2 is absent or a C-terminal modification.
[024] Described herein are peptides of Formula (II) or (III) comprising an N-terminal half-life extension moiety. In some embodiments of a peptide of Formula (II) or Formula (III), the peptide is not identical to SEQ ID NO: 1. In some embodiments a peptide of Formula (II) or Formula (III) , the peptide is not identical to a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 is absent or an N-terminal modification. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 is a half-life extending moiety or broadens the antibiotic spectrum. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 comprises a water-soluble polymer, a lipid, or a peptide. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 comprises a polyethylene glycol polymer. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 is acyl. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 is C1-C6 acyl. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 is hexenoyl. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 is acetyl. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 comprises an XTEN peptide. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 comprises an XTEN peptide polymer comprising 700-900 repeats. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 comprises an XTEN peptide polymer comprising 20-50, 20-100, 100-300, 200-500, 400-800, 500-1000, or 750-1500 repeats. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 comprises a PAS peptide. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 comprises a PAS peptide polymer comprising 500-900 repeats. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 compress an PAS peptide polymer comprising 20-50, 20-100, 100-300, 200-500, 400-800, 500-1000, or 750-1500 repeats. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 comprises a protein. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 is serum albumin. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 comprises an antibody or antibody fragment. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 comprises an Fc. In some embodiments of a peptide of Formula (II) or Formula (III), Y1 comprises IgG.
[025] Described herein are peptides of Formula (II) or Formula (III) comprising a C- terminal half-life extension moiety or a moiety which broadens the antibiotic spectrum. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises a half-life extension moiety. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 is absent or a C-terminal modification. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises a peptide. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises a water-soluble polymer, a lipid, or a peptide. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises a polyethylene glycol polymer. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises an XTEN peptide. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises an XTEN peptide polymer comprising 700-900 repeats. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises an XTEN peptide polymer comprising 20-50, 20-100, 100-300, 200-500, 400-800, 500-1000, or 750-1500 repeats. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises a PAS peptide. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises a PAS peptide polymer comprising 500-900 repeats. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 compress an PAS peptide polymer comprising 20-50, 20-100, 100-300, 200-500, 400-800, 500-1000, or 750-1500 repeats. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises a protein. In some embodiments of a peptide of Formula (II) or Formula (III), Y2is serum albumin. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises an antibody or antibody fragment. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises an Fc. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises IgG. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises at least one K. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises at least one Y. In some embodiments of a peptide of Formula (II) or Formula (III), Y2 comprises YYKK. In some embodiments of a peptide of Formula (II) or Formula
(III), Y2 comprises a peptide comprising at least two positively charged amino acids. In some embodiments a peptide of Formula (II) or Formula (III), one or more both cysteines are replaced with penicillamine or other amino acid capable of forming a disulfide bond.
[026] Described herein are peptides comprising the sequence of Formula (IV):
Y1-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-Y2
Formula (IV); wherein:
Y1 is absent or an N-terminal modification;
X1 and X2 are independently present or absent, and when present are any amino acid;
X3 X4 X5 c6 X9 X11 X13 c14 c15 c16 c18 c19 and c21 are any aminO acid; and
Y2 is absent or a C-terminal modification.
[027] Described herein are peptides of Formula (IV) comprising an N-terminal half-life extension moiety. In some embodiments of a peptide of Formula (IV), the peptide is not identical to SEQ ID NO: 1. In some embodiments a peptide of Formula (IV), the peptide is not identical to a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. In some embodiments of a peptide of Formula (IV), Y1 is absent or an N-terminal modification. In some embodiments of a peptide of Formula (IV), Y1 is a half-life extending moiety or broadens the antibiotic spectrum. In some embodiments of a peptide of Formula
(IV), Y1 comprises a water-soluble polymer, a lipid, or a peptide. In some embodiments of a peptide of Formula (IV), Y1 comprises a polyethylene glycol polymer. In some embodiments of a peptide of Formula (IV), Y1 is acyl. In some embodiments of a peptide of Formula (IV), Y1 is Ci-C6 acyl. In some embodiments of a peptide of Formula (IV), Y1 is hexenoyl. In some embodiments of a peptide of Formula (IV), Y1 is acetyl. In some embodiments of a peptide of Formula (IV), Y1 comprises an XTEN peptide. In some embodiments of a peptide of Formula (IV), Y1 comprises an XTEN peptide polymer comprising 700-900 repeats. In some embodiments of a peptide of Formula (IV), Y1 comprises an XTEN peptide polymer comprising 20-50, 20-100, 100-300, 200-500, 400-800, 500-1000, or 750-1500 repeats. In some embodiments of a peptide of Formula (IV), Y1 comprises a PAS peptide. In some embodiments of a peptide of Formula (IV), Y1 comprises a PAS peptide polymer comprising 500-900 repeats. In some embodiments of a peptide of Formula (IV), Y1 compress an PAS peptide polymer comprising 20-50, 20-100, 100-300, 200-500, 400-800, 500-1000, or 750- 1500 repeats. In some embodiments of a peptide of Formula (IV), Y1 comprises a protein. In some embodiments of a peptide of Formula (IV), Y1 is serum albumin. In some embodiments of a peptide of Formula (IV), Y1 comprises an antibody or antibody fragment. In some embodiments of a peptide of Formula (IV), Y1 comprises an Fc. In some embodiments of a peptide of Formula (IV), Y1 comprises IgG. In some embodiments a peptide of Formula (IV), at least one of X3 , X4 , X5 , X6 , X9 , X11 , X13 , X14 , X15 , X16 , X18 , X19 , or X21 is a non-canonical amino acid. In some embodiments a peptide of Formula (IV), at least one of X3 , X4 , X5 , X6 , X9 , X11 , X13 c14 c15 c16 c18 c19 or c21 K, DAB, or DAP. In some embodiments a peptide of Formula (IV), one or more both cysteines are replaced with penicillamine or other amino acid capable of forming a disulfide bond.
[028] Described herein are peptides having a sequence shown in Table 1.
[029] Table 1. Antimicrobial peptides described herein.
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
{Dab}=2,4-diaminobutryic acid; lowercase = D/dextrorotatory amino acids; {aMeR}=alpha-methylarginine; unless indicated otherwise, peptides are in an oxidized state (intramolecular disulfide bond formation is present, if applicable); {Dap}=Diaminopropionic acid; {Acetyl-G}=N-acetylglycine; (6-AHA}=6-aminohexanoic acid; {GABA}=gamma-aminobutyric acid; {D-S}=D-serine; {aMeS}=alpha-methylserine; {D-P}=D-proline; {aMeY}=alpha-methyltyrosine; {4-CF3-F}=4-(trifluoromethyl)-L-phenylalanine; {4-NH2-F}=4-amino-L- phenylalanine; {Pen}=Penicillamine; {D-T}=D-threonine; {D-K}=D -lysine; {D-R}=D -arginine; {D-F}=D- phenylalanine; {bHomoF}=beta-homophenylalanine; {bHomoI}=beta-homoisoleucine; {bHomoL}=beta- homoleucine; {f5f}=pentafluorophenylalanine.
[030] Peptides disclosed herein include peptides as well as structurally similar compounds (i.e., small molecules) that are in some embodiments formulated to mimic the key portions of a peptide. In certain embodiments, the peptide or peptide has at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 99% sequence identity with any one of SEQ ID NOs: 1-313. In certain embodiments, the peptide or peptide has at least 70, 75, 80, 85, 90, 95, or 98% sequence identity with any one of SEQ ID NOs: 1-313. In some embodiments, the peptide comprises an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99% sequence identity with SEQ ID NO: 1. In some embodiments, the peptide or peptide has a sequence identity of 70-80%, 75-85%, 80-90%, 85-95%, or 90-100% with any one of SEQ ID NOs: 1-313. In some embodiments, the peptide or peptide has a sequence identity of 70- 80% with any one of SEQ ID NOs: 1-313. In some embodiments, the peptide or peptide has a sequence identity of 80-89% with any one of SEQ ID NOs: 1-313. In some embodiments, the peptide or peptide has a sequence identity of 90-99% with any one of SEQ ID NOs: 1-313. In some embodiments, the peptide or peptide has a sequence identity of 70-79% with any one of SEQ ID NOs: 1-313. In certain embodiments, the peptide or peptide has at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 99% sequence similarity with any one of SEQ ID NOs: 1-313.
In some embodiments, the peptide comprises an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99% sequence similarity with SEQ ID NO: 1. In some embodiments, the peptide or peptide has a sequence similarity of 70-80%, 75-85%, 80-90%, 85-95%, or 90-100% with any one of SEQ ID NOs: 1-313. In some embodiments, the peptide or peptide has a sequence similarity of 70-80% with any one of SEQ ID NOs: 1-313. In some embodiments, the peptide or peptide has a sequence similarity of 80-89% with any one of SEQ ID NOs: 1-313. In some embodiments, the peptide or peptide has a sequence similarity of 90-99% with any one of SEQ ID NOs: 1-313. In some embodiments, the peptide or peptide has a sequence similarity of 70-79% with any one of SEQ ID NOs: 1-313.
[031] Described herein are peptides comprising any one of SEQ ID NOs: 1-313, wherein one or more residues susceptible to proteolytic cleavage is replaced with a residue less susceptible to proteolytic cleavage. In some embodiments, one or more lysine residues of a peptide comprising any one of SEQ ID NOs: 1-313, is replaced with an non-canonical amino acid. In some embodiments, one or more arginine residues of a peptide comprising any one of SEQ ID NOs: 1-313, is replaced with an non-canonical amino acid. In some embodiments, all lysine and arginine residues of a peptide comprising any one of SEQ ID NOs: 1-313, is replaced with an non-canonical amino acid. In some embodiments, one or more arginine or lysine residues of a peptide comprising any one of SEQ ID NOs: 1-313, is replaced with a conservative non-canonical amino acid. In some embodiments, one or more arginine or lysine residues of a peptide comprising any one of SEQ ID NOs: 1-313, is replaced with an non- canonical amino acid, such as DAP or DAB. In some embodiments, one or more arginine or lysine residues of a peptide comprising any one of SEQ ID NOs: 1-313, is replaced with a D- amino acid.
[032] Peptide lengths described herein are in some embodiments about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 residues. In some embodiments, peptide lengths are 10-50 residues, 15-50 residues, 20-25 residues 21-25, residues, 20-30 residues, 30-40 residues, and 35-45 residues, or 25-35 residues. In some embodiments, peptide lengths are less than 30, 25, 20, 15, or less than 10 amino residues. In some embodiments, peptides described herein comprise L-configuration amino acids, D-configuration amino acids, non- canonical amino acids, or a mixture thereof. In some embodiments, peptides comprise at least 1, 2, 3, 4, 5, 7, 9, 12, 15, 20, or more than 20 D-amino acids. In some embodiments, peptides comprise not more than 1, 2, 3, 4, 5, 7, 9, 12, 15, 20, or more than 20 D-amino acids. In some embodiments, peptides comprise 1-5, 2-7, 2-10, 5-10, 7-15, or 4-8 D-amino acids. In some embodiments, all amino acids in a peptide described herein are D-amino acids.
[033] Peptides described herein comprise amino acids. In some embodiments, amino acids comprise naturally occurring and synthetic amino acids (e.g., non-canonical amino acids such as beta-amino acids, homo-amino acids, or others), as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O- phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid. Peptides described herein are often isolated from natural sources of from recombinant organisms. In some embodiments, a peptide or peptide has been separated from any natural environment, such as a body fluid, e.g., blood, and separated from the components that naturally accompany the peptide.
[034] Described herein are variants of anti-microbial peptides, wherein the peptide differs from another peptide by one or more amino acid or nucleic acid deletions, additions, substitutions or side-chain modifications, yet retains one or more specific functions or biological activities of the molecule. Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring or a nonconventional/non- canonical amino acid residue. Such substitutions in some embodiments are classified as conservative, in which case an amino acid residue contained in a peptide or peptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present disclosure may also be non-conservative, in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties, such as naturally-occurring amino acid from a different group (e.g, substituting a charged or hydrophobic amino; acid with alanine), or alternatively, in which a naturally occurring amino acid is substituted with a non- conventional amino acid. In some embodiments, amino acid substitutions are conservative. Also encompassed within the term variant when used with reference to a polynucleotide or peptide, refers to a polynucleotide or peptide that can vary in primary, secondary, or tertiary structure, as compared to a reference polynucleotide or peptide, respectively (e.g, as compared to a wild- type polynucleotide or peptide).
[035] Peptides described herein may comprise insertions, deletions, or substitutions. In some embodiments, insertions and deletions are in the range of about 1 to 5 amino acids. The variation allowed in some embodiments is experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques. In some embodiments, substitution comprises a change in an amino acid for a different entity, for example another amino acid or amino-acid moiety. Substitutions can be conservative or non-conservative substitutions. In some embodiments, the peptide is a variant comprising at least one amino acid substitution, deletion, or insertion relative to the amino acid sequence of any one of SEQ ID NOS: 1-313. Variants in some embodiments are synthetic, recombinant, or chemically modified peptides isolated or generated using methods well known in the art. Variants can include conservative or non-conservative amino acid changes, as described below. Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the peptide encoded by the reference sequence. Variants can also include insertions, deletions or substitutions of amino acids, including insertions and substitutions of amino acids and other molecules that do not normally occur in the peptide sequence that is the basis of the variant, for example but not limited to insertion of ornithine which do not normally occur in human proteins. The term conservative substitution, when describing a peptide, refers to a change in the amino acid composition of the peptide that does not substantially alter the peptide's activity. For example, a conservative substitution refers to substituting an amino acid residue for a different amino acid residue that has similar chemical properties. Conservative amino acid substitutions include replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Conservative amino acid substitutions result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Thus, a conservative substitution of a particular amino acid sequence refers to substitution of those amino acids that are not critical for peptide activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or nonpolar) such that the substitution of even critical amino acids does not reduce the activity of the peptide. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Groups of amino acids are categorized in some embodiments based on polarity or charge of their respective side chains. In some embodiments, non-polar amino acids include but are not limited to Glycine, Alanine, Valine, Leucine, Isoleucine, Methionine, Phenylalanine, Tryptophan, or Proline. In some embodiments polar amino acids include but are not limited to Serine, Threonine, Cysteine, Tryptophan, Asparagine, or Glutamine. In some embodiments positively charged amino acids include but are not limited to Lysine, Arginine, or Histidine. In some embodiments negatively charged amino acid include but are not limited to Aspartic acid or Glutamic acid. Non- canonical amino acids are also in some embodiments are conservatively substituted for such amino acids. For example, non-canonical amino acids comprising a basic side chain (e.g., DAB or DAP) in some embodiments are positively charged amino acids.
[036] In some embodiments, an amino acid is a negatively charged amino acid. In some embodiments, negatively charged amino acids comprise side-chain functional groups which are negatively charged under aqueous physiological conditions (e.g., pH ~7). In some embodiments, negatively charged amino acids comprise acidic functional group side chains. In some embodiments, acidic functional groups include but are not limited to carboxylic acids, sulfonic acids, sulfmic acids, thioacids, dithioacids, sulfamates, sulfimates, phosphates, diketoacids, phosphites, boronic acid, phenols, or other acidic functional group.
[037] In some embodiments, an amino acid is a positively charged amino acid. In some embodiments, positively charged amino acids comprise side-chain functional groups which are positively charged under aqueous physiological conditions (e.g., pH ~7). In some embodiments, positively charged amino acids comprise basic functional group side chains. In some embodiments, basic functional groups include but are not limited to heteroaryl rings (e.g., pyridines, pyrimidines, imidazoles, pyrroles), amines (substituted or unsubstituted), guanidines, piperidines, pyrrolidines, morpholines, hydrazines, other basic functional group. [038] In some embodiments, amino acids are non-canonical amino acids.
[039] In some embodiments, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids can also be considered conservative substitutions if the change does not significantly reduce the activity of the peptide. Insertions or deletions are typically in the range of about 1 to 5 amino acids. The choice of conservative amino acids in some embodiments is selected based on the location of the amino acid to be substituted in the peptide, for example if the amino acid is on the exterior of the peptide and expose to solvents, or on the interior and not exposed to solvents. [040] In alternative embodiments, one can select the amino acid which will substitute an existing amino acid based on the location of the existing amino acid, i.e. its exposure to solvents (i.e. if the amino acid is exposed to solvents or is present on the outer surface of the peptide or peptide as compared to internally localized amino acids not exposed to solvents). Selection of such conservative amino acid substitutions are well known in the art. Accordingly, one can select conservative amino acid substitutions suitable for amino acids on the exterior of a protein or peptide (i.e. amino acids exposed to a solvent). For example, but not limited to, the following substitutions can be used: substitution of Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with Tor A, R with K, G with N or A, K with R, A with S, K or P.
[041] In alternative embodiments, one can also select conservative amino acid substitutions suitable for amino acids on the interior of a protein or peptide, for example one can use suitable conservative substitutions for amino acids is on the interior of a protein or peptide (i.e. the amino acids are not exposed to a solvent). For example but not limited to, one can use the following conservative substitutions: where Y is substituted with F, T with A or S, I with L or V, W with Y, M with L, N with D, G with A, T with A or S, D with N, I with L or V, F with Y or L, S with A or T and A with S, G, Tor V. In some embodiments, nonconservative amino acid substitutions are also encompassed within the term of variants. [042] In some aspects, the peptides or peptides disclosed herein are derivatives of the SEQ ID NOs:1-313. The term derivative in some embodiments comprises peptides which have been chemically modified, for example but not limited to by techniques such as ubiquitination, labeling, pegylation (i.e., derivatization with polyethylene glycol), lipidation, glycosylation, or addition of other molecules. A molecule is also in some embodiments a derivative of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties can improve the molecule's potency, solubility, absorption, biological half-life, etc. In some embodiments, a peptide described herein comprises a half-life extending moiety (e.g., water soluble polymer, lipid, protein, or peptide). The moieties can alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, increase antibiotic spectrum, or have other effects.
[043] In some aspects, amino acid substitutions can be made in a peptide at one or more positions wherein the substitution is for an amino acid having a similar hydrophilicity. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. In some embodiments, the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Thus such conservative substitution can be made in a peptide and will likely only have minor effects on their activity. For example, the following hydrophilicity values may be assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine ( -0.4); proline ( -0.5 ± 1); alanine ( 0.5); histidine -0.5); cysteine ( -1.0); methionine ( -1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). These values can be used as a guide and thus substitution of amino acids whose hydrophilicity values are within ±2 are preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. Thus, any of the peptides or peptides described herein in some embodiments are modified by the substitution of an amino acid, for a different, but homologous amino acid with a similar hydrophilicity value. Amino acids with hydrophilicities within+/- 1.0, or+/- 0.5 points are considered homologous.
[044] The antimicrobial peptides or peptides described herein may comprise modifications. In some embodiments, a modification comprises a co-translational and/or post- translational (C-terminal peptide cleavage) modification. In some embodiments, a modification comprises, for example, disulfide bond formation, backbone cyclization, glycosylation, acetylation, phosphorylation, and proteolytic cleavage (e.g., cleavage by furins or metalloproteases). Such modifications in some instances are enabled by bridging amino acids. In some intances, modification comprise intramolecular cyclizations to form intramolecular linkages. In some instances, intramolecular linkages comprise amide, ether, disulfide, sulfide, alkyl, ester, or other bond. Non-limiting examples of bridging amino acids include aspartic acid, glutamic acid, serine, cysteine, penicillamine, dehydroalanine, or other amino acid capable of intramolecular reaction to form one or more rings. Bridging amino acids in some instances comprise canonical or non-canonical amino acids described herein. [045] In some aspects, the antimicrobial peptide (or peptide) comprises nonnaturally occurring or non-canonical amino acids. The antimicrobial peptides can comprise a combination of naturally occurring and non-naturally occurring amino acids, or may comprise only nonnaturally occurring amino acids. The non-naturally occurring amino acids optionally include synthetic non-native amino acids, substituted amino acids, or one or more D-amino acids into the peptides (or other components of the composition, with exception for protease recognition sequences) is desirable in certain situations. D-amino acid-containing peptides exhibit increased stability in vitro or in vivo compared to L-amino acid-containing forms. Thus, the construction of peptides incorporating D-amino acids can be particularly useful when greater in vivo or intracellular stability is desired or required. More specifically, D-peptides are resistant to endogenous peptidases and proteases, thereby providing better oral trans-epithelial and transdermal delivery of linked drugs and conjugates, improved bioavailability of membrane -permanent complexes, and prolonged intravascular and interstitial lifetimes when such properties are desirable. The use of D isomer peptides can also enhance trans dermal and oral trans-epithelial delivery of linked drugs and other cargo molecules. Additionally, D-peptides are often not processed efficiently for major histocompatibility complex class II-restricted presentation to T helper cells, and are therefore less likely to induce humoral immune responses in the whole organism Peptide conjugates can therefore be constructed using, for example, D-isomer forms of cell penetrating peptide sequences, L-isomer forms of cleavage sites, and D-isomer forms of therapeutic peptides. [046] In addition to the standard L-amino acids, D-amino acids or non-standard (or non- canonical), modified or unusual amino acids which are wel1-defined in the art are also contemplated for use in the present disclosure including phosphorylated amino acids (Ser,
Thr, Tyr), glycosylated amino acids (Ser, Thr, Asn), P-amino acids, GABA, and omega- amino acids.
[047] Peptides described herein often comprise non-standard (or non-canonical) amino acids. Selenocysteine is incorporated into some proteins at a UGA codon, which is normally a stop codon. Pyrrolysine is used by some methanogenic archaea in enzymes that they use to produce methane. It is coded for with the codon UAG. Examples of non-standard amino acids that are not found in proteins include lanthionine, 2-aminoisobutyric acid, dehydroalanine and the neurotransmitter gammaaminobutyric acid. Non-standard amino acids often occur as intermediates in the metabolic pathways for standard amino acids; for example ornithine and citrulline occur in the urea cycle, part of amino acid catabolism. Non-standard amino acids are usually formed through modifications to standard amino acids. For example, homocysteine is formed through the transsulfuration pathway or by the demethylation of methionine via the intermediate metabolite S-adenosyl methionine, while hydroxyproline is made by a posttranslational modification of proline.
[048] Described herein are peptide analogs, comprising a molecule similar in function to either the entire molecule or to a fragment thereof. In some embodiments, analogs comprise allelic species and induced variants. Analogs typically differ from naturally occurring peptides at one or a few positions, often by virtue of conservative substitutions. Analogs typically exhibit at least 80% or 90% sequence identity with natural peptides. Some analogs also include non-canonical amino acids or modifications of N or C terminal amino acids. Examples of non-canonical amino acids are, for example but not limited to, disubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, gamma-carboxyglutamate, epsi lon-N,NN-tri methyl 1 ysi ne, epilson-N-acetyllysine, O-phosphoserine, N-acetylserine, N- formylmethionine, 3-methylhistidine, 5-hydroxylysine, and si gm a-A-m ethyl argi nine. In some embodiments, a non-canonical amino acid is selected from 2,4-diaminobutryic acid; alpha- methylarginine; homophenylalanine; homoleucine; homoisoleucine; Diaminopropionic acid; N-acetylglycine; 6-aminohexanoic acid; gamma-aminobutyric acid; alpha-methylserine; alpha-methyltyrosine; 4-(trifluoromethyl)-L-phenylalanine; 4-amino-L-phenylalanine; Penicillamine; beta-homophenylalanine; beta-homoisoleucine; and beta-homoleucine.
[049] In some embodiments, non-canonical amino acids comprise similar structures to natural amino acids, and/or are derivatives thereof. In some embodiments, non-canonical amino acids include but are not limited to alanine derivatives; alicyclic amino acids; arginine derivatives; aromatic amino acids; asparagine derivatives; aspartic acid derivatives; beta- amino acids; cysteine derivatives; dab (2,4-diaminobutyric acid); dap (2,3-diaminopropionic acid); glutamic acid derivatives; glutamine derivatives; glycine derivatives; homo-amino acids; isoleucine derivatives; leucine derivatives; linear core amino acids; lysine derivatives; methionine derivatives; n-methyl amino acids; norleucine derivatives; norvaline derivatives; ornithine derivatives; penicillamine derivatives; phenylalanine derivatives; phenylglycine derivatives; proline derivatives; pyroglutamine derivatives; serine derivatives; threonine derivatives; tryptophan derivatives; tyrosine derivatives; valine derivatives; or other non- canonical amino acid or derivative.
[050] In some embodiments, a non-canonical amino acid is an alanine derivative. In some embodiments, alanine derivatives include but are not limited to: (-)-3-(3,4-dihydroxyphenyl)- 2-methy1-L-alanine; (R)-(+)-α-allylalanine; (R)-2-(2-propenyl)Ala-OH; (R)-2-(pentenyl)Ala- OH; (R)-propargy1-Ala-OH; (S)-(-)-α-allylalanine; (S)-(+)-2-Amino-4-phenylbutyric acid; (S)-2-(2-azidoethane)Ala-OH; (S)-2-(4-azidobutane)Ala-OH; (S)-2-(4-pentenyl)Ala-OH; (S)-
2-(5-azidopentane)Ala-OH; (S)-2-(6-azidohexane)Ala-OH; (S)-2-(7-octenyl)Ala-OH; (S)-3- (2-naphthyl)-β-Ala-OH; (S)-3-(3-pyridyl)-β-Ala-OH; (S)-propargy1-Ala-OH; 1-Na1-OH; 2- Aminoisobutyric acid; 2-Na1-OH; 3-(2-Oxo-l,2-dihydro-4-quinolinyl)alanine; 3-(2-pyridyl)- Ala-OH; 3-(2-Pyridyl)-D-alanine; 3-(2-Pyridyl)-L-alanine; 3-(2-quinolyl)-Ala-OH; 3-(2- quinolyl)-Ala-OH; 3-(2-quinoxalyl)-Ala-OH; 3-(2-Thienyl)-alanine; 3-(2-Thienyl)-L-alanine;
3-(3-pyridyl)-Ala-OH; 3-(3-Pyridyl)-L-alanine; 3-(3-quinolyl)-Ala-OH; 3-(4-pyridyl)-Ala- OH; 3-(4-Pyridyl)DD-alanine; 3-(4-pyridyl)-D-Ala-OH; 3-(4-Pyridyl)-L-alanine; 3-(9- anthryl)-Ala-OH; 3,3,3-Trifluoro-alanine; 3,3-Dipheny1-D-alanine; 3, 3 -Diphenyl -L-alanine; 3-Chloro-D-alanine; 3 -Cyclopenty1-alanine; 3 -cyclopenty1- Ala-OH; 3-iodo-D-Ala-OMe; 3- Ureidopropionic acid; Abu-OH; Aib-OH; Benzyl a-aminoisobutyrate; Cha-OH; D-1-Na1-OH; D-1-Na1-OH; D-2-Aminobutyric acid; D-2-Na1-OH; dehydro- Ala; 2-aminobutyric acid; D- styrylalanine; L-2-aminobutyric acid; L-styrylalanine; N-(3-fluorobenzyl)alanine; N-(3- Indolylacetyl)-L-alanine; N-[(S)-(+)-1-(Ethoxycarbonyl)-3-phenylpropyl]-L-alanine; N- Acety1-3-(3,4-dimethoxyphenyl)-D-alanine; a-Me-Ala-OH; β-(2-furyl)-Ala-OH; β-(2-furyl)- Ala-OH; β-(2-furyl)-D-Ala-OH; β-(2-furyl)-D-Ala-OH; β-(2-pyridyl)-Ala-OH; β-(2- quinolyl)-Ala-OH; β-(2-thienyl)- Ala-OH; β-(2-thienyl)-Ala-OH; β-(2-thienyl)-D- Ala-OH; β- (3-benzothienyl)-Ala-OH; β-(3-benzothienyl)-D-Ala-OH; β-(3-pyridyl)-Ala-OH; β-(3- pyridyl)-D-Ala-OH; β-(3 -thienyl)- Ala-OH; β-(3-thienyl)-D-Ala-OH; β-(4-pyridyl)-D-AH- OH; β-(4-thiazolyl)-Ala-OH; β-(4-thiazolyl)-Ala-OH; β-(9-anthryl)-Ala-OH; β-azido-Ala- OH; β-iodo-Ala-OBzl; β-iodo-Ala-OMe; β-iodo-Ala-OMe; β-iodo-D-Ala-OBzl; or β-pheny1- Phe-OH.
[051] In some embodiments, a non-canonical amino acid is an alicyclic amino acids. In some embodiments, alicyclic amino acids include but are not limited to: (cis)-3- aminobicyclo[2.2.1]heptane-2-carboxylic acid; exo-cis-3-aminobicyclo[2.2.1]hept-5-ene-2- carboxylic acid; 1 -amino- 1-cy cl obutanecarboxylic acid; cis-2-aminocycloheptanecarboxylic acid; 1-aminocyclohexanecarboxylic acid; trans-2-aminocyclohexanecarboxylic acid; cis-6- amino-3 -cyclohexene- 1 -carboxylic acid; 2-(1-aminocyclohexyl)acetic acid; cis-2-amino-1- cyclooctanecarboxylic acid; cis-2-amino-3-cyclooctene-1-carboxylic acid; cis-2-amino-2- methylcyclohexanecarboxylic acid; 3 -amino-3 -(4-nitrophenyl)propionic acid; 3- Azetidinecarboxylic acid; 1-aminocyclobutane carboxylic acid; 1- (amino)cyclohexanecarboxylic acid; cis-2-(amino)-cyclohexanecarboxylic acid; trans-2- (amino)-cyclohexanecarboxylic acid; cis-4-(amino)cyclohexanecarboxylic acid; trans-4- (amino)cyclohexanecarboxylic acid; (±)-cis-2-(amino)-3-cyclohexene-1-carboxylic acid; (±)- cis-6-(amino)-3-cyclohexene-1-carboxylic acid; 2-(1-aminocyclohexyl)acetic acid; cis-[4- (amino)cyclohexyl]acetic acid; (±)-cis-2-(amino)-3-cyclopentene-1-carboxylic acid; 2-(1- aminocyclopentyl)acetic acid; 1-(amino)cyclopropanecarboxylic acid; 1- aminocyclopropanecarboxylic acid; 1-(amino)cycl obutanecarboxylic acid; cis-2-(amino)- cyclohexanecarboxylic acid; trans-2-(amino)cyclohexanecarboxylic acid; trans-4- (amino)cyclohexanecarboxylic acid; cis-[4-(amino)cyclohexyl]acetic acid; (lR,4S)-(+)-4- (amino)-2-cyclopentene-1-carboxylic acid; (lS,4R)-(-)-4-(amino)-2-cyclopentene-1- carboxylic acid; 1-(amino)cyclopropanecarboxylic acid; trans-4- (aminomethyl)cyclohexanecarboxylic acid. [052] In some embodiments, a non-canonical amino acid is an arginine derivative. In some embodiments, arginine derivatives include but are not limited to L-2-amino-3- guanidinopropionic acid or 4-guanidinobutyric acid.
[053] In some embodiments, a non-canonical amino acid is an asparagine derivative. In some embodiments, arginine derivatives include but are not limited to β-acety1-L-asparagine, β-methy1-L-asparagine, or β-xantheny1-L-asparagine.
[054] In some embodiments, a non-canonical amino acid is an aspartic acid derivative. In some embodiments, aspartic acid derivatives include but are not limited to esters, amides, or anhydrides of the aspartic acid side chain.
[055] In some embodiments, a non-canonical amino acid is a beta amino acid. In some embodiments, beta amino acids include but are not limited to: (lS,3R)-(+)-3- (amino)cyclopentanecarboxylic acid; (2R,3R)-3-(amino)-2-hydroxy-4-phenylbutyric acid; (R)-2-methyl-β-Phe-OH; (R)-3-(amino)-2-methylpropionic acid; (R)-3-(amino)-4-(2- naphthyl)butyric acid; (R)-3-(amino)-5-phenylpentanoic acid; (R)-3-(trifluoromethyl)-β-Phe- OH; (R)-3,4-dimethoxy-β-Phe-OH; (R)-3-methoxy-β-Phe-OH; (R)-3-methyl-β-Phe-OH; (R)- 4-(3-pyridyl)-β-Homoala-OH; (R)-4-(4-pyridyl)-β-Homoala-OH; (R)-4-(trifluoromethyl)-β- Phe-OH; (R)-4-bromo-β-Phe-OH; (R)-4-chloro-β-Phe-OH; (R)-4-cyano-β-Homophe-OH;
(R)-4-cyano-β-Phe-OH; (R)-4-fluoro-β-Phe-OH; (R)-4-methoxy-β-Phe-OH; (R)-4-methyl-β- Phe-OH; (R)-β-Tyr-OH; (S)-(+)-Pyrrolidine-3-carboxylic acid; (S)-2-(trifluoromethyl)-β- Homophe-OH; (S)-2-(trifluoromethyl)-β-Phe-OH; (S)-2-cyano-β-Homophe-OH; (S)-2- methy1-β-Homophe-OH; (S)-2-methyl-β-Phe-OH; (S)-3-(amino)-2-methylpropionic acid;
(S)-3,4-difluoro-β-Homophe-OH; (S)-3,4-dimethoxy-β-Phe-OH; (S)-3-methoxy-β-Phe-OH; (S)-3-methy1-β-Homophe-OH; (S)-4-(4-pyridyl)-β-Homoala-OH; (S)-4-bromo-β-Phe-OH; (S)-4-chloro-β-Homophe-OH; (S)-4-chloro-β-Phe-OH; (S)-4-cyano-β-Homophe-OH; (S)-4- cyano-β-Phe-OH; (S)-4-fluoro-β-Phe-OH; (S)-4-methyl-β-Phe-OH; (S)-β-Tyr-OH; (S)-γ,γ- dipheny1-β-Homoala-OH; 2-(aminomethyl)phenylacetic acid; 3,4-Dehydro-proline; 3- Amino-3-(3-bromophenyl)propionic acid; 3-Aminobutanoic acid; β-Leu-OH; β-Leu-OH; cis- 3-(amino)cyclohexanecarboxylic acid; 3-Aminoisobutyric acid; β-Homoleucine; β- Homophenylalanine; β-Phenylalanine; D-β-Phe-OH; Hyp-OH; L-β3 -homoproline; L-β- Homohydroxyproline; L-β-Homoleucine; L-β-Homolysine; L-β-Homomethionine; L-β- Homophenylalanine; L-β-Homoproline; L-β-Homoserine; L-β-Homothreonine; L-β-Leucine; N-Acety1-β-phenylalanine; N-cis-4-hydroxy-L-proline; N-trans-4-hydroxy-L-proline; Pipecolinic acid; trans-4-Hydroxy-L-proline; β-3-Homopro-OH; β-Alanine; β-Ala-OH; β- Dab(Boc)-OH; β-Dab(Fmoc)-OH; β-Dab(Me)-OH; β-Dab-OH; β-D-Phe-OH; β-Gln-OH; β- Glu(OBzl)-OH; β-Glu(0Me)-0H; β-Glu(OtBu)-0H; β-Homoala-OH; β-Homoarg(Pmc)-OH; β-Homoarg(Tos)-OH; β-Homoarg-OH; β-Homogln(Trt)-OH; β-Homogln-OH; β- Homoglu(OtBu)-OH; β-Homohyp(Bzl)-OH; β-Homohyp(tBu)-OH; β-Homohyp-OH; β- Homoile-OH; β-Homoleu-OH; β-Homolys(Z)-OH; β-Homolys-OH; β-Homomet-OH; β- Homophe-OH; β-Homoser(Bzl)-OH; β-Homoser-OH; β-Homothr(Bzl)-OH; β- Homothr(tBu)-OH; β-Homothr-OH; β-Homotrp-OH; β-Homotyr(Bz1)-OH; β-Homotyr(tBu)- OH; β-Homotyr-OH; or β-Phe-OH.
[056] In some embodiments, a non-canonical amino acid is an aromatic amino acid. In some embodiments, aromatic amino acids include but are not limited to: 4-Acetamidobenzoic acid; 4-Acetamido-2-methylbenzoic acid; N-Acetylanthranilic acid; 3-Aminobenzoic acid; 4- Aminobenzoic acid; 2-Aminobenzophenone-2'-carboxylic acid; 2-Amino-4-bromobenzoic acid; 2-Amino-5-bromobenzoic acid; 3- Amino-2 -bromobenzoic acid; 3-Amino-4- bromobenzoic acid; 3-Amino-5-bromobenzoic acid; 4- Amino-3 -bromobenzoic acid; 5- Amino-2 -bromobenzoic acid; 2-Amino-3-bromo-5-methylbenzoic acid; 2-Amino-3- chlorobenzoic acid; 2-Amino-4-chlorobenzoic acid; 2-Amino-5-chlorobenzoic acid; 2- Amino-6-chlorobenzoic acid; 3-Amino-2-chlorobenzoic acid; 3-Amino-4-chlorobenzoic acid; 4-Amino-2-chlorobenzoic acid; 4-Amino-3-chlorobenzoic acid; 5-Amino-2-chlorobenzoic acid; 4-Amino-5-chloro-2-methoxybenzoic acid; 2-Amino-5-chloro-3-methylbenzoic acid; 3- Amino-2,5-dichlorobenzoic acid; 4-Amino-3,5-dichlorobenzoic acid; 2 -Amino-4, 5- dimethoxybenzoic acid; 4-(2-Aminoethyl)benzoic acid; 2-Amino-4-fluorobenzoic acid; 2- Amino-5-fluorobenzoic acid; 2-Amino-6-fluorobenzoic acid; 4-Amino-2-fluorobenzoic acid; 2-Amino-5-hydroxybenzoic acid; 3-Amino-4-hydroxybenzoic acid; 4-Amino-3- hydroxybenzoic acid; 2-Amino-5-iodobenzoic acid; 5-Aminoisophthalic acid; 2-Amino-3- methoxybenzoic acid; 2-Amino-4-methoxybenzoic acid; 2-Amino-5-methoxybenzoic acid; 3- Amino-2-methoxybenzoic acid; 3-Amino-5-methoxybenzoic acid; 4-Amino-2- methoxybenzoic acid; 4- Amino-3 -methoxybenzoic acid; 5-Amino-2-methoxybenzoic acid; 2- Amino-3-methylbenzoic acid; 2-Amino-5-methylbenzoic acid; 2-Amino-6-methylbenzoic acid; 3-Amino-2-methylbenzoic acid; 3-Amino-4-methylbenzoic acid; 4- (Aminomethyl)benzoic acid; 4-Amino-2-methylbenzoic acid; 4-Amino-3-methylbenzoic acid; 5-Amino-2-methylbenzoic acid; 3-Amino-2-naphthoic acid; 6-Amino-2-naphthoic acid; 2-Amino-3-nitrobenzoic acid; 2-Amino-5-nitrobenzoic acid; 4-Amino-3-nitrobenzoic acid; 5- Amino-2-nitrobenzoic acid; 3-(4-Aminophenyl)propionic acid; 4-Aminophthalic acid; 3- Aminosalicylic acid; 4-Aminosalicylic acid; 5-Aminosalicylic acid; 2-Aminoterephthalic acid; 4-Amino-2,3,5,6-tetrafluorobenzoic acid; (R)-2-Amino-l,2,3,4-tetrahydronaphthalene- 2-carboxylic acid; 2-Amino-3-(trifluoromethyl)benzoic acid; 3-Amino-5- (trifluoromethyl)benzoic acid; 5-Amino-2,4,6-triiodoisophthalic acid; 2 -Amino-3, 4, 5- trimethoxybenzoic acid; 2-Anilinophenylacetic acid; 2-Abz-OH; 3-Abz-OH; 4-Abz-OH; 3- (aminomethyl)benzoic acid; tert-Butyl 2-aminobenzoate; tert-Butyl 3-aminobenzoate; tert- Butyl 4-aminobenzoate; 4-(Butylamino)benzoic acid; 3,4-Diaminobenzoic acid; 3,5- Diaminobenzoic acid; 3,5-Dichloroanthranilic acid; 4,5-Difluoroanthranilic acid; 4- (Dimethylamino)benzoic acid; 3,5-Dimethylanthranilic acid; 5-Fluoro-2-methoxybenzoic acid; 4-(2-hydrazino)benzoic acid; 3-Hydroxyanthranilic acid; Methyl 3-aminobenzoate; 3- (Methylamino)benzoic acid; 4-(Methylamino)benzoic acid; Methyl 2-amino-4,5- dimethoxybenzoate; 4-Nitroanthranilic acid; N-Phenylanthranilic acid; or 4-aminosalicylate. [057] In some embodiments, a non-canonical amino acid is a cysteine derivative. In some embodiments, cysteine derivatives include but are not limited to: methylselenocysteine; L- cysteic acid; L-cysteinesulfmic acid; D-ethionine; or seleno-L-cystine.
[058] In some embodiments, a non-canonical amino acid is a Dab or Dap derivative. In some embodiments, Dab or Dap derivatives include but are not limited to: L-2,4- Diaminobutyric acid; Dab(Alloc)-OH; Dab(Boc)-OH; Dap(Alloc)-OH; Dap(Fmoc)-OH; Dap-OH; Dap(Z)-OH; D-2,3-Diaminopropionic acid; L-2,3-Diaminopropionic acid; 2,3- Diaminopropionic acid; Dap(Boc)-OH; or D-Dap-OH.
[059] In some embodiments, a non-canonical amino acid is a glutamic acid or glutamine derivative. In some embodiments, glutamic acid or glutamine derivatives include but are not limited to: g-Carboxy-glutamic acid, (4S)-4-(4-Trifluoromethy1-benzyl)-L-glutamic acid, or D-Citrulline.
[060] In some embodiments, a non-canonical amino acid is a glycine derivative. In some embodiments, glycine derivatives include but are not limited to: D-Allylglycine; N- [Bis(methylthio)methylene]glycine; ally1-Gly-OH; Chg-OH; D-Chg-OH; D- cyclopropylglycine; L-cyclopropylglycine; N-iminodiacetic acid; (2-indanyl)-Gly-OH; (±)-α- phosphonoglycine; D-propargylglycine; propargy1-Gly-OH; (R)-2-thienylglycine; (S)-2- thienylglycine; (R)-3 -thienyl glycine; (2S,3R,4S)-α-(Carboxycyclopropyl)glycine; D-α- Cyclohexylglycine; Ethyl acetamidocyanoacetate; ally1-Gly-OH; D-ally1-Gly-OH; N-(4- aminobutyl)-Gly-OH; N-(2-aminoethyl)-Gly-OH; N-(2,4-dimethoxybenzyl)-Gly-OH; N- iminodiacetic acid; D-propargy1-Gly-OH; N-(2-Hydroxyethyl)iminodiacetic acid; N-(4- Hydroxyphenyl)glycine; Iminodiacetic acid; N-Lauroylsarcosine; L-α-Neopentyl glycine; N- (Phosphonomethyl)glycine; L-C-Propargylglycine; Sarcosine; or a-Phosphonoglycine.
[061] In some embodiments, a non-canonical amino acid is a homo-amino acid. In some embodiments, homo-amino acids include but are not limited to: (R)-2-(Boc-amino)-5- hexynoic acid; Homophe-OH; Homophe-OH; Homoser(Trt)-OH; piperidine-2-carboxylic acid; L-Homoarginine; Homocysteine; L-Homocystine; L-Homophenylalanine; D- Homophenylalanine; D-Homoserine; or L-homoserine.
[062] In some embodiments, a non-canonical amino acid is a leucine, isoleucine, or valine derivative. In some embodiments, leucine, isoleucine, or valine derivatives include but are not limited to: 3-Fluoro-valine; 4,4,4,4',4',4'-Hexafluoro-valine; (R)-(+)-α-Methylvaline; (S)- (-)-α-Methylvaline; N-(2,2,2-trifluoromethyl)-L-Valine; D-allo-Isoleucine; N-[(2S,3R)-3- Amino-2-hydroxy-4-phenylbutyryl]-L-leucine; Cycloleucine; N-(3,5-Dinitrobenzoyl)- leucine; tBu-Gly-OH; N-Formy1-Leu-OH; N-(3-Indolylacetyl)-L-isoleucine; D-tert-Leucine; L-tert-Leucine; or 5,5,5-Trifluoro-leucine.
[063] In some embodiments, a non-canonical amino acid is a linear core amino acid. In some embodiments, linear core amino acids include but are not limited to: L-Allysine ethylene acetal; 12-Aminododecanoic acid; 2-Aminoheptanoic acid; 7-Aminoheptanoic acid;
2-Aminohexadecanoic acid technical,; 6-Aminohexanoic acid; (R)-3-Amino-5-hexynoic acid; (S)-3-Amino-5-hexynoic acid; 4-Amino-3-hydroxybutyric acid; (R)-3- Amino-2 - (hydroxym ethyl )propionic acid; (S)-3-Amino-2-(hydroxymethyl)propionic acid; 3 -Amino-3 - (3-methoxyphenyl)propionic acid; (R)-2-(Aminomethyl)-3-methylbutyric acid; (S)-2- (Aminomethyl)-3-methylbutyric acid; 8-Aminooctanoic acid; (R)-3-Aminopentanoic acid; (S)-3-Aminopentanoic acid; 11-Aminoundecanoic acid; 5-Aminovaleric acid; Boc-12-Ado- OH; Boc-7-Ahp-OH; Boc-6-Ahx-OH; Boc-6-Ahx-OSu; (R)-3-(amino)-5-hexenoic acid; (S)-
3-(amino)-5-hexenoic acid; (R)-3-(amino)-5-hexynoic acid; (S)-3-(amino)-5-hexynoic acid; (2R, 3R)-3-(amino)-2-m ethy1-3 -(4-chlorophenyl)propionic acid; (2S, 3S)-3-(amino)-2- methy1-3-(4-chlorophenyl)propionic acid; (2R, 3R)-3-(amino)-2-m ethy1-3 -phenylpropionic acid; (2S,3S)-3-(amino)-2-methy1-3-phenylpropionic acid; (R)-2-(amino)octanedioic acid; 8- Aoc-OH; 11-Aun-OH; 5-Ava-OH; GABA-OH; 4-(Dimethylamino)butyric acid; 12-Ado-OH; 6-Ahx-OH; 8-Aoc-OH; 11-Aun-OH; 5-Ava-OH; GABA-OH; 4-(Methylamino)butyric acid; 12-(Methylamino)dodecanoic acid; or 6-Aminohexanoic acid.
[064] In some embodiments, a non-canonical amino acid is a lysine derivative. In some embodiments, lysine derivatives include but are not limited to: Lys(Me,Boc)-OH; Lys(palmitoyl)-OH; 5-Hydroxylysine; Lys(Me)2-OH; Lys(Ac)-OH; Lys(C(O)CF3)-0H; or Lys(Me)-OH.
[065] In some embodiments, a non-canonical amino acid is a methionine derivative. In some embodiments, methionine derivatives include but are not limited to: (2S)-2-[[[4-[[(2R)- 2-amino-3 -mercaptopropyl] amino] -2-phenylphenyl] -oxom ethyl] amino] -4- (methylthio)butanoic acid; 2-(1,3-benzothiazo1-2-ylamino)-4-(methylthio)butanoic acid; 2- [(6-bromo-4-quinazolinyl)amino]-4-(methylthio)butanoic acid; 2-[[(4-ethylphenyl)- oxomethyl]amino]-4-(methylthio)butanoic acid; 2-amino-4-(methylsulfanyl)butanoic acid; N- acetylmethionine; methionine S-oxide; methionine sulfone; or methionine sulfoximine.
[066] In some embodiments, a non-canonical amino acid is an N-methyl amino acid. In some embodiments, N-methyl amino acids include but are not limited to: L-Abrine; N-Me- Aib-OH; N-Me-Ala-OH; N-Me-D-Ala-OH; N-Me-Leu-OH; N-Me-D-Leu-OH; N-Me-Phe- OH; N-Me-Thr(Bzl)-OH; N-Me-Thr-OH; N-Me-Tyr(Bzl)-OH; N-Me-Tyr-OH; N-Me-Va1- OH; N,N-Dimethylglycine; N-methy1-L-phenylalanine; N-Me-Aib-OH; N-Me-Ala-OH; N- Me-Asp(OtBu)-OH;; N-Me-Asp-OH; N-Me-Cys(Trt)-OH; N-Me-Cys-OH; N-Me- Glu(OtBu)-OH; N-Me-Glu-OH N-Me-His(Trt)-OH; N-Me-His-OH; N-Me-Ile-OH; N-Me- Lys(Boc)-OH; N-Me-Lys-OH; N-Me-Met-OH; N-Me-D-Phe-OH; N-Me-Ser(Bzl)-OH; N- Me-Ser-OH; N-Me-Ser(tBu)-OH; N-Me-Thr(tBu)-OH; N-Me-Thr-OH; N-Me-Tyr(tBu)-OH; N-Me-Tyr-OH; N-Me-Va1-OH; N-Methy1-L-alanine; N-Methy1-L-leucine; N-Methy1-L- phenylalanine; or N-Methy1-L-proline.
[067] In some embodiments, a non-canonical amino acid is a norleucine, norvaline, ornithine, or penicillamine derivative. In some embodiments, norleucine, norvaline, ornithine, or penicillamine derivatives include but are not limited to: 6-azido-L-norleucine; Nle-OH; D- Norleucine; L-Norleucine; Norvaline; (S)-5-Azido-2-(amino)pentanoic acid; Orn(Alloc)-OH; Om(Z)-OH; D-Ornithine; L-Omithine; Pen(Mob)-OH; Pen(Trt)-OH; D-Penicillamine; L- Penicillamine; or D-Penicillamine disulfide.
[068] In some embodiments, a non-canonical amino acid is a phenylalanine derivative. In some embodiments, phenylalanine derivatives include but are not limited to: 2-fluoro- phenylalanine; 4-fluoro-phenylalanine; 4-Amino-L-phenylalanine; 3 -[3, 4- bis(trifluoromethyl)phenyl]-L-alanine; Bpa-OH; D-Bpa-OH; 4-tert-buty1-Phe-OH; 4-tert- buty1-D-Phe-OH; 4-(Fmoc-amino)-L-phenylalanine; β2-homophenyl alanine; 2-methoxy-L- phenylalanine; 2-nitro-L-phenylalanine; pentafluoro-D-phenylalanine; pentafluoro-L- phenylalanine; Phe(4-Br)-OH; D-Phe(4-Br)-OH; D-Phe(2-CF3)-OH; Phe(3-CF3)-OH; Phe(4- CF3)-OH; Phe(2-Cl)-OH; D-Phe(2-Cl)-OH; Phe(2,4-Cl2)-OH; D-Phe(2,4-Cl2)-OH; D-Phe(3- Cl)-OH; Phe(3,4-Cl2)-OH; D-Phe(3,4-Cl2)-OH; D-Phe(4-Cl)-OH; D-Phe(2-CN)-OH; Phe(3- CN)-OH; D-Phe(3 -CN)-OH; Phe(4-CN)-OH; D-Phe(4-CN)-OH; Phe(2-Me)-OH; D-Phe(2- Me)-OH; Phe(3-Me)-OH; D-Phe(3-Me)-OH; Phe(4-Me)-OH; Phe(4-NH2)-OH; Phe(4-NO2)- OH; D-Phe(4-NO2)-0H; Phe(2-F)-OH; Phe(3-F)-OH; D-Phe(3-F)-OH; Phe(3,4-F2)-OH; D- Phe(3,4-F2)-OH; Phe(3,5-F2)-OH; Phe(4-F)-OH; Phe(4-I)-OH; D-Phe(4-I)-OH; 4-Borono-D- phenylalanine; 4-Borono-L-phenylalanine; p-Bromo-phenylalanine; 4-Bromo-L- phenylalanine; β-pheny1-D-phenylalanine; 4-Chloro-L-phenylalanine; 2,3- Difluorophenylalanine; 3,5-Difluorophenylalanine; 3,4-Dihydroxy-L-phenylalanine; 3 -(3 ,4- Dimethoxyphenyl)-L-alanine; 2-methoxy-L-phenylalanine; o-Fluoro-phenylalanine; m- Fluoro-L-phenylalanine; m-Fluoro-phenylalanine; p-Fluoro-D-phenylalanine; p-Fluoro- phenylalanine; 4-Fluoro-D-phenylalanine; 2-fluoro-L-phenylalanine; D-4- bromophenylalanine; L-3-fluorophenylalanine; L-4-fluorophenylalanine; Phe(4-Boc2- guanidino)-OH; phe(3-Br)-OH; Phe(2-CF3)-OH; D-Phe(3-CF3)-OH; D-Phe(4-CF3)-OH; Phe(4-Cl)-OH; D-Phe(2-F)-OH; D-Phe(4-F)-OH; 4-(phosphonomethyl)-Phe-OH; L-4- trifluoromethylphenylalanine; 3,4,5-trifluoro-D-phenylalanine; 6-Hydroxy-DOPA; N-(3- Indolylacetyl)-L-phenylalanine; p-Iodo-D-phenylalanine; a-Methy1-D-phenylalanine; a- Methy1-L-phenylalanine; a-Methy1-phenylalanine; 4-Nitro-D-phenylalanine; 4-Nitro-L- phenylalanine; 2-(Trifluoromethyl)-D-phenylalanine; 2-(Trifluoromethyl)-L-phenylalanine; 3-(Trifluoromethyl)-L-phenylalanine; 4-(Trifluoromethyl)-D-phenylalanine; or L-Phe chloromethyl ketone.
[069] In some embodiments, a non-canonical amino acid is a phenylglycine derivative. In some embodiments, phenylglycine derivatives include but are not limited to: 2-(piperazino)- 2-(2-fluorophenyl)acetic acid; 2-(piperazino)-2-(3-pyridyl)acetic acid; 2-(piperazino)-2-[4- (trifluorom ethyl )phenyl] acetic acid; 2-Chlorophenyl glycine; 4-Chlorophenyl glycine; (R)-(-)- 2-(2,5-Dihydrophenyl)glycine; (R)-(-)-N-(3,5-Dinitrobenzoyl)-α-phenylglycine; (S)-(+)-N- (3,5-Dinitrobenzoyl)-α-phenylglycine; 2,2-Diphenylglycine; 2-Fluoro-α-phenylglycine; 4- Fluoro-D-α-phenylglycine; 4-Hydroxy-D-phenylglycine; 4-Hydroxy-L-phenylglycine; (R)- (-)-2-Phenylglycine; (S)-(+)-2-Phenylglycine; 3-(Trifluoromethyl)-phenylglycine; or 4- (Trifluoromethyl)-L-phenylglycine.
[070] In some embodiments, a non-canonical amino acid is a pyroglutamine derivative.
In some embodiments, pyroglutamine derivatives include but are not limited to: (4R)-4- benzy1-Pyr-OH (4R)-4-(4-bromobenzyl)-Pyr-OH; (4R)-4-(4-methylbenzyl)-Pyr-OH; (R)-5- oxopyrrolidine-2-carboxylic acid; or L-Pyroglutamic acid.
[071] In some embodiments, a non-canonical amino acid is a serine or threonine derivative. In some embodiments, serine or threonine derivatives include but are not limited to: (2R,3S)-3-phenylisoserine; Ser[GalNAc(Ac)3-α-D]-OH; Isoserine; 3-Phenylserine; Thr[GalNAc(Ac)3-α-D]-OH; or L-allo-Threonine.
[072] In some embodiments, a non-canonical amino acid is a tryptophan derivative. In some embodiments, tryptophan derivatives include but are not limited to: 5-Fluoro-L- tryptophan; 5-Hydroxy-L-tryptophan; 5-Methoxy-tryptophan; or 5 -Methyl -tryptophan.
[073] In some embodiments, a non-canonical amino acid is a tyrosine derivative. In some embodiments, tyrosine derivatives include but are not limited to: 3-Amino-L-tyrosine; Tyr(3,5-l2)-OH; 3-Chloro-L-tyrosine; [CpRu(tyrosin)]CF3CO2; α-Methy1-tyrosine; 3-Nitro-L- tyrosine; or o-tyrosine.
[074] In some embodiments, exemplary non-canonical amino acids include but are not limited to: (S)-α-Amino-γ-butyrolactone; 2-Aminocaprylic acid; 7-Aminocephalosporanic acid; (S)-(+)-α-Aminocyclohexanepropionic acid; (R)-Amino-(4-hydroxyphenyl)acetic acid; 5-Aminolevulinic acid; 4-Amino-nicotinic acid; 3-Aminophenylacetic acid; 4- Aminophenylacetic acid; 2-Amino-2-phenylbutyric acid; 4-(4-Aminophenyl)butyric acid; 2- (4-Aminophenylthio)acetic acid; 5-Aminovaleric acid; 8-Benzyl (S)-2-aminooctanedioate; (lR,3S,4S)-2-azabicyclo[2.2.1]heptane-3-carboxylic acid; 1-L-azetidine-2-carboxylic acid; 1- azetidine-3-carboxylic acid; N-N'-Fmoc-diaminoacetic acid; N-N'-diaminoacetic acid; Boc- Inp-OH; (S)-1-4-oxopiperidine-2-carboxylic acid; 2-(4-Boc-piperazino)-2-(4- fluorophenyl)acetic acid; 2-(4-piperazino)-2-(4-fluorophenyl)acetic acid; 2-(4-piperazino)-2- phenylacetic acid; 2-(4-Boc-piperazino)-2-phenylacetic acid; 1-4-piperidylacetic acid; (-)-L- thioproline; Tle-OH; 3-piperidinecarboxylic acid; L-(+)-Canavanine; Carnitine; 2,6- Diaminopimelic acid; 2,6-Diaminopimelic acid; meso-2,3-Diaminosuccinic acid; 4- (Dimethylamino)cinnamic acid; 4-(Dimethylamino)phenylacetic acid; Ethyl (S)-piperidine-3- carboxylate; Ethyl piperazinoacetate; Inp-OH; Nip-OH; 4-pheny1-piperidine-4-carboxylic acid; 1-piperazineacetic acid; 4-piperidineacetic acid; (R)-piperidine-2-carboxylic acid; (S)- piperidine-2-carboxylic acid; (S)-2- 1,2, 3, 4-tetrahydronorharmane-3 -carboxylic acid; Tic-OH; Iminodiacetic acid; Indoline-2-carboxylic acid; Kynurenine; L-aziridine-2-carboxylate; Methyl 4-aminobutyrate; (S)-2-Piperazinecarboxylic acid; (R)-(-)-3-Piperidinecarboxylic acid; 2-Pyrrolidone-5-carboxylic acid; (R)-(+)-2-Pyrrolidone-5-carboxylic acid; (S)-l,2,3,4- Tetrahydro-3-isoquinolinecarboxylic acid; L-4-Thiazolidinecarboxylic acid; (4R)-(-)-2- Thioxo-4-thiazolidinecarboxylic acid; hydrazinoacetic acid; or Tle-OH.
[075] In some embodiments, peptides described herein comprise amino acids which promote secondary structure formation, such as hairpin formation (constrained amino acids). In some embodiments, constrained amino acids include but are not limited to analogs of proline. For example, analogs of proline include but are not limited to proline, proline analogs, Om(i-PrCO-Hao), 5-hydrazino-2-methoxybenzoic acid (Hao), N-alkyl amino acid (e.g., N-( C1-C6 alkyl)), or an alpha, alpha-disubstituted amino acid (e.g., alpha, alpha-(C1-C6 dialkyl)). In some embodiments two or more amino acids are replaced with a constrained amino acid or analog or isostere thereof.
[076] Analogs of amino acids may comprise non-canonical amino acids with similar shape, charge, or properties. In some embodiments, peptides described herein comprise proline analogs. Proline include but are not limited to: (R)-α-ally1-proline; (R)-(+)4- oxazolidinecarboxylic acid; (S)-(-)-4-oxazolidinecarboxylic acid; (S)-α-ally1-proline; L- azetidine-2-carboxylic acid; α-(2-bromobenzyl)-Proline; α-(4-bromobenzyl)-Proline; α-(2- chlorobenzyl)-Proline; α-(3-chlorobenzyl)-Proline; α-(diphenylmethyl)-proline; (R)-α-(4- fluorobenzyl)-Proline; (S)-α-(4-fluorobenzyl)-Pro; α-(4-fluorobenzyl)-proline; cis-4-amino- proline; trans-4-amino-proline; b3 -Homoproline; cis-4-hydroxy-proline; trans-4-hydroxy- proline; a-Me-proline; α-(4-methylbenzyl)-proline; α-(1-naphthylmethyl)-proline; thioproline; 4-[2-(trifluoromethyl)benzyl]-proline; 4-[4-(trifluoromethyl)benzyl]-proline; a- (4-trifluoromethylbenzyl)-Proline; trans-3-tertbutoxyproline; piperidine-2-carboxylic acid; D- Proline; a-Methy1-proline; or thiazolidine-2-carboxylic acid.
[077] The amino acids of the antimicrobial peptides of the present disclosure may also be modified. For example, amino groups in some embodiments are acylated, alkylated, or arylated. Benzyl groups in some embodiments are halogenated, nitrosylated, alkylated, sulfonated or acylated.
[078] Carboxy terminal modifications include acylation with carboxylic acids: formic, acetic, propionic, fatty acids (myristic, palmitic, stearic), succinic, benzoic, carbobenzoxy (Cbz); and biotinylation. Amino terminal modifications include: (i) acylation with carboxylic acids: formic, acetic, propionic, fatty acids (myristic, palmitic, stearic, etc.) succinic, benzoic, carbobenzoxy (Cbz); (ii) biotinylation; (iii) attachment of dyes such as fluorescein (FITC, FAM, etc.), 7-hydroxy-4-methylcoumarin-3-acetic acid, 7-hydroxycoumarin-3-acetic acid, 7- methoxycoumarin-3 -acetic acid and other coumarins; rhodamines (5-carboxyrhodamine 110 or 6G, 5(6)-TAMRA, ROX); N-4-(4-dimethylamino)phenylazo]benzoic acid (Dabcyl), 2,4- dinitrobenzene (Dnp), 5-dimethylaminonaphthalene-1-sulfonic acid (Dansyl) and other dyes; and (iv) polyethylene glycol. These include, for example, beta-alanine (beta-Ala) and other omega-amino acids such as 3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4- aminobutyric acid and so forth; alpha-aminoisobutyric acid (Aib); epsilon-aminohexanoic acid (Aha); sigma-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Om); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); norleucine (Nle); 4-chlorophenylalanine (Phe(4-Cl)); 2- fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); homoarginine (hArg); epsilon-N-acetyllysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,4- diaminobutyric acid (Dab); p-aminophenylalanine (Phe(pNH2)); N-methyl valine (MeVal); homocysteine (hCys), homophenylalanine (hPhe) and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro), beta-homocysteine (bHomoCys), beta-homophenylalanine (bHomoPhe) and beta-homoserine (bHomoSer); N-methylated amino acids and peptoids (N- substituted glycines). In some embodiments, a peptide described herein comprises a non- canonical amino acid with a free thiol, such as homocysteine, penicillamine (Pen), or other thio1-containing amino acid.
[079] The peptide in some embodiments is modified at its N and/or C termini (“capping” groups) with an acyl (abbreviated "Ac") and/or an amido (abbreviated "Am") group, respectively, for example acetyl (CH3CO-) at the N terminus and amido (-NH2) at the C terminus. A broad range of N-terminal capping functions, preferably in a linkage to the terminal amino group, is contemplated, for example: formyl; alkanoyl, having from 1 to 10 carbon atoms (such as acetyl, propionyl, or butyryl); cycloalkanoyl, having from 1 to 10 carbon atoms; alkenoyl, having from 1 to 10 carbon atoms (such as hex-3 -enoyl); alkynoyl, having from 1 to 10 carbon atoms, such as (hex-5 -ynoyl); aroyl, (such as benzoyl or 1- naphthoyl); heteroaroyl (such as 3-pyrroyl or 4-quinoloyl); alkylsulfonyl (such as methanesulfonyl); arylsulfonyl (such as benzenesulfonyl or sulfanilyl); heteroaryl sulfonyl (such as pyridine-4-sulfonyl); substituted alkanoyl, having from 1 to 10 carbon atoms (such as 4-aminobutyryl); substituted alkenoyl, having from 1 to 10 carbon atoms, (such as 6- hydroxy-hex-3 -enoyl); substituted alkynoyl, having from 1 to 10 carbon atoms (such as 3- hydroxy-hex-5-ynoyl) substituted aroyl (such as 4-chlorobenzoyl or 8-hydroxy-naphth-2- oyl); substituted heteroaroyl (such as 2,4-dioxo-1,2,3,4-tetrahydro-3-methylquinazolin-6- oyl); substituted alkyl sulfonyl (such as 2-aminoethanesulfonyl); substituted arylsulfonyl (such as 5-dimethylamino-1-naphthalenesulfonyl); substituted heteroaryl sulfonyl (such as 1- methoxy-6-isoquinolinesulfonyl); carbamoyl (-C(O)NH2) or thiocarbamoyl (-C(S)NH2); substituted carbamoyl (-C(O)NR'R") or substituted thiocarbamoyl (-C(S)NR'R") wherein R' and R" are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl, or substituted heteroaryl; substituted carbamoyl (-C(O)NR'R") and substituted thiocarbamoyl (-C(S)NR'R") wherein R' and R" are each independently hydrogen, alkanoyl, alkenoyl, alkynoyl, aroyl, heteroaroyl, substituted alkanoyl, substituted alkenoyl, substituted alkynoyl, substituted aroyl, or substituted heteroaroyl.
[080] In some embodiments, a peptide described herein has a C-terminal modification. In some embodiments, the C-terminal modification is an amide or ester. In some embodiments, C-terminal modifications that provide for an amide bond are designated as -NR'R2 wherein R1 and R2 are independently hydrogen; alkyl, preferably having from 1 to 10 carbon atoms, such as methyl, ethyl, or isopropyl; cycloalkyl, preferably having from 1 to 10 carbon atoms, such as cyclopropyl, cyclobutyl, or cyclopentyl; alkenyl, preferably having from 1 to 10 carbon atoms, such as prop-2-enyl; alkynyl, preferably having from 1 to 10 carbon atoms, such as prop-2-ynyl; substituted alkyl having from 1 to 10 carbon atoms, such as hydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl, haloalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkanoylalkyl, carboxyalkyl , carbamoylalkyl; substituted alkenyl having from 1 to 10 carbon atoms, such as hydroxyalkenyl, alkoxyalkenyl, mercaptoalkenyl, alkylthioalkenyl, haloalkenyl, cyanoalkenyl, aminoalkenyl, alkylaminoalkenyl, dialkylaminoalkenyl, alkanoylalkenyl, carboxyalkenyl, carbamoylalkenyl; substituted alkynyl having from 1 to 10 carbon atoms, such as hydroxyalkynyl, alkoxyalkynyl, mercaptoalkynyl, alkylthioalkynyl, halogenoalkynyl, cyanoalkynyl, aminoalkynyl, alkylaminoalkynyl, dialkylaminoalkynyl, alkanoylalkynyl, carboxy alkynyl, carbamoylalkynyl; aroylalkyl having up to 10 carbon atoms, such as phenacyl or 2-benzoyl ethyl; aryl, such as phenyl or 1 -naphthyl; heteroaryl, such as 4- quinolyl; alkanoyl having from 1 to 10 carbon atoms, such as acetyl or butyryl; aroyl, such as benzoyl; heteroaroyl, such as 3-quinoloyl; OR' or NR'R" where R' and R" are independently hydrogen, alkyl, aryl, heteroaryl, acyl including aroyl, sulfonyl, sulfmyl, or SO2-R'" or SO- R" where R'" is substituted or unsubstituted alkyl, aryl, heteroaryl, alkenyl, or alkynyl. [081] Alternatively, R1 and R2 in some embodiments are taken together to form a heterocycloalkyl group or a substituted version thereof containing from 3 to 10 ring members including heteroatoms.
[082] C-terminal modifications providing an ester bond are designated as OR, wherein R may comprise an alkoxy; aryloxy; heteroaryloxy; aralkoxy; heteroaralkoxy; substituted alkoxy; substituted aryloxy; substituted heteroaryloxy; substituted aralkoxy; or substituted heteroaralkoxy group.
[083] N-terminal or the C-terminal modifications, or both, are in some embodiments of such a structure that the peptide functions as a prodrug (a pharmacologically inactive derivative of the parent drug molecule) that undergoes spontaneous or enzymatic transformation within the body in order to release the active drug and that has improved delivery properties over the parent drug molecule.
[084] In some embodiments, N or C terminal modification results in attachment of additional chemical groups. For example, the presence of a sulfhydryl group linked to the N- or C-modification permits conjugation of the derivatized peptide to other molecules.
[085] In yet a further aspect, the peptides or variants or derivatives thereof can be "retro- inverso peptides." A "retro-inverso peptide" refers to a peptide with a reversal of the direction of the peptide bond on at least one position, i.e., a reversal of the amino- and carboxy- termini with respect to the side chain of the amino acid. Thus, a retro-inverso analogue has reversed termini and reversed direction of peptide bonds while approximately maintaining the topology of the side chains as in the native peptide sequence. The retro-inverso peptide can contain L-amino acids or D-amino acids, or a mixture of L-amino acids and D-amino acids, up to all of the amino acids being the D-isomer. Partial retro-inverso peptide analogues are peptides in which only part of the sequence is reversed and replaced with enantiomeric amino acid residues. Since the retro- inverted portion of such an analogue has reversed amino and carboxyl termini, the amino acid residues flanking the retro-inverted portion are replaced by side-chain-analogous alpha-substituted geminal-diaminomethanes and malonates, respectively. Retro-inverso forms of cell penetrating peptides have been found to work as efficiently in translocating across a membrane as the natural forms.
[086] Embodiments of the present disclosure also include longer peptides built from repeating units of an antimicrobial peptide. A peptide multimer may comprise different combinations of peptide. Such multimeric peptides can be made by chemical synthesis or by recombinant DNA techniques as discussed herein. When produced by chemical synthesis, the oligomers preferably have from 2-5 repeats of a core peptide sequence, and the total number of amino acids in the multimer should not exceed about 160 residues, preferably not more than 100 residues (or their equivalents, when including linkers or spacers).
[087] Peptidomimetics
[088] In addition to the peptides disclosed herein, the present disclosure also contemplates that structurally similar compounds in some embodiments are formulated to mimic the key portions of peptide or peptides of the present disclosure. Such compounds, which may be termed peptidomimetics, in some embodiments are used in the same manner as the peptides of the present disclosure and, hence, also are functional equivalents.
[089] In some embodiments peptides and proteins describe a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits in some embodiments are linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g. ester, ether, etc. In some embodiments, peptides comprise a peptidomimetic or peptide mimic wherein the peptide is modified in such a way that it includes at least one non-peptidic bond such as, for example, urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond. A peptidomimetic agent in some embodiments is an unnatural peptide or a non-peptide agent that recreates the stereospatial properties of the binding elements of the antimicrobial peptide such that it has the binding activity and biological activity of the unmodified peptide. In some aspects, the present disclosure also includes compounds that retain partial peptide characteristics. For example, any proteolytically unstable bond within a peptide of the present disclosure could be selectively replaced by a non-peptidic element such as an isostere (N-methylation; D-amino acid) or a reduced peptide bond while the rest of the molecule retains its peptidic nature. In some embodiments, one, two, three, four, or five peptide bonds are reduced in the antimicrobial peptides described herein. These reduced peptide bonds result in the conversion of an amide into a amine.
[090] Additionally contemplated are azapeptide analogs wherein the alpha-carbon atom is replaced with an isoelectronic nitrogen atom within the azapeptide, the side chains remain unchanged but the hydrogen atom on the alpha-carbon atom is missing. It is contemplated the entire peptide in some embodiments is constructed of azapeptide linkages or only one, two, three, four, five, six, seven, eight, nine, ten, fifteen, or all alpha-carbon atoms are replaced with azapeptide linkages. [091] In yet another aspect, the antimicrobial peptides described herein in some embodiments are chemically prepared as a comparable peptoid. The peptoids in some embodiments are prepared using a glycine backbone in which the respective side chain of the peptide has been attached to the nitrogen atom rather than the alpha-carbon atom. In some aspects, the conversion of the peptides into isomerically similar peptoids results in the production of compound which is more resistant to the activity of proteases or peptidases. It is contemplated one or more side chain from one or more amino acid has been converted into the corresponding peptoid molecule. In some embodiments, the number of amino acid residues converted into their peptoid counterpart is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or all of the amino acid residues.
[092] Peptidomimetic compounds, either agonists, substrates or inhibitors, have been described for a number of bioactive peptides/peptides such as opioid peptides, VIP, thrombin, HIV protease, etc. These methods are used to make peptidomimetics that possess at least the binding capacity and specificity of the desired antimicrobial peptide and also possess the biological activity. Knowledge of peptide chemistry and general organic chemistry available to those skilled in the art are sufficient, in view of the present disclosure, for designing and synthesizing such compounds.
[093] For example, such peptidomimetics in some embodiments are identified by inspection of the three-dimensional structure of a peptide or peptide of the present disclosure either free or bound in complex with a ligand. Alternatively, the structure of a peptide of the present bound to its ligand can be gained by the techniques of nuclear magnetic resonance spectroscopy. Greater knowledge of the stereochemistry of the interaction of the peptide with its ligand or receptor will permit the rational design of such peptidomimetic agents. The structure of a peptide or peptide of the invention in the absence of ligand could also provide a scaffold for the design of mimetic molecules.
[094] Stabilized Peptides
[095] Peptides described herein may be stabilized by one or more bonds, such as covalent bonds. In some embodiments, stabilization of peptides comprises use of "Stapled Peptide" technology. The general approach for "stapling" a peptide is that two key residues within the peptide are modified by attachment of linkers through the amino acid side chains. Once synthesized, the linkers are connected, optionally through a catalyst, thereby creating a bridge (or linkage) that physically constrains the peptide into its native alpha-helical shape. In addition to helping retain the native structure needed to interact with a target molecule, this conformation also provides stability against peptidases as well as promotes cell permeating properties.
[096] More particularly, peptide stapling in some embodiments encompasses the joining of two double bond-containing sidechains, two triple bond-containing sidechains, or one double bond-containing and one triple bond-containing side chain, which may be present in a peptide chain, using any number of reaction conditions and/or catalysts to facilitate such a reaction, to provide a singly "stapled" peptide. In a specific embodiment, the introduction of a staple entails a modification of standard peptide synthesis, with alpha-methyl, alpha-alkenyl amino acids being introduced at two positions along the peptide chain, separated by either three or six intervening residues (i + 4 or i + 7). These spacings place the stapling amino acids on the same face of the alpha-helix, straddling either one (i + 4) or two (i + 7) helical turns. The fully elongated, resin-bound peptide can be exposed to a ruthenium catalyst that promotes cross-linking of the alkenyl chains through olefin metathesis, thereby forming an al1-hydrocarbon macrocyclic cross-link. Additionally, the term "peptide stitching" refers to multiple and tandem "stapling" events in a single peptide chain to provide a "stitched" (multiply stapled) peptide.
[097] Water-Soluble Polymers
[098] In some embodiments, a peptide described herein is attached to a water-soluble polymer. In some embodiments, the water-soluble polymer is a nonpeptidic, nontoxic, and biocompatible. As used herein, a substance is considered biocompatible if the beneficial effects associated with use of the substance alone or with another substance (e.g., an antimicrobial peptide) in connection with living tissues (e.g., administration to a patient) outweighs any deleterious effects as evaluated by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist. In some embodiments, a water-soluble polymer is further non-immunogenic. In some embodiments, a substance is considered non-immunogenic if the intended use of the substance in vivo does not produce an undesired immune response (e.g., the formation of antibodies) or, if an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist.
[099] The antimicrobial peptides in some embodiments are conjugated with heterologous peptide segments or polymers, such as polyethylene glycol (PEG). The peptides in some embodiments are linked to PEG to increase the hydrodynamic radius and hence increase the serum persistence. The peptides in some embodiments are conjugated to any targeting agent, such as a ligand having the ability to specifically and stably bind to an external receptor. [0100] In certain aspects, methods and compositions of the embodiments relate to PEGylation of disclosed peptides. PEGylation is the process of covalent attachment of poly(ethylene glycol) polymer chains to another molecule, normally a drug or therapeutic protein. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target macromolecule. The covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent from the host's immune system (reduced immunogenicity and antigenicity) or increase the hydrodynamic size (size in solution) of the agent, which prolongs its circulatory time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins.
[0101] The first step of the PEGylation is the suitable functionalization of the PEG polymer at one or both terminals. PEGs that are activated at each terminus with the same reactive moiety are known as "homobifunctional," whereas if the functional groups present are different, then the PEG derivative is referred as "heterobifunctional" or "heterofunctional." The chemically active or activated derivatives of the PEG polymer are prepared to attach the PEG to the desired molecule.
[0102] The choice of the suitable functional group for the PEG derivative is based on the type of available reactive group on the molecule that will be coupled to the PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. The N-terminal amino group and the C- terminal carboxylic acid in some embodiments are sites of PEGylation.
[0103] The techniques used to form first generation PEG derivatives are generally reacting the PEG polymer with a group that is reactive with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates, and carbonates. In the second generation PEGylation chemistry more efficient functional groups, such as aldehyde, esters, amides, etc., are made available for conjugation.
[0104] As applications of PEGylation have become more and more advanced and sophisticated, there has been an increase in need for heterobifunctional PEGs for conjugation. These heterobifunctional PEGs are very useful in linking two entities, where a hydrophilic, flexible, and biocompatible spacer is needed. Preferred end groups for heterobifunctional PEGs are maleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids, and NHS esters. [0105] The most common modification agents, or linkers, are based on methoxy PEG (mPEG) molecules. Their activity depends on adding a protein-modifying group to the alcohol end. In some embodiments polyethylene glycol (PEG diol) is used as the precursor molecule. The diol is subsequently modified at both ends in order to make a hetero- or homo- dimeric PEGlinked molecule.
[0106] Proteins are generally PEGylated at nucleophilic sites, such as unprotonated thiols (cysteinyl residues) or amino groups. Examples of cysteinyl -specific modification reagents include PEG maleimide, PEG iodoacetate, PEG thiols, and PEG vinylsulfone. All four are strongly cysteiny1-specific under mild conditions and neutral to slightly alkaline pH.
[0107] In some embodiments, the water-soluble polymer is characterized as having from about 2 to about 300 termini. Exemplary water soluble polymers include, but are not limited to, poly(alkylene glycols) such as polyethylene glycol (“PEG”), polypropylene glycol) (“PPG”), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefmic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polydimethylacrylamide (PDAAm), polyphosphazene, polyoxazolines (“POZ”), poly(N-acryloylmorpholine), and combinations thereof.
[0108] In some embodiments, the water-soluble polymer is not limited to a particular structure. In some embodiments, the water-soluble polymer is linear (e.g., an end capped, e.g., alkoxy PEG or a bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), a dendritic (or star) architecture, each with or without one or more degradable linkages. Moreover, the internal structure of the water-soluble polymer can be organized in any number of different repeat patterns and can be selected from the group consisting of homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer.
[0109] In some embodiments, the weight-average molecular weight of the water-soluble polymer attached to a peptide described herein is from about 100 Daltons to about 150,000 Daltons. Exemplary ranges include, for example, weight-average molecular weights in the range of greater than 5,000 Daltons to about 100,000 Daltons, in the range of from about 6,000 Daltons to about 90,000 Daltons, in the range of from about 10,000 Daltons to about 85,000 Daltons, in the range of greater than 10,000 Daltons to about 85,000 Daltons, in the range of from about 20,000 Daltons to about 85,000 Daltons, in the range of from about 53,000 Daltons to about 85,000 Daltons, in the range of from about 25,000 Daltons to about 120,000 Daltons, in the range of from about 29,000 Daltons to about 120,000 Daltons, in the range of from about 35,000 Daltons to about 120,000 Daltons, and in the range of from about 40,000 Daltons to about 120,000 Daltons.
[0110] PEGs will typically comprise a number of (OCH2CH2) monomers [or (CH2CH2O) monomers, depending on how the PEG is defined]. As used herein, the number of repeating units is identified by the subscript “n” in “(OCH2CH2)n.” Thus, the value of (n) typically falls within one or more of the following ranges: from 2 to about 3400, from about 100 to about 2300, from about 100 to about 2270, from about 136 to about 2050, from about 225 to about 1930, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1,200 to about 1,900. For any given polymer in which the molecular weight is known, it is possible to determine the number of repeating units (i.e., “n”) by dividing the total weight-average molecular weight of the polymer by the molecular weight of the repeating monomer.
[0111] In some embodiments, the water-soluble polymer is an end-capped polymer, that is, a polymer having at least one terminus capped with a relatively inert group, such as a lower C1-6 alkoxy group, or a hydroxyl group. When the polymer is PEG, for example, a methoxy-PEG (commonly referred to as mPEG) may be used, which is a linear form of PEG wherein one terminus of the polymer is a methoxy ( — OCH3) group, while the other terminus is a hydroxyl or other functional group that can be optionally chemically modified. In some embodiments, exemplary water-soluble polymers include, but are not limited to, linear or branched discrete PEG (dPEG); linear, branched, or forked PEGs; and Y-shaped PEG derivatives.
[0112] In some embodiments, a water-soluble polymer comprises a polyglycerol (PG). In some embodiments, the polyglycerol is a hyperbranched PG (HPG). In other cases, the polyglycerol is a linear PG (LPG). In additional cases, the polyglycerol is a midfunctional PG, a linear-block-hyperbranched PG or a side-chain functional PG.
[0113] In some embodiments, a water-soluble polymer is a degradable synthetic PEG alternative. Exemplary degradable synthetic PEG alternatives include, but are not limited to, poly[oligo(ethylene glycol)methyl methacrylate] (POEGMA); backbone modified PEG derivatives generated by polymerization of telechelic, or di-end-functionalized PEG-based macromonomers; PEG derivatives comprising comonomers comprising degradable linkage such as poly [(ethylene oxide)-co-(methylene ethylene oxide)] [P(EO-co-MEO)], cyclic ketene acetals such as 5,6-benzo-2-methylene-1,3-dioxepane (BMDO), 2-methylene- 1,3- dioxepane (MDO), and 2-methyl ene-4-pheny1-1,3-dioxolane (MPDL) copolymerized with OEGMA; or poly-(ε-caprolactone)-graft-poly(ethylene oxide) (PCL-g-PEO).
[0114] In some embodiments, a peptide described herein is conjugated to a degradable synthetic PEG alternative, such as for example, POEGM; backbone modified PEG derivatives generated by polymerization of telechelic, or di-end-functionalized PEG-based macromonomers; P(EO-co-MEO); cyclic ketene acetals such as BMDO, MDO, and MPDL copolymerized with OEGMA; or PCL-g-PEO.
[0115] In some embodiments, a water-soluble polymer comprises a poly(zwitterions). Exemplary poly(zwitterions) include, but are not limited to, poly(sulfobetaine methacrylate) (PSBMA), poly(carboxybetaine methacrylate) (PCBMA), and poly(2-methyacryloyloxyethyl phosphorylcholine) (PMPC). In some embodiments, a water-soluble polymer comprises a polycarbonate. Exemplary polycarbones include, but are not limited to, pentafluorophenyl 5- methy1-2-oxo-1,3-dioxane-5-carboxylate (MTC-OC6F5). In some embodiments, a water- soluble polymer comprises a polymer hybrid, such as for example, a polycarbonate/PEG polymer hybrid, a peptide/protein-polymer conjugate, or a hydroxyl containing and/or zwitterionic derivatized polymer (e.g., a hydroxyl containing and/or zwitterionic derivatized PEG polymer). In some embodiments, a water-soluble polymer comprises a polysaccharide. Exemplary polysaccharides include, but are not limited to, dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethy1- starch (HES). In some embodiments, a water-soluble polymer comprises a glycan. Exemplary classes of glycans include A-linked glycans, O-linked glycans, glycolipids, O-GlcNAc, and glycosainoglycans. In some embodiments, a water-soluble polymer comprises a polyoxazoline polymer. A polyoxazoline polymer is a linear synthetic polymer, and similar to PEG, comprises a low polydispersity. In some embodiments, a polyoxazoline polymer is a polydispersed polyoxazoline polymer, characterized with an average molecule weight. In some embodiments, the average molecule weight of a polyoxazoline polymer includes, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, 100,000, 200,000, 300,000, 400,000, or 500,000 Da.
[0116] In some embodiments, a polyoxazoline polymer comprises poly(2 -methyl 2- oxazoline) (PMOZ), poly(2-ethyl 2-oxazoline) (PEOZ), or poly(2 -propyl 2-oxazoline) (PPOZ). In some embodiments, a water-soluble polymer comprises a polyacrylic acid polymer.
[0117] In some embodiments, a water-soluble polymer comprises polyamine. Polyamine is an organic polymer comprising two or more primary amino groups. In some embodiments, a polyamine includes a branched polyamine, a linear polyamine, or cyclic polyamine. In some embodiments, a polyamine is a low-molecular-weight linear polyamine. Exemplary polyamines include putrescine, cadaverine, spermidine, spermine, ethylene diamine, 1,3- diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, and piperazine.
[0118] Lipids
[0119] In some embodiments, a peptide described herein is conjugated to a moiety described herein is a lipid. In some embodiments, the lipid is a fatty acid. In some embodiments, the fatty acid is a saturated fatty acid. In other cases, the fatty acid is an unsaturated fatty acid. Exemplary fatty acids include, but are not limited to, fatty acids comprising from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms.
In some embodiments, the lipid binds to one or more serum proteins, thereby increasing serum stability and/or serum half-life.
[0120] In some embodiments, the lipid is conjugated to a peptide described herein. In some embodiments, the lipid is a fatty acid, e.g., a saturated fatty acid or an unsaturated fatty acid. In some embodiments, the fatty acid is from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms. In some embodiments, the fatty acid comprises about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbon atoms in length. In some embodiments, the fatty acid comprises caproic acid (hexanoic acid), enanthic acid (heptanoic acid), caprylic acid (octanoic acid), pelargonic acid (nonanoic acid), capric acid (decanoic acid), undecylic acid (undecanoic acid), lauric acid (dodecanoic acid), tridecylic acid (tridecanoic acid), myristic acid (tetradecanoic acid), pentadecylic acid (pentadecanoic acid), palmitic acid (hexadecanoic acid), margaric acid (heptadecanoic acid), stearic acid (octadecanoic acid), nonadecylic acid (nonadecanoic acid), arachidic acid (eicosanoic acid), heneicosylic acid (heneicosanoic acid), behenic acid (docosanoic acid), tricosylic acid (tricosanoic acid), lignoceric acid (tetracosanoic acid), pentacosylic acid (pentacosanoic acid), or cerotic acid (hexacosanoic acid). In some embodiments, the antimicrobial peptide-lipid conjugate enhances serum stability and/or serum half-life.
[0121] Fusion Proteins
[0122] Certain embodiments of the present disclosure concern fusion proteins of the antimicrobial peptides or peptides. These molecules may have the peptides of the embodiments linked at the N- or C-terminus to a heterologous domain. For example, fusions may also employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Fusion proteins can comprise a half-life extender. Another useful fusion includes the addition of a protein affinity tag, such as a serum albumin affinity tag or six histidine residues, or an immunologically active domain, such as an antibody epitope, preferably cleavable, to facilitate purification of the fusion protein. Non-limiting affinity tags include polyhistidine, chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST).
[0123] In a particular embodiment, the peptides or peptides of the embodiments in some embodiments are linked to a peptide that increases the in vivo half-life, such as an XTEN peptide, IgG Fe domain, albumin, or albumin binding peptide.
[0124] Methods of generating fusion proteins are well known to those of skill in the art. Such proteins can be produced, for example, by de novo synthesis of the complete fusion protein, or by attachment of the DNA sequence encoding the heterologous domain, followed by expression of the intact fusion protein.
[0125] Production of fusion proteins that recover the functional activities of the parent proteins in some embodiments are facilitated by connecting genes with a bridging DNA segment encoding a peptide linker that is spliced between the peptides connected in tandem. The linker would be of sufficient length to allow proper folding of the resulting fusion protein.
[0126] In some embodiments, a peptide described herein is attached to a protein or a binding fragment thereof. Exemplary proteins include albumin, transferrin, or transthyretin.
In some embodiments, the protein or a binding fragment thereof comprises an antibody, or its binding fragments thereof. In some embodiments, a peptide comprises a protein or a binding fragment thereof. In some embodiments, a peptide described herein comprising a protein or a binding fragment thereof has an increased serum half-life, and/or stability. In some embodiments, a peptide described herein comprising a protein or a binding fragment thereof has an increased antibiotic spectrum.
[0127] In some embodiments, the protein is albumin. Albumin is a family of water-soluble globular proteins. It is commonly found in blood plasma, comprising about 55-60% of all plasma proteins. Human serum albumin (HSA) is a 585 amino acid polypeptide in which the tertiary structure is divided into three domains, domain I (amino acid residues 1-195), domain II (amino acid residues 196-383), and domain III (amino acid residues 384-585). Each domain further comprises a binding site, which can interact either reversibly or irreversibly with endogenous ligands such as long- and medium-chain fatty acids, bilirubin, or hemin, or exogenous compounds such as heterocyclic or aromatic compounds.
[0128] In some embodiments, the protein is albumin is transferrin. Transferrin is a 679 amino acid polypeptide that is about 80 kDa in size and comprises two Fe3+ binding sites with one at the N-terminal domain and the other at the C-terminal domain. In some embodiments, human transferrin has a half-life of about 7-12 days.
[0129] In some embodiments, the protein is transthyretin (TTR). Transthyretin is a transport protein located in the serum and cerebrospinal fluid which transports the thyroid hormone thyroxine (T4) and retino1-binding protein bound to retinol.
[0130] In some embodiments, the protein is an antibody, or its binding fragments thereof. In some embodiments, an antibody or its binding fragments thereof comprise a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab’, divalent Fab2, F(ab)'3 fragments, single-chain variable fragment (scFv), bis- scFv, (SCFV)2, diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof.
[0131] In some embodiments, the protein comprises a scFv, bis-scFv, (scFv)2, dsFv, or sdAb. In some embodiments, the protein comprises a scFv. In some embodiments, the protein comprises a bis-scFv. In some embodiments, the protein comprises a (scFv)2. In some embodiments, the protein comprises a dsFv. In some embodiments, the protein comprises a sdAb. [0132] In some embodiments, the protein comprises an Fc portion of an antibody, e.g., of IgG, IgA, IgM, IgE, or IgD. In some embodiments, the moiety comprises an Fc portion of IgG (e.g, IgGi, IgG3, or IgG4).
[0133] Linkers
[0134] In certain embodiments, the antimicrobial peptides or peptides of the embodiments in some embodiments are chemically conjugated using bifunctional cross-linking reagents or fused at the protein level with peptide linkers. Bifunctional cross-linking reagents have been extensively used for a variety of purposes, including preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies. Suitable peptide linkers may also be used to link the peptide or peptide of the embodiments, such as Gly-Ser linkers.
[0135] Homobifunctional reagents that carry two identical functional groups induce cross- linking between identical and different macromolecules or subunits of a macromolecule, and linking of peptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By taking advantage of the differential reactivities of the two different functional groups, cross-linking can be controlled both selectively and sequentially. The bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g., amino-, sulfhydryl' guanidine-, indole-, carboxy1-specific groups. Of these, reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis, and the mild reaction conditions under which they can be applied. A majority of heterobifunctional cross-linking reagents contain a primary amine-reactive group and a thio1- reactive group.
[0136] In another example, heterobifunctional cross-linking reagents are used to generate peptides described herein. The cross-linking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing coupling, in one example, of aldehydes to free thiols. The cross-linking reagent can be modified to crosslink various functional groups.
[0137] Additionally, any other linking/coupling agents and/or mechanisms known to those of skill in the art are in some embodiments used to combine peptides of the embodiments, such as, for example, antibody-antigen interaction, avidin biotin linkages, amide linkages, ester linkages, thioester linkages, ether linkages, thioether linkages, phosphoester linkages, phosphoramide linkages, anhydride linkages, disulfide linkages, ionic and hydrophobic interactions, bispecific antibodies and antibody fragments, or combinations thereof. [0138] It is preferred that a cross-linker having reasonable stability in blood or other relevant tissue site of infection will be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo. These linkers are thus one group of linking agents.
[0139] In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP, and 2-iminothiolane. The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.
[0140] Once chemically conjugated, the peptide generally will be purified to separate the conjugate from unconjugated agents and from other contaminants. A large number of purification techniques are available for use in providing conjugates of a sufficient degree of purity to render them clinically useful.
[0141] Purification methods based upon size separation, such as gel filtration, gel permeation, or high performance liquid chromatography, will generally be of most use. Other chromatographic techniques, such as Blue-Sepharose separation, may also be used. Conventional methods to purify the fusion proteins from inclusion bodies in some embodiments are useful, such as using weak detergents, such as sodium N-lauroy1-sarcosine (SLS).
[0142] Cell Penetrating and Membrane Translocation Peptides [0143] Furthermore, in certain aspects, the present disclosure contemplates fusing or conjugating a cel1-penetrating domain (also called a cell delivery domain, or cell transduction domain) to an antimicrobial peptide. Such domains are well known in the art and are generally characterized as short amphipathic or cationic peptides and peptide derivatives, often containing multiple lysine and arginine resides. Of particular interest are the TAT sequence from HIV1 (YGRKKRRQRRR), and poly-D-Arg and poly-D-Lys sequences (e.g, dextrorotary residues, eight residues in length). As used herein the terms "cell penetrating peptide" and "membrane translocation domain" are used interchangeably and refer to segments of peptide sequence that allow a peptide to cross the cell membrane (e.g, the plasma membrane in the case a eukaryotic cell).
[0144] Examples of CPP segments include, but are not limited to, segments derived from HIV Tat (e.g, GRKKRRQRRRPPQ), herpes virus VP22, the Drosophila Antennapedia homeobox gene product, protegrin I, Penetratin (RQIKIWFQNRRMKWKK) or melittin (GIGAVLKVLTTGLPALISWIKRKRQQ). In certain aspects, the CPP comprises the T1 (TKIESLKEHG), T2 (TQIENLKEKG), 26 (AALEALAEALEALAEALEALAEAAAA) or INF7 (GLFEAIEGFIENGWEGMIEGW Y GCG) CPP sequence.
[0145] Peptides in some embodiments are modified for in vivo use by the addition, at the amino- and/or carboxy1-terminal ends, of a blocking agent to facilitate survival of the peptide in vivo are contemplated. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. These agents can be added either chemically during the synthesis of the peptide, or by recombinant DNA technology by methods familiar in the art. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues. In addition, nanoparticles could be used for the packaging and delivery of the peptide.
[0146] Peptide Delivery
[0147] A nucleic acid encoding a peptide of the present disclosure may be made by any technique known to one of ordinary skill in the art. Non-limiting examples of a synthetic nucleic acid, particularly a synthetic oligonucleotide, include a nucleic acid made by in vitro chemical synthesis using phosphotriester, phosphite or phosphoramidite chemistry and solid phase techniques, or via deoxynucleoside H-phosphonate intermediates. A non-limiting example of enzymatically produced nucleic acids includes one produced by enzymes in amplification reactions such as PCR™. A non-limiting example of a biologically produced nucleic acid includes recombinant nucleic acid production in living cells, such as recombinant DNA vector production in bacteria.
[0148] The nucleic acid(s), regardless of the length of the sequence itself, is in some embodiments combined with other nucleic acid sequences, including but not limited to, promoters, enhancers, polyadenylation signals, restriction enzyme sites, multiple cloning sites, coding segments, and the like, to create one or more nucleic acid construct(s). The overall length may vary considerably between nucleic acid constructs. Thus, a nucleic acid segment of almost any length may be employed, with the total length preferably being limited by the ease of preparation or use in the intended recombinant nucleic acid protocol.
[0149] Targeting Agents [0150] Peptides described herein may comprise other peptides, small molecules, antibodies, nucleic acids, or other targeting agent. In some embodiments, the N-terminal modification or C-terminal modification comprises at least one targeting agent. In some embodiments, the targeting agent increases the local concentration of the peptide. In some embodiments, the targeting agent broadens or broadens the antibiotic spectrum.
[0151] Pharmaceutical Compositions
[0152] Disclosed herein are pharmaceutical compositions comprising a peptide disclosed herein and a pharmaceutically acceptable excipient, vehicle, or second therapeutic agent. The compositions may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides described herein. The peptides in some embodiments are different. Alternatively, the peptides in some embodiments are the same or similar. The compositions may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides. The peptides in some embodiments are different. The peptides may comprise different therapeutic agents, different peptides, or a combination thereof. The peptides in some embodiments are the same or similar. In some embodiments a pharmaceutical composition comprises a peptide having any one of SEQ ID NOS: 1-313. In some embodiments a pharmaceutical composition comprises a peptide having any one of SEQ ID NOS: 2-313.
[0153] In some embodiments, compositions comprise an anti-microbial peptide described herein are a pharmaceutically acceptable vehicle such as a diluent, adjuvant, excipient, or carrier with which at least one antibody is administered. The compositions disclosed herein may further comprise one or more pharmaceutically acceptable salts, excipients or vehicles. Pharmaceutically acceptable salts, excipients, or vehicles in some embodiments include carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, aerosolizing agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants. In some embodiments, compositions described herein comprise a pharmaceutically acceptable excipient, carrier or adjuvant, such as an excipient, carrier or adjuvant that may be administered to a subject, together with at least one antibody of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.
[0154] Neutral buffered saline or saline mixed with serum albumin are exemplary appropriate carriers. The pharmaceutical compositions may include antioxidants such as ascorbic acid; low molecular weight peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, pluronics, or polyethylene glycol (PEG). Also by way of example, suitable tonicity enhancing agents include alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol, and the like. Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and the like. Hydrogen peroxide in some embodiments is used as a preservative. Suitable cosolvents include glycerin, propylene glycol, and PEG. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxy- propy1-beta-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal, and the like. The buffers in some embodiments are conventional buffers such as acetate, borate, citrate, phosphate, bicarbonate, or Tris-HCl. Acetate buffer in some embodiments is about pH 4-5.5, and Tris buffer can be about pH 7-8.5.
[0155] The composition comprising a peptide described herein often is in liquid form or in a lyophilized or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and/or bulking agents. In one embodiment, a lyoprotectant is included, which is a non-reducing sugar such as sucrose, lactose or trehalose. The amount of lyoprotectant generally included is such that, upon reconstitution, the resulting formulation will be isotonic, although hypertonic or slightly hypotonic formulations also are suitable in some embodiments. In addition, the amount of lyoprotectant should be sufficient to prevent an unacceptable amount of degradation and/or aggregation of the protein upon lyophilization. Exemplary lyoprotectant concentrations for sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilized formulation are from about 10 mM to about 400 mM. In another embodiment, a surfactant is included, such as for example, nonionic surfactants and ionic surfactants such as polysorbates (e.g., polysorbate 20, polysorbate 80); poloxamers (e.g., poloxamer 188); poly(ethylene glycol) phenyl ethers (e.g., Triton); sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; laury1-, myristy1-, linoley1-, or steary1-sulfobetaine; laury1-, myristy1-, linoley1-or steary1-sarcosine; linoleyl, myristy1-, or cety1-betaine; lauroamidopropy1-, cocamidopropy1-, linoleamidopropy1- , myristamidopropy1-, palmidopropy1-, or isostearamidopropy1-betaine (e.g., lauroamidopropyl); myristamidopropy1-, palmidopropy1-, or isostearamidopropy1- dimethylamine; sodium methyl cocoy1-, or disodium methyl oleoy1-taurate; and the MONAQUAT™. series (Mona Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68 etc). Exemplary amounts of surfactant present in the pre-lyophilized formulation are from about 0.001-0.5%. High molecular weight structural additives (e.g., fillers, binders) may include for example, acacia, albumin, alginic acid, calcium phosphate (dibasic), cellulose, carboxymethylcellulose, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, dextran, dextrin, dextrates, sucrose, tylose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylates, povidone, sodium alginate, tragacanth microcrystalline cellulose, starch, and zein. Exemplary concentrations of high molecular weight structural additives are from 0.1% to 10% by weight. In other embodiments, a bulking agent (e.g., mannitol, glycine) is included.
[0156] Compositions in some embodiments are suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into an animal by any route available to the skilled worker, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, or intralesional routes. A parenteral formulation in some embodiments is a sterile, pyrogen-free, isotonic aqueous solution, optionally containing pharmaceutically acceptable preservatives.
[0157] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringers' dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, anti-microbials, anti -oxidants, chelating agents, inert gases and the like. [0158] Pharmaceutical compositions described herein may be formulated for controlled or sustained delivery in a manner that provides local concentration of the product (e.g., bolus, depot effect) and/or increased stability or half-life in a particular local environment. The compositions can include the formulation peptides, nucleic acids, or vectors disclosed herein with particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., as well as agents such as a biodegradable matrix, injectable microspheres, microcapsular particles, microcapsules, bioerodible particles beads, liposomes, and implantable delivery devices that provide for the controlled or sustained release of the active agent which then can be delivered as a depot injection. Techniques for formulating such sustained-or controlled-delivery means are known and a variety of polymers have been developed and used for the controlled release and delivery of drugs. Such polymers are typically biodegradable and biocompatible. Polymer hydrogels, including those formed by complexation of enantiomeric polymer or peptide segments, and hydrogels with temperature or pH sensitive properties, may be desirable for providing drug depot effect because of the mild and aqueous conditions involved in trapping bioactive protein agents (e.g., antibodies comprising an ultralong CDR3). In some embodiments, pharmaceutical compositions comprise controlled release porous polymeric microparticles.
[0159] Suitable materials for this purpose include polylactides, polymers of poly-(a- hydroxycarboxylic acids), such as poly-D-(-)-3-hydroxybutyric acid, copolymers of L- glutamic acid and gamma ethy1-L-glutamate, poly(2-hydroxyethy1-methacrylate), and ethylene vinyl acetate, or poly-D-(-)-3-hydroxybutyric acid. Other biodegradable polymers include poly(lactones), poly(acetals), poly(orthoesters), and poly(orthocarbonates). Sustained-release compositions also may include liposomes, which can be prepared by any of several methods known in the art. The carrier itself, or its degradation products, should be nontoxic in the target tissue and should not further aggravate the condition. This can be determined by routine screening in animal models of the target disorder or, if such models are unavailable, in normal animals.
[0160] Microencapsulation of recombinant proteins for sustained release has been performed successfully with human growth hormone (rhGH), interferon-(rhlFN-), interleukin-2, and MN rgpl20. The sustained-release formulations of these proteins were developed using poly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids can be cleared quickly within the human body. Moreover, the degradability of this polymer can be depending on its molecular weight and composition.
[0161] Bioadhesive polymers are also contemplated for use in or with compositions of the present disclosure. Bioadhesives are synthetic and naturally occurring materials able to adhere to biological substrates for extended time periods. For example, Carbopol and polycarbophil are both synthetic cross-linked derivatives of poly(acrylic acid). Bioadhesive delivery systems based on naturally occurring substances include for example hyaluronic acid, also known as hyaluronan. Hyaluronic acid is a naturally occurring mucopolysaccharide consisting of residues of D-glucuronic and N-acety1-D-glucosamine. Hyaluronic acid is found in the extracellular tissue matrix of vertebrates, including in connective tissues, as well as in synovial fluid and in the vitreous and aqueous humor of the eye. Esterified derivatives of hyaluronic acid have been used to produce microspheres for use in delivery that are biocompatible and biodegradable.
[0162] Both biodegradable and non-biodegradable polymeric matrices in some embodiments are used to deliver compositions of the present disclosure, and such polymeric matrices may comprise natural or synthetic polymers. Biodegradable matrices are preferred. The period of time over which release occurs is based on selection of the polymer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. Exemplary synthetic polymers used to form the biodegradable delivery system include: polymers of lactic acid and glycolic acid, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkyl ene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyanhydrides, polyurethanes and copolymers thereof, poly(butyric acid), poly(valeric acid), alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), polyethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone. Exemplary natural polymers include alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.
[0163] Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and triglycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the product is contained in a form within a matrix and (b) diffusional systems in which a product permeates at a controlled rate. Liposomes containing the product are often prepared by methods known by those skilled in the art.
[0164] Peptides described herein may be administered to a subject to treat a disease or condition, such as an infection. In one embodiment, the compositions described herein are administered enterically to the small intestine. Routes of delivery may include non-invasive peroral (through the mouth), topical (skin), transmucosal (nasal, buccal/sublingual, vaginal, ocular and rectal) and inhalation routes, as well as parenteral routes, and other methods known in the art. Parenteral refers to a route of delivery that is generally associated with injection, including intraorbital, infusion, intraarterial, intracarotid, intracap sular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrastemal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions in some embodiments are in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. In some embodiments, administration of peptides described herein comprises inhalation (e.g., vaporization).
[0165] Alternatively or additionally, the compositions in some embodiments are administered locally via implantation into the affected area of a membrane, sponge, or other appropriate material on to which a peptide disclosed herein has been absorbed or encapsulated. Where an implantation device is used, the device in some embodiments is implanted into any suitable tissue or organ, and delivery of an antibody comprising an ultralong CDR3 antibody fragment, nucleic acid, or vector disclosed herein can be directly through the device via bolus, or via continuous administration, or via catheter using continuous infusion.
[0166] A pharmaceutical composition comprising a peptide disclosed herein may be formulated for inhalation, such as for example, as a dry powder. Inhalation solutions in some embodiments are formulated in a liquefied propellant for aerosol delivery. In yet another formulation, solutions in some embodiments are nebulized. Additional pharmaceutical compositions for pulmonary administration include pulmonary delivery of chemically modified proteins. For pulmonary delivery, the particle size should be suitable for delivery to the distal lung. For example, the particle size is 1 pm to 5 pm; however, larger particles in some embodiments are used, for example, if each particle is fairly porous.
[0167] Certain formulations containing antibodies comprising a peptide disclosed herein may be administered orally. Formulations administered in this fashion in some embodiments are formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents in some embodiments are included to facilitate absorption of a selective binding agent. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders also can be employed.
[0168] Another preparation may involve an effective quantity of an antibody comprising a peptide disclosed herein in a mixture with non-toxic excipients which are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions in some embodiments are prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
[0169] Suitable and/or preferred pharmaceutical formulations in some embodiments are determined in view of the present disclosure and general knowledge of formulation technology, depending upon the intended route of administration, delivery format, and desired dosage. Regardless of the manner of administration, an effective dose is often calculated according to patient body weight, body surface area, or organ size. Further refinement of the calculations for determining the appropriate dosage for treatment involving each of the formulations described herein are routinely made in the art and is within the ambit of tasks routinely performed in the art. Appropriate dosages in some embodiments are ascertained through use of appropriate dose-response data.
[0170] Disinfectant Compositions
[00171] The antimicrobial peptides of the present disclosure are useful in a variety of environments including industrial, clinical, the household, and personal care. The peptide compositions of the present disclosure for industrial, pharmaceutical, household and personal care use may comprise at least one active ingredient, of which the peptide of the present disclosure is an active ingredient acting alone, additively, or synergistically against the target microbe.
[00172] Accordingly, the antimicrobial compositions of the present disclosure may be used to form contact-killing coatings or layers on a variety of substrates including personal care products (e.g., toothbrushes, contact lens cases and dental equipment), healthcare products, household products, food preparation surfaces and packaging, and laboratory and scientific equipment. Further, other substrates include medical devices such as catheters, urological devices, blood collection and transfer devices, tracheotomy devices, intraocular lenses, wound dressings, sutures, surgical staples, membranes, shunts, gloves, tissue patches, prosthetic devices (e.g., heart valves) and wound drainage tubes. Still further, other substrates include textile products such as carpets and fabrics, paints and joint cement. A further use is as an antimicrobial soil fumigant. In some embodiments, the substrate is a shunt, a catheter, artificial protheses, screw, plate, or pin. In some embodiments, a device configured for external fixation of a surgical device is coated with a composition comprising a peptide described herein.
[0173] Methods of treatment
[0174] Described herein are peptides comprising antimicrobial activity, wherein the peptide is capable of killing a microbial organism or inhibiting its growth. The antimicrobial activities of the antimicrobial peptides can include, without limitation, antibacterial, antiviral, or antifungal activities. In particular aspects, the present disclosure provides antimicrobial peptides with activity against gram-negative bacteria. The present disclosure also provides methods of using the antimicrobial peptides and antimicrobial compositions of the present disclosure to prevent, inhibit or terminate the growth of at least one microbe which may include, for example, bacteria, archaea, fungi, algae, protozoa, multicellular parasites, and viruses.
[0175] Described herein are therapeutic agents which may comprise a protein or peptide that modulates the activity of another protein, peptide, cell or tissue. Modulating the activity in some embodiments comprises increasing, decreasing, stimulating, or preventing the activity or expression of the protein, peptide, cell or tissue. Therapeutic agents may modulate the activity of proteins or peptides involved in the etiology of a disease or disorder. Exemplary proteins may include, but are not limited to, at least a portion of a hormone, kinase, receptor, ligand, growth factor, regulatory protein, metabolic protein, cytokine, chemokine, interferon, phosphatase, antibody or any combination thereof.
[0176] Peptides and compositions of the present disclosure may result in antimicrobial effects on a target microbial organism. In some embodiments, peptides and compositions disclosed herein are used to treat a disease or infection associated with the target microbial organism. An antimicrobial effect includes preventing or inhibiting the growth of or killing the target microbial organisms, or interfering with any biological functions of the target microbial organisms. In general, the compositions of the present disclosure can be used to treat or prevent a disease or infection at any place in a host, e.g., at any tissue including surfaces of any tissue or implant. In one embodiment, the compositions are used to specifically kill or inhibit bacterial target microbial organisms in body fluid (e.g, blood, sputum). In some embodiments, peptides described herein are bacteriostatic.
[0177] Peptides described herein may be used for the treatment or alleviation of diseases, including but not limited to: 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder; and/or 2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. In some embodiments treatment comprises clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. Thus those in need of treatment may include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In some embodiments, treatment of a disease or condition comprises executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. In some embodiments, the administration of one or more antimicrobial peptides provided herein is used to prevent, treat or relieve the symptoms of a microbial infection. In some embodiments, peptides described herein comprise antimicrobial activity, including prevention, inhibition or termination of a microbe. In some aspects, prevention is considered to be the obstruction or hindrance of any potential microbial growth, and inhibition is considered to be a reduction in microbial growth. This may occur via, but is not limited to, a microbiostatic mechanism such as interference in the synthesis of the cell wall or binding to ribosomal subunits to prevent production of microbial proteins. In some embodiments, termination is considered to be the actual killing of the microbes by the presence of the composition. This may occur via, but is not limited to, a microbiocidal mechanism such as a change in osmotic pressure leading to bursting of the cell or formation of leaky channels in the cell wall and membrane causing loss of cellular material. In some embodiments, peptides described herein treat organisms comprising the phylogenetic domains bacteria and archaea, as well as unicellular and filamentous fungi (e.g., yeasts and molds), unicellular and filamentous algae, unicellular and multicellular parasites, and viruses.
[0178] In some embodiments, compositions of the present disclosure are effective against bacteria including Gram-positive and Gram-negative cocci, Gram-positive and Gram negative straight, curved and helical/vibroid and branched rods, sheathed bacteria, sulfur- oxidizing bacteria, sulfur or sulfate-reducing bacteria, spirochetes, actinomycetes and related genera, myxobacteria, mycoplasmas, rickettsias and chlamydias, cyanobacteria, archea, fungi, parasites, viruses and algae. For example, the target microbial organisms of the present disclosure include, without limitation, Enter obacteriaceae, including Escherichia and Klebsiella spp., Enterococcus spp., Acinetobacter spp., Candida spp., Salmonella spp., Staphylococcus spp., Streptococcus spp. and Pseudomonas spp., especially Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Campylobacter jejuni, Candida albicans, Candida krusei, Chlamydia trachomatis, Clostridium difficile, Cryptococcus neoformans, Haempohilus injluenzae, Helicobacter pylori, Moraxella catarrhalis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella disenteriae, Staphylococcus aureus, and Streptococcus pneumoniae. In some embodiments, compositions are effective against drug-resistant and multi-drug resistant bacterial species.
[0179] In particular embodiments, the compositions of the present disclosure are effective against Gram-negative bacteria. Gram-positive and Gram-negative cocci include, but are not limited to, Aerococcus, Enterococcus, Halococcus, Leuconostoc, Micrococcus, Mobiluncus, Moraxella catarrhalis, Neisseria (including N gonorrheae and N. meningitidis), Pediococcus, Peptostreptococcus, Staphylococcus species (including S. aureus, methicillin resistant S. aureus, coagulase-negative S. aureus, and S. saprophyticus), Streptococcus species (including S. pyogenes, S. agalactiae, S. bovis, S. pneumoniae, S. mutans, S. sanguis, S. equi, S. equinus, S. thermophilus, S. morbillorum, S. hansenii, S. pleomorphus, and S. parvulus), and Veillonella.
[0180] The Gram -positive and Gram-negative straight, curved, helical/vibrioid and branched rods include, but are not limited to, Acetobacter, Acinetobacter species (including A. baumannii), Actinobacillus equuli, Aeromonas, Agrobacterium, Alcaligenes, Aquaspirillum, Arcanobacterium haemolyticum, Bacillus species (including B. cereus and B. anthracis), Bacteroides species (including B. fragilis), Bartonella, Bordetella species (including B. pertussis), Brochothrix, Brucella, Burkholderia cepacia, Calymmatobacterium granulomatis, Campylobacter species (including C. jejuni), Capnocytophaga, Caulobacter, Chromobacterium violaceum, Citrobacter, Clostridium species (including C . perfringens, C. tetani and C. difficile), Comamonas, Curtobacterium, Edwardsiella, Eikenella, Enterobacter, Enterococcus species (including E.faecium and E. faecal is), Erwinia, Erysipelothrix, Escherichia species (including E. coli), Flavobacterium species (including E. meninosepticum), Francisella species (including E. tularensis), Fusobacterium (including E. nucleatum), Gardnerella species (including G. vaginalis), Gluconobacter, Haemophilus species (including//, influenzae and H. ducreyi), Haftiia, Helicobacter (including//. pylori), Herpetosiphon, Klebsiella species (including K. pneumoniae), Kluyvera, Lactobacillus, Legionella species (including E. pneumophila), Leptotrichia, Listeria species (including E. monocytogenes), Microbacterium, Morganella, Nitrobacter, Nitrosomonas, Pasteurella species (including/1, multocida), Pectinatus, Porphyromonas gingivalis, Proteus species (including E. mirabilis), Providencia, Pseudomonas species (including P. aeruginosa, P. mallei, P. pseudomallei and E. solanacearum), Rahnella, Renibacterium salmoninarum, Salmonella, Serratia, Shigella, Spirillum, Streptobacillus species (including S. moniliformis), Vibrio species (including V cholerae and V vulnificus), Wolinella, Xanthobacter, Xenorhabdus, Yersinia species (including Y. pestis and Y. enter ocolitica), Zanthomonas and Zymomonas. In some embodiments, compositions are effective against drug-resistant and multi-drug resistant bacterial species.
[0181] The clinical diseases or infections caused by Gram-positive and/or Gram negative bacteria, treatable with the peptides described herein include abscesses, bacteremia, blood stream infection, contamination of peritoneal dialysis fluid, endocarditis, pneumonia, meningitis, osteomyelitis, cellulitis, pharyngitis, otitis media, sinusitis, scarlet fever, arthritis, urinary tract infection, laryngotracheitis, erysipeloid, gas gangrene, tetanus, typhoid fever, acute gastroenteritis, bronchitis, bronchiolitis, epiglottitis, plague, sepsis, chancroid, wound and bum infection, cholera, glanders, periodontitis, genital infections, empyema, granuloma inguinale, Legionnaire's disease, paratyphoid, bacillary dysentery, brucellosis, diphtheria, pertussis, botulism, toxic shock syndrome, mastitis, rheumatic fever, cystic fibrosis, eye infections, plaque, and dental caries. Other uses include swine erysipelas, peritonitis, intraabdominal infection, abortion, encephalitis, anthrax, nocardiosis, pericarditis, mycetoma, peptic ulcer, melioidosis, Haverhill fever, tularemia, Moko disease, galls (e.g., crown, cane and leaf), hairy root, bacterial rot, bacterial blight, bacterial brown spot, bacterial wilt, bacterial fin rot, dropsy, columnaris disease, pasteurellosis, furunculosis, enteric redmouth disease, vibriosis, tuberculosis, offish, and fouling of medical devices. In some cases, the infection may be acute. In other cases, the infection may be chronic. In still other cases, infections may be community-acquired or hospita1-acquired. In some embodiments, peptides are used to treat chronic skin ulcers, infected acute wounds or bum wounds, infected skin eczema, impetigo, atopic dermatitis, acne, external otitis, vaginal infections, seborrheic dermatitis, oral infections, parodontitis, conjunctivitis or pneumonia.
[0182] Another embodiment of the present disclosure relates to administering an antimicrobial peptide provided herein in combination with an antibiotic. Antibiotics suitable for co-administration with the antimicrobial peptides disclosed herein include substances, produced synthetically or naturally, which can inhibit the growth of or kill microbial organisms. Such antibiotics include, without any limitation, beta-lactam antibiotics (e.g, ampicillin, aziocillin, aztreonam, carbenicillin, cefoperazone, ceftriaxone, cephaloridine, cephalothin, cloxacillin, moxalactam, penicillin, piperacillin, and ticarcillin), amoxicillin, bacitracin, chloramphenicol, clindamycin, capreomycin, colistimethate, ciprofloxacin, doxycycline, erythromycin, fusidic acid, fosfomycin, fusidate sodium, gramicidin, gentamycin, lincomycin, minocycline, macrolides, monobactams, nalidixic acid, novobiocin, ofloxcin, rifamycins, tetracyclines, vancomycin, tobramycin, fluoroquinolones, polymyxins, DNA gyrase inhibitors, bacterial polymerase inhibitors, folate synthesis inhibitors, and trimethoprim.
[0183] Another aspect of the present disclosure relates to a composition comprising an antimicrobial peptide and an agent which can enhance, maintain, or facilitate the function or activity of the peptide. In one embodiment, the chemical is a protease inhibitor. The peptide is exposed to a protease-present environment where the presence of the protease may reduce the antimicrobial activity of the peptide via, for example, enzymatic degradation. The combination of a protease inhibitor and the peptide stabilizes the peptide from the protease degradation and thus enhances the activity of the antimicrobial peptide. The protease-present environment includes, for example, body fluid (e.g., urine, blood, serum, salvia, sputum, and mucosal fluid). The protease includes, for example, neutrophil elastase, proteinase-3, cysteine protease, metalloprotease, serine-protease, or other proteases derived from bacteria and/or hosts. The protease inhibitor includes, for example, BMF, EDTA, PMSF, benzamidine, and/or recombinant alpha-1 antitrypsin (rAAT).
[0184] Vectors, Host Cells and Recombinant Methods
[0185] A peptide, as disclosed herein, may be expressed by recombinant methods. Generally, a nucleic acid encoding a peptide is often isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the peptide may be prepared by PCR amplification and sequenced using conventional procedures (e.g, by using oligonucleotide probes that are capable of binding specifically to nucleotides encoding a peptide). In an exemplary embodiment, nucleic acid encoding a peptide is PCR amplified, restriction enzyme digested and gel purified. The digested nucleic acid in some embodiments is inserted into a replicable vector. The replicable vector containing the nucleic acid insertion in some embodiments is transformed or transduced into a host cell for further cloning (amplification of the DNA) or for expression of the peptide. Host cells in some embodiments are prokaryotic or eukaryotic cells. In some embodiments, a vector comprises an expression cassette for a peptide described herein. In some embodiments, an expression cassette comprises at least one promoter and a polynucleotide sequence encoding for the peptide. In some embodiments, an expression cassette comprises at least one terminator. [0186] Polynucleotide sequences encoding peptide components disclosed herein may be obtained by PCR amplification with overlapping oligonucleotide primers. Polynucleotide sequences in some embodiments are isolated and sequenced from peptide-producing cells. Altematively, polynucleotides in some embodiments are synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the peptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic and/or eukaryotic hosts.
[0187] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism may be used as transforming vectors in connection with these hosts. For example, bacteriophage such as XGEM™- 11 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392. Peptides in some embodiments are expressed in intracellularly (e.g., cytoplasm) or extracellularly (e.g., secretion). For extracellular expression, the vector may comprise a secretion signal which enables translocation of the peptide to the outside of the cell. Suitable host cells for cloning or expression of peptide-encoding vectors include prokaryotic or eukaryotic cells. The host cell in some embodiments is a eukaryotic cell. Examples of eukaryotic cells include, but are not limited to, Human Embryonic Kidney (HEK) cell, Chinese Hamster Ovary (CHO) cell, fungi, yeasts, invertebrate cells (e.g., plant cells and insect cells), lymphoid cell (e.g., YO, NSO, Sp20 cell). In some embodiments, the host cell is Pichia pastoris or S. cerevisiae. Other examples of suitable mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); baby hamster kidney cells (BHK); mouse Sertoli cells; monkey kidney cells (CV1); African green monkey kidney cells (VERO- 76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TR1 cells; MRC 5 cells; and FS4 cells. The host cell in some embodiments is a prokaryotic cell (e.g., E. coli).
[0188] Host cells in some embodiments are transformed with vectors containing nucleotides encoding a peptide. Transformed host cells in some embodiments are cultured in media. The media may be supplemented with one or more agents for inducing promoters, selecting transformants, or amplifying or expressing the genes encoding the desired sequences. Methods for transforming host cells are known in the art and may include electroporation, calcium chloride, or polyethylene glycol/DMSO. Alternatively, host cells in some embodiments are transfected or transduced with vectors containing nucleotides encoding a peptide. Transfected or transduced host cells in some embodiments are cultured in media. In some embodiments the media is supplemented with one or more agents for inducing promoters, selecting transfected or transduced cells, or expressing genes encoding the desired sequences.
[0189] The expressed peptides in some embodiments are secreted into and recovered from the periplasm of the host cells or transported into the culture media. Protein recovery from the periplasm may involve disrupting the host cell. Disruption of the host cell may comprise osmotic shock, sonication or lysis. Centrifugation or filtration is often used to remove cell debris or whole cells. The peptides in some embodiments are further purified, for example, by affinity resin chromatography.
[0190] Alternatively, peptides that are secreted into the culture media may be isolated therein. Cells in some embodiments are removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced. The expressed peptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.
[0191] Peptide production may be conducted in large quantity by a fermentation process. Various large-scale fed-batch fermentation procedures are available for production of recombinant proteins. Large-scale fermentations have at least 1000 liters of capacity, preferably about 1,000 to 100,000 liters of capacity. These fermenters use agitator impellers to distribute oxygen and nutrients, especially glucose (a preferred carbon/energy source). Small scale fermentation refers generally to fermentation in a fermenter that is no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters. In a fermentation process, induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD550 of about 180-220, at which stage the cells are in the early stationary phase. A variety of inducers in some embodiments are used, according to the vector construct employed, as is known in the art and described above. Cells in some embodiments are grown for shorter periods prior to induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction time may be used.
[0192] To improve the production yield and quality of the peptides disclosed herein, various fermentation conditions can be modified. For example, to improve the proper assembly and folding of the secreted peptides, additional vectors overexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis,trans-isom erase with chaperone activity) may be used to co-transform the host prokaryotic cells. The chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells.
[0193] To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains deficient for proteolytic enzymes can be used for the present disclosure. For example, host cell strains in some embodiments are modified to effect genetic mutation(s) in the genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof. Some E. coli protease-deficient strains are available.
[0194] Standard protein purification methods known in the art can be employed. The following procedures are exemplary of suitable purification procedures: fractionation on immunoaffmity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, hydroxyl apatite chromatography, gel electrophoresis, dialysis, and affinity chromatography and gel filtration using, for example, Sephadex G-75.
[0195] Peptides in some embodiments are concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon® ultrafiltration unit.
[0196] Protease inhibitors or protease inhibitor cocktails in some embodiments are included in any of the foregoing steps to inhibit proteolysis of the peptide.
[0197] In some cases, a peptide or fragment thereof may not be biologically active upon isolation. Various methods for "refolding" or converting a peptide to its tertiary structure and generating disulfide linkages, can be used to restore biological activity. Such methods include exposing the solubilized peptide to a pH usually above 7 and in the presence of a particular concentration of a chaotrope. The selection of chaotrope is very similar to the choices used for inclusion body solubilization, but usually the chaotrope is used at a lower concentration and is not necessarily the same as chaotropes used for the solubilization. In most cases the refolding/oxidation solution will also contain a reducing agent or the reducing agent plus its oxidized form in a specific ratio to generate a particular redox potential allowing for disulfide shuffling to occur in the formation of the protein's cysteine bridge(s). Some of the commonly used redox couples include cystein/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride, dithiothreitol(DTT)/dithiane DTT, and 2-mercaptoethanol(bME)/di-thio-b(ME). In some embodiments, a cosolvent may be used to increase the efficiency of the refolding, and common reagents used for this purpose include glycerol, polyethylene glycol of various molecular weights, arginine and the like. In some embodiments, peptides described herein comprise disulfide linkages, or their equivalent. Such linkages in some instances are enabled by bridging amino acids. In some instances, intramolecular linkages comprise amide, ether, disulfide, sulfide, alkyl, ester, or other bond. Non-limiting examples of bridging amino acids include aspartic acid, glutamic acid, serine, cysteine, penicillamine, dehydroalanine, or other amino acid capable of intramolecular reaction to form one or more rings. Bridging amino acids in some instances comprise canonical or non-canonical amino acids described herein. For example, alternative linkages include but are not limited to alkenyl linkages (formed by olefin metathesis), alkyl linkages, lactam formation (amide linkages), lanthionine linkages, thio linkages (formed through displacement of electrophiles or conjugate addition), ether linkages, sulfate linkages, sulfmyl linkages, sulfamide linkages, ester linkages, amine linkages, or other linkage. In some embodiments, the linkage comprises the structure of
Figure imgf000076_0001
,or ; wherein R is a substituent, Ar is aryl, and HA is heteroaryl. In some embodiments R is alkyl, aralkyl, or cycloalkyl. In some embodiments R is C1-C6 alkyl. In some embodiments Ar is optionally substituted phenyl or naphthyl. In some embodiments HA is optionally substituted: pyridinyl, imidazolyl, thiophenyl, pyrrol yl, thiazolyl, oxazolyl, or furanyl. In some instances, two amino acids from a peptide described herein are cyclized to form an intramolecular linkage.
[0198] Definitions [0199] The terms below, as used herein, have the following meanings, unless indicated otherwise:
[0200] “Amino” refers to the -NH2 radical.
[0201] “Cyano” or “nitrile” refers to the -CN radical.
[0202] “Hydroxy” or “hydroxyl” refers to the -OH radical.
[0203] “Nitro” refers to the -NO2 radical.
[0204] “Oxo” refers to the =O substituent.
[0205] “Oxime” refers to the =N-OH substituent.
[0206] “Thioxo” refers to the =S substituent.
[0207] “Alkyl” refers to a straight or branched hydrocarbon chain radical, has from one to thirty carbon atoms, and is attached to the rest of the molecule by a single bond. Alkyls comprising any number of carbon atoms from 1 to 30 are included. An alkyl comprising up to 30 carbon atoms is referred to as a C1-C30 alkyl, likewise, for example, an alkyl comprising up to 12 carbon atoms is a C1-C12 alkyl. Alkyls (and other moieties defined herein) comprising other numbers of carbon atoms are represented similarly. Alkyl groups include, but are not limited to, C1-C30 alkyl, C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, C1-C8 alkyl, C1-C6 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C2-C8 alkyl, C3-C8 alkyl and C4-C8 alkyl. Representative alkyl groups include, but are not limited to, methyl, ethyl, «-propyl,
1 -methyl ethyl (/50-propyl), «-butyl, /'-butyl, 5-butyl, «-pentyl, 1,1 -dimethyl ethyl (/-butyl), 3-methylhexyl, 2-methylhexyl, vinyl, allyl, propynyl, and the like. Alkyl comprising unsaturations include alkenyl and alkynyl groups. Unless stated otherwise specifically in the specification, an alkyl group may be optionally substituted as described below.
[0208] “Alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain, as described for alkyl above. Unless stated otherwise specifically in the specification, an alkylene group may be optionally substituted as described below.
[0209] “Alkoxy” refers to a radical of the formula -ORa where Ra is an alkyl radical as defined. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted as described below.
[0210] “Aryl” refers to a radical derived from a hydrocarbon ring system comprising hydrogen, 6 to 30 carbon atoms and at least one aromatic ring. The aryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. Aryl radicals include, but are not limited to, aryl radicals derived from the hydrocarbon ring systems of aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, .v-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless stated otherwise specifically in the specification, the term “aryl” or the prefix “ar-“ (such as in “aralkyl”) is meant to include aryl radicals that are optionally substituted.
[0211] “Cycloalkyl” or “carbocycle” refers to a stable, non-aromatic, monocyclic or polycyclic carbocyclic ring, which may include fused or bridged ring systems, which is saturated or unsaturated. Representative cycloalkyls or carbocycles include, but are not limited to, cycloalkyls having from three to fifteen carbon atoms, from three to ten carbon atoms, from three to eight carbon atoms, from three to six carbon atoms, from three to five carbon atoms, or three to four carbon atoms. Monocyclic cycloalkyls or carbocycles include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyls or carbocycles include, for example, adamantyl, norbornyl, decalinyl, bicyclo[3.3.0]octane, bicyclo[4.3.0]nonane, cis-decalin, trans-decalin, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, and bicyclo[3.3.2]decane, and 7,7-dimethy1-bicyclo[2.2.1]heptanyl. Unless otherwise stated specifically in the specification, a cycloalkyl or carbocycle group may be optionally substituted. Illustrative examples of cycloalkyl groups include, but are not limited to, the following moieties:
Figure imgf000078_0001
[0213] “Fused” refers to any ring structure described herein which is fused to an existing ring structure. When the fused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atom on the existing ring structure which becomes part of the fused heterocyclyl ring or the fused heteroaryl ring may be replaced with a nitrogen atom.
[0214] “Halo” or “halogen” refers to bromo, chloro, fluoro or iodo.
[0215] “Haloalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g ., trifluoromethyl, difluoromethyl, fluoromethyl, tri chi orom ethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like. Unless stated otherwise specifically in the specification, a haloalkyl group may be optionally substituted. [0216] “Haloalkoxy” similarly refers to a radical of the formula -ORa where Ra is a haloalkyl radical as defined. Unless stated otherwise specifically in the specification, a haloalkoxy group may be optionally substituted as described below.
[0217] “Heterocycloalkyl” or “heterocyclyl” or “heterocyclic ring” or “heterocycle” refers to a stable 3- to 24-membered non-aromatic ring radical comprising 2 to 23 carbon atoms and from one to 8 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorous and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quatemized; and the heterocyclyl radical may be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, azetidinyl, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, 12-crown-4, 15-crown-5, 18-crown-6, 21 -crown-7, aza-18-crown-6, diaza-18-crown-6, aza-21 -crown-7, and diaza-21 -crown-7. Unless stated otherwise specifically in the specification, a heterocyclyl group may be optionally substituted. Illustrative examples of heterocycloalkyl groups, also referred to as non-aromatic heterocycles, include:
Figure imgf000079_0001
Figure imgf000080_0001
and the like. The term heterocycloalkyl also includes all ring forms of the carbohydrates, including but not limited to the monosaccharides, the disaccharides and the oligosaccharides. Unless otherwise noted, heterocycloalkyls have from 2 to 10 carbons in the ring. It is understood that when referring to the number of carbon atoms in a heterocycloalkyl, the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including the heteroatoms) that make up the heterocycloalkyl (i.e. skeletal atoms of the heterocycloalkyl ring). Unless stated otherwise specifically in the specification, a heterocycloalkyl group may be optionally substituted. [0218] The term "heteroaryl" as used herein, alone or in combination, refers to optionally substituted aromatic monoradicals containing from about five to about twenty skeletal ring atoms, where one or more of the ring atoms is a heteroatom independently selected from among oxygen, nitrogen, sulfur, phosphorous, silicon, selenium and tin but not limited to these atoms and with the proviso that the ring of said group does not contain two adjacent O or S atoms. In embodiments in which two or more heteroatoms are present in the ring, the two or more heteroatoms can be the same as each another, or some or all of the two or more heteroatoms can each be different from the others. The term heteroaryl includes optionally substituted fused and non-fused heteroaryl radicals having at least one heteroatom. The term heteroaryl also includes fused and non-fused heteroaryls having from five to about twelve skeletal ring atoms, as well as those having from five to about ten skeletal ring atoms. Bonding to a heteroaryl group can be via a carbon atom or a heteroatom. Thus, as a nonlimiting example, an imidiazole group may be attached to a parent molecule via any of its carbon atoms (imidazo1-2-yl, imidazo1-4-yl or imidazo1-5-yl), or its nitrogen atoms (imidazo1- 1-yl or imidazo1-3-yl). Likewise, a heteroaryl group may be further substituted via any or all of its carbon atoms, and/or any or all of its heteroatoms. A fused heteroaryl radical may contain from two to four fused rings where the ring of attachment is a heteroaromatic ring and the other individual rings in some embodiments are alicyclic, heterocyclic, aromatic, heteroaromatic or any combination thereof. A non-limiting example of a single ring heteroaryl group includes pyridyl; fused ring heteroaryl groups include benzimidazolyl, quinolinyl, acridinyl; and a non-fused bi-heteroaryl group includes bipyridinyl. Further examples of heteroaryls include, without limitation, furanyl, thienyl, oxazolyl, acridinyl, azepinyl, phenazinyl, benzimidazolyl, benzindolyl, benzofuranyl, benzofuranonyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzothiophenyl, benzoxadiazolyl, benzodioxolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzotriazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzothienyl (benzothiophenyl), benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanonyl, imidazolyl, indolyl, isoxazolyl, isoquinolinyl, indolizinyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, indolizinyl, isothiazolyl, isoindolyloxadiazolyl, indazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl,
1 -pheny1- liT-pyrrolyl, phenothiazinyl, phenoxazinyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl, pyrazinyl, pyrazolyl, purinyl, phthalazinyl, pteridinyl, quinolinyl, quinazolinyl, quinoxalinyl, quinuclidinyl, triazolyl, tetrazolyl, thiazolyl, triazinyl, thiadiazolyl, tetrahydroquinolinyl, thiazolyl, and thiophenyl and the like, and their oxides, such as for example pyridy1-N-oxide. Illustrative examples of heteroaryl groups include the following moieties:
Figure imgf000081_0001
and the like.
[0219] All the above groups in some embodiments are either substituted or unsubstituted. The term “substituted” as used herein means any of the above groups ( e.g ., alkyl, alkylene, alkoxy, aryl, cycloalkyl, haloalkyl, heterocyclyl and/or heteroaryl) may be further functionalized wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atom substituent. Unless stated specifically in the specification, a substituted group may include one or more substituents selected from: oxo, amino, -CO2H, nitrile, nitro, hydroxyl, thiooxy, alkyl, alkylene, alkoxy, aryl, cycloalkyl, heterocyclyl, heteroaryl, dialkylamines, arylamines, alkylarylamines, diarylamines, trialkylammonium (-N+R.3), N-oxides, imides, and enamines; a silicon atom in groups such as trialkyl silyl groups, di al ky 1 aryl si 1 y 1 groups, al ky 1 di aryl si 1 y 1 groups, triarylsilyl groups, perfluoroalkyl or perfluoroalkoxy, for example, trifluoromethyl or trifluoromethoxy. “Substituted” also means any of the above groups in which one or more hydrogen atoms are replaced by a higher-order bond (e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, “substituted” includes any of the above groups in which one or more hydrogen atoms are replaced with -NH2, -NRgC (=O)NRgRh, -NRgC(=O)ORh, -NRgSO2Rh, -OC (=O)NRgRh, -ORg, -SRg, -S ORg, -SO2Rg, -0SO2Rg, -SO2ORg, =NSO2Rg, and -SO2NRgRh. In the foregoing, Rg and Rh are the same or different and independently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, /V-heterocyclyl, heterocyclylalkyl, heteroaryl, A'-heteroaryl and/or heteroaryl alkyl. In addition, each of the foregoing substituents may also be optionally substituted with one or more of the above substituents. Furthermore, any of the above groups in some embodiments are substituted to include one or more internal oxygen, sulfur, or nitrogen atoms. For example, an alkyl group may be substituted with one or more internal oxygen atoms to form an ether or polyether group. Similarly, an alkyl group may be substituted with one or more internal sulfur atoms to form a thioether, disulfide, etc.
[0220] The term “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, "optionally substituted alkyl" means either "alkyl" or "substituted alkyl" as defined above. Further, an optionally substituted group may be un-sub stituted (e.g., -CH2CH3), fully substituted (e.g., -CF2CF3), mono- sub stituted (e.g., -CH2CH2F) or substituted at a level anywhere in-between fully substituted and mono-substituted
(e.g., -CH2CHF2, -CH2CF3, -CF2CH3, -CFHCHF2, etc). It will be understood by those skilled in the art with respect to any group containing one or more substituents that such groups are not intended to introduce any substitution or substitution patterns (e.g., substituted alkyl includes optionally substituted cycloalkyl groups, which in turn are defined as including optionally substituted alkyl groups, potentially ad infinitum) that are sterically impractical and/or synthetically non-feasible. Thus, any substituents described should generally be understood as having a maximum molecular weight of about 1,000 daltons, and more typically, up to about 500 daltons.
[0221] A “tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule. The compounds presented herein may exist as tautomers. Tautomers are compounds that are interconvertible by migration of a hydrogen atom, accompanied by a switch of a single bond and adjacent double bond. In bonding arrangements where tautomerization is possible, a chemical equilibrium of the tautomers will exist. All tautomeric forms of the compounds disclosed herein are contemplated. The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH. Some examples of tautomeric interconversions include:
Figure imgf000083_0001
[0222] A “metabolite” of a compound disclosed herein is a derivative of that compound that is formed when the compound is metabolized. The term “active metabolite” refers to a biologically active derivative of a compound that is formed when the compound is metabolized. The term “metabolized,” as used herein, refers to the sum of the processes (including, but not limited to, hydrolysis reactions and reactions catalyzed by enzymes, such as, oxidation reactions) by which a particular substance is changed by an organism. Thus, enzymes may produce specific structural alterations to a compound. For example, cytochrome P450 catalyzes a variety of oxidative and reductive reactions while uridine diphosphate glucuronyl transferases catalyze the transfer of an activated glucuronic-acid molecule to aromatic alcohols, aliphatic alcohols, carboxylic acids, amines and free sulfhydryl groups. Metabolites of the compounds disclosed herein can be identified either by administration of compounds to a host and analysis of tissue samples from the host, or by incubation of compounds with hepatic cells in vitro and analysis of the resulting compounds. Both methods are well known in the art. Metabolites of a compound may be formed by oxidative processes and correspond to the corresponding hydroxy-containing compound. A compound may be metabolized to one or more pharmacologically active metabolites.
[0223] As used herein, a derivative of a peptide refers to a fragment, analog, homolog, complex and/or aggregate of the peptide.
[0224] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.
[0225] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.
[0226] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
[0227] The term "identity" or "homology" shall be construed to mean the percentage of amino acid residues in the candidate sequence that are identical with the residue of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Conservative substitutions in some embodiments involve substitution of one amino acid of similar shape or charge for another, including but not limited to substitution of a natural amino acid for a different natural amino acid, substitution of a non-canonical amino acid for a different non- canonical amino acid, substitution of a non-canonical amino acid for a natural amino acid, or substitution of a natural amino acid for a non-canonical amino acid. A polynucleotide or polynucleotide region (or a peptide or peptide region) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" or "homology" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. Neither N- or C-terminal extensions nor insertions shall be construed as reducing identity or homology. Alignment and the percent homology or sequence identity can be determined using software programs known in the art. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand= both; cutoff=60; expect= 10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non- redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Similarity, or percent similarity in some embodiments of two sequences is the sum of both identical and similar matches (residues that have undergone conservative substitution). In some embodiments, similarity is measured using the program BLAST “Positives.”
[0228] By isolated and "substantially pure" is meant a peptide or peptide that has been separated and purified to at least some degree from the components that naturally accompany it. Typically, a peptide or peptide is substantially pure when it is at least about 60%, or at least about 70%, at least about 80%, at least about 90%, at least about 95%, or even at least about 99%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. For example, a substantially pure peptide or peptide may be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in a cell that does not normally express that protein, or by chemical synthesis.
[0229] As used herein, "essentially free," in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
[0230] "Disorder" or "disease" refers to a condition that would benefit from treatment with a substance/molecule (e.g., a peptide as disclosed herein) or method disclosed herein. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
[0231] "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, rodents (e.g., mice and rats), and monkeys; domestic and farm animals; and zoo, sports, laboratory, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. In some embodiments, the mammal is selected from a human, rodent, or monkey.
[0232] The terms "subject" and "individual" and "patient" are used interchangeably herein, and refer to an animal, for example a human or non-human animal (e.g., a mammal), to whom treatment, including prophylactic treatment, with a pharmaceutical composition as disclosed herein, is provided. The term "subject" as used herein refers to human and nonhuman animals. The term "non-human animals" includes all vertebrates, e.g, mammals, such as nonhuman primates (particularly higher primates), sheep, dogs, rodents (e.g. mouse or rat), guinea pigs, goats, pigs, cats, rabbits, cows, and non-mammals such as chickens, amphibians, reptiles etc. In one embodiment, the subject is human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. Non-human mammals include mammals such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g. mouse or rat), guinea pigs, goats, pigs, cats, rabbits and cows.
[0233] Numbered embodiments [0234] Described herein are the following numbered embodiments 1-169. Embodiment 1. An isolated peptide, wherein the peptide has a sequence comprising Formula (I): Y1-X1-X2- X3 -X4-X5 -X6-X7 -X8-X9-X 10-X 11 -X 12-X 13 -X 14-X 15 -X 16-X 17-X 18-X 19-X20-X21 - Y2 Formula (I); wherein Y1 is absent or an N-terminal modification; X1 and X2 are independently absent, or any amino acid; X3 is absent or any amino acid; X4 is positively charged amino acid; X5 is any amino acid; X6 is a non-polar amino acid; X7 is any amino acid; X8 is a non-polar amino acid; X9 is any amino acid; X10 is any amino acid; X11 is a bridging amino acid; X12 is a polar amino acid or a positively charged amino acid; X13 is any amino acid; X14 is any amino acid; X15 is a polar amino acid; X16 is a non-polar amino acid; X17 is any amino acid; X18 is a bridging amino acid; X19 is a polar amino acid; X20 is a positively charged amino acid; X21 is absent or any amino acid; Y2 is absent or a C- terminal modification, wherein if: X1 and X16 are G; X2 is S, X3, X4, X17 are K; X5 and X7 are P; X6 is V; X8 and X9 are I; X10 is Y; X11 is C; X12 is N; X13, X14, and X20 are R; X15 is T, X16 is G, X18 is C, X19 is Q; then X21 is not M; and the peptide is not identical to SEQ ID NO: 1 Embodiment 2. The peptide of embodiment 1, wherein X1 is absent. Embodiment 3. The peptide of embodiment 1 or 2, wherein X2 is absent. Embodiment 4. The peptide of any one of embodiments 1-3, wherein X1 and X2 if present are independently a non-polar amino acid. Embodiment 5. The peptide of any one of embodiments 1-3, wherein X1 and X2 if present are independently G, A, V, L, I, M, F, W, or P. Embodiment 6. The peptide of any one of embodiments 1-5, wherein X3 is absent. Embodiment 7. The peptide of any one of embodiments 1-6, wherein X4 is K, R, or H. Embodiment 8. The peptide of any one of embodiments 1-7, wherein X5 is a constrained amino acid. Embodiment 9. The peptide of any one of embodiments 1-8, wherein X7 is a constrained amino acid.
Embodiment 10. The peptide of any one of embodiments 1-7, wherein X5 and X7 are each independently a constrained amino acid. Embodiment 11. The peptide of any one of embodiments 1-7, wherein X5 is P. Embodiment 12. The peptide of any one of embodiments 1-11, wherein X6 is G, A, V, L, I, M, F, W, or P. Embodiment 13. The peptide of any one of embodiments 1-11, wherein X6 is V, L, I, F. Embodiment 14. The peptide of embodiment 8 or 9, wherein the constrained amino acid is proline, a proline analog, Orn(i-PrCO-Hao), 5- hydrazino-2-methoxybenzoic acid (Hao), an N-alkyl amino acid, or an alpha, alpha- disubstituted amino acid. Embodiment 15. The peptide of any one of embodiments 1-14, wherein X7 is P. Embodiment 16. The peptide of any one of embodiments 1-15, wherein X8 is G, A, V, L, I, M, F, W, or P. Embodiment 17. The peptide of any one of embodiments 1- 15, wherein X8 is I or F. Embodiment 18. The peptide of any one of embodiments 1-17, wherein X9 is G, A, V, L, I, T, N, S, V, E, Y, M, F, W, or P. 19. The peptide of any one of embodiments 1-17, wherein X9 is S, T, C, Y, N, or Q. Embodiment 20. The peptide of any one of embodiments 1-17, wherein X9 is I, T, N, S, V, E, Y, or F. Embodiment 21. The peptide of any one of embodiments 1-20, wherein X10 is F, W, or Y. Embodiment 22. The peptide of any one of embodiments 1-20, wherein X10 is F, H, L, or Y. Embodiment 23. The peptide of any one of embodiments 1-22, wherein X11 and X18 are taken together to form an intramolecular linkage. Embodiment 24. The peptide of embodiment 23, wherein at least one of X11 and X18 is C. Embodiment 25. The peptide of embodiment 23 or 24, wherein at least one of X11 and X18 is dehydroalanine. Embodiment 26. The peptide of embodiment 23, wherein X11 and X18 are both C. Embodiment 27. The peptide of embodiment 23, wherein the intramolecular linkage comprises the structure:
Figure imgf000087_0001
Figure imgf000087_0002
, or ; wherein R is a substituent, Ar is aryl, and HA is heteroaryl.
Embodiment 28. The peptide of embodiment 27, wherein R is alkyl, aralkyl, or cycloalkyl. Embodiment 29. The peptide of embodiment 28, wherein R is C1-C6 alkyl. Embodiment 30. The peptide of embodiment 27, wherein Ar is optionally substituted phenyl or naphthyl. Embodiment 31. The peptide of embodiment 27, wherein HA is optionally substituted: pyridinyl, imidazolyl, thiophenyl, pyrrolyl, thiazolyl, oxazolyl, or furanyl. Embodiment 32. The peptide of any one of embodiments 1-31, wherein X12 is S, T, C, Y, N, or Q. Embodiment 33. The peptide of any one of embodiments 1-31, wherein X12 is S, N, H, or R. Embodiment 34. The peptide of any one of embodiments 1-33, wherein X13 is G, A, V, L, I, M, F, W, or P. Embodiment 35. The peptide of any one of embodiments 1-33, wherein X13 is R, Q, L, or F. Embodiment 36. The peptide of any one of embodiments 1-35, wherein X14 is a positively charged amino acid. Embodiment 37. The peptide of embodiment 36, wherein X14 is K, R, or H. Embodiment 38. The peptide of any one of embodiments 1-37, wherein X15 is S, T, C, Y, N, or Q. Embodiment 39. The peptide of any one of embodiments 1-37, wherein X15 is S or T. Embodiment 40. The peptide of any one of embodiments 1-39, wherein X16 is G, A, V, L, I, M, F, W, or P. Embodiment 41. The peptide of any one of embodiments 1-40, wherein X17 is a positively charged amino acid. Embodiment 42. The peptide of embodiment 41, wherein X17 is K, R, or H. Embodiment 43. The peptide of embodiment 41, wherein X17 is Q or T. Embodiment 44. The peptide of any one of embodiments 1-43, wherein X19 is S, T, C, Y, N, or Q. Embodiment 45. The peptide of any one of embodiments 1-44, wherein X20 is K, R, or H. Embodiment 46. The peptide of any one of embodiments 1-45, wherein X21 is M, L, V, or F. Embodiment 47. The peptide of any one of embodiments 1-45, wherein X21 is absent. Embodiment 48. The peptide of any one of embodiments 1-45, wherein X21 is a beta-homoamino acid analog. Embodiment 49. The peptide of embodiment 48, wherein the beta-homoamino acid analog is selected from the group consisting of (lS,3R)-(+)-3-(amino)cyclopentanecarboxylic acid; (2R,3R)-3-(amino)-
2-hydroxy-4-phenylbutyric acid; (R)-2-m ethyl -β-Phe-OH; (R)-3-(amino)-2-methylpropionic acid; (R)-3-(amino)-4-(2-naphthyl)butyric acid; (R)-3-(amino)-5-phenylpentanoic acid; (R)-
3-(trifluoromethyl)-β-Phe-OH; (R)-3,4-dimethoxy-β-Phe-OH; (R)-3-methoxy-β-Phe-OH; (R)-3-methy1-β-Phe-OH; (R)-4-(3-pyridyl)-β-Homoala-OH; (R)-4-(4-pyridyl)-β-Homoala- OH; (R)-4-(trifluoromethyl)-β-Phe-OH; (R)-4-bromo-β-Phe-OH; (R)-4-chloro-β-Phe-OH;
(R)-4-cyano-β-Homophe-OH; (R)-4-cyano-β-Phe-OH; (R)-4-fluoro-β-Phe-OH; (R)-4- methoxy-β-Phe-OH; (R)-4-methy1-β-Phe-OH; (R)-β-Tyr-OH; (S)-(+)-Pyrrolidine-3- carboxylic acid; (S)-2-(trifluorom ethyl )-β-Homophe-OH; (S)-2-(trifluoromethyl)-β-Phe-OH;
(S)-2-cyano-β-Homophe-OH; (S)-2-methy1-β-Homophe-OH; (S)-2-methy1-β-Phe-OH; (S)-3- (amino)-2-methylpropionic acid; (S)-3,4-difluoro-β-Homophe-OH; (S)-3,4-dimethoxy-β-Phe- OH; (S)-3-methoxy-β-Phe-OH; (S)-3-methy1-β-Homophe-OH; (S)-4-(4-pyridyl)-β-Homoala- OH; (S)-4-bromo-β-Phe-OH; (S)-4-chloro-β-Homophe-OH; (S)-4-chloro-β-Phe-OH; (S)-4- cyano-β-Homophe-OH; (S)-4-cyano-β-Phe-OH; (S)-4-fluoro-β-Phe-OH; (S)-4-methy1-β-Phe- OH; (S)-β-Tyr-OH; (S)-γ,γ-di phenyl -β-Homoala-OH; 2-(aminomethyl)phenylacetic acid; 3,4-Dehydro-proline; 3-Amino-3-(3-bromophenyl)propionic acid; 3-Aminobutanoic acid; Boc-D-β-Leu-OH; Boc-β-Leu-OH; cis-3-(amino)cyclohexanecarboxylic acid; 3- Aminoisobutyric acid; β-Homoleucine; β-Homophenylalanine; β-Phenylalanine; D-β-Phe- OH; Hyp-OH; L-β3 -homoproline; L-β-Homohydroxyproline; L-β-Homoleucine; L-β- Homolysine; L-β-Homomethionine; L-β-Homophenylalanine; L-β-Homoproline; L-β- Homoserine; L-β-Homothreonine; L-β-Leucine; N-Acety1-β-phenylalanine; N-cis-4-hydroxy- L-proline; N-trans-4-hydroxy-L-proline; Pipecolinic acid; trans-4-Hydroxy-L-proline; β-3- Homopro-OH; β-Alanine; β-Ala-OH; β-Dab(Boc)-OH; β-Dab(Fmoc)-OH; β-Dab(Me)-OH; β-Dab-OH; β-D-Phe-OH; β-Gln-OH; β-Glu(OBzl)-OH; β-Glu(OMe)-OH; β-Glu(OtBu)-OH; β-Homoala-OH; β-Homoarg(Pmc)-OH; β-Homoarg(Tos)-OH; β-Homoarg-OH; β- Homogln(Trt)-OH; β-Homogln-OH; β-Homoglu(OtBu)-OH; β-Homohyp(Bzl)-OH; β- Homohyp(tBu)-OH; β-Homohyp-OH; β-Homoile-OH; β-Homoleu-OH; β-Homolys(Z)-OH; β-Homolys-OH; β-Homomet-OH; β-Homophe-OH; β-Homoser(Bzl)-OH; β-Homoser-OH; β- Homothr(Bzl)-OH; β-Homothr(tBu)-OH; β-Homothr-OH; β-Homotrp-OH; β-Homotyr(Bzl)- OH; β-Homotyr(tBu)-OH; β-Homotyr-OH; and β-Phe-OH. Embodiment 50. The peptide of embodiment 48, wherein the beta-homo amino acid analog is selected from the group consisting of beta-homophenylalanine, beta-homoleucine, beta-homocysteine, beta- homomethionine, and beta-homoisoleucine. Embodiment 51 The peptide of any one of embodiments 1-50, wherein X21 is G, A, V, L, I, M, F, W, or P. Embodiment 52. The peptide of any one of embodiments 1-51, wherein at least one of X1, X2, X3, X4, X5, X6,
X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, or X21 is an non- canonical amino acid. Embodiment 53. The peptide of any one of embodiments 1-51, wherein at least two of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, or X21 are independently a no-canonical amino acid. Embodiment 54. The peptide of any one of embodiments 1-51, wherein at least three of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19,
X20, or X21 are independently a no-canonical amino acid. Embodiment 55. The peptide of any one of embodiments 1-51, wherein at least four of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, or X21 are independently a no- canonical amino acid. Embodiment 56. The peptide of any one of embodiments 1-51, wherein at least five of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, or X21 are independently a non-canonical amino acid. Embodiment 57. The peptide of any one of embodiments 52-56, wherein the non-canonical amino acid is selected from the group consisting of alanine derivatives; alicyclic amino acids; arginine derivatives; aromatic amino acids; asparagine derivatives; aspartic acid derivatives; beta-amino acids; cysteine derivatives; dab (2,4-diaminobutyric acid); dap (2,3- diaminopropionic acid); glutamic acid derivatives; glutamine derivatives; glycine derivatives; homo-amino acids; isoleucine derivatives; leucine derivatives; linear core amino acids; lysine derivatives; methionine derivatives; n-methyl amino acids; norleucine derivatives; norvaline derivatives; ornithine derivatives; penicillamine derivatives; phenylalanine derivatives; phenylglycine derivatives; proline derivatives; pyroglutamine derivatives; serine derivatives; threonine derivatives; tryptophan derivatives; tyrosine derivatives; and valine derivatives. Embodiment 58. The peptide of any one of embodiments 52-56, wherein the non-canonical amino acid is selected from 2,4-diaminobutryic acid; alpha-methylarginine; homophenylalanine; homoleucine; homoisoleucine; diaminopropionic acid; N-acetylglycine; 6-aminohexanoic acid; gamma-aminobutyric acid; alpha-methyl serine; alpha-methyltyrosine; 4-(trifluoromethyl)-L-phenylalanine; 4-amino-L-phenylalanine; Penicillamine, t-butyl D- serine, Statine, N-methyl glycine, N-methyl serine, N-methyl lysine, beta-phenylalanine, L- alpha-cyclohexyl glycine, L-cyclopropylglycine, or beta-homoleucine. Embodiment 59. The peptide of embodiment 1, wherein at least one of X3, X4, X17, or X20 are DAB (2,4- diaminobutryic acid) or DAP (diaminopimelic acid). Embodiment 60. The peptide of embodiment 1, wherein at least one of X3, X4, X17, are DAB (2,4-diaminobutryic acid) or DAP (diaminopimelic acid). Embodiment 61. The peptide of embodiment 1, wherein at least one of X1, X2, X3, and Y2 are non-canonical amino acids. Embodiment 62. The peptide of embodiment 1, wherein at least one of X20, X21, or Y1 are non-canonical amino acids. Embodiment 63. The peptide of embodiment 1, wherein Y1 is attached to X1, X2, or X3. Embodiment 64. The peptide of embodiment 1, wherein Y2 is attached to X20 or X21. Embodiment 65. The peptide of any one of embodiments 52-56, wherein the non-canonical amino acid is linked to a half-life extending moiety or a moiety which broadens the antibiotic spectrum. Embodiment 66. The peptide of embodiment 65, wherein the half-life extending moiety comprises a water-soluble polymer. 67. The peptide of embodiment 66, wherein the half-life extending moiety comprises PEG (polyethylene glycol). Embodiment 68. The peptide of embodiment 67, wherein PEG has an average molecular weight of 10,000-85,000 daltons. Embodiment 69. The peptide of embodiment 65, wherein the half-life extending moiety comprises a peptide or protein. Embodiment 70. The peptide of embodiment 69, wherein the half-life extending moiety comprises XTEN or PAS. Embodiment 71. The peptide of embodiment 70, comprises a polymer having 100-1000 XTEN or PAS repeats. Embodiment 72. The peptide of embodiment 65, wherein the half-life extending moiety comprises an antibody or antibody fragment. Embodiment 73. The peptide of embodiment 72, wherein the half-life extending moiety comprises an Fc. Embodiment 74. The peptide of embodiment 72, wherein the half-life extending moiety comprises an IgG. Embodiment 75. The peptide of embodiment 1, wherein Y1 is absent or an N-terminal modification; X1 is absent, or G; X2 is absent, or S; X3 is absent, K, DAB, or DAP; X4 is K, DAB, or DAP; X5 is P; X6 is V, I, L, or F; X7 is P; X8 is I or F; X9 is I, T, N, S, V, E, Y, F; X10 is Y, F, H, or L; X11 is C; X12 is N, S, H, or R; X13 is R, Q, L, F, DAB, or DAP; X14 is any amino acid; X15 is T or S; X16 is G; X17 is any amino acid; X18 is C; X19 is Q or T; X20 is R, DAB or DAP; X21 is absent, I, L, V, M, F, or a non-canonical amino acid; and Y2 is absent or a C- terminal modification. Embodiment 76. The peptide of any one of embodiments 1-75, wherein the N-terminal modification or C-terminal modification comprises at least one targeting agent. 77. The peptide of embodiment 76, wherein the targeting agent increases the local concentration of the peptide. 78. The peptide of embodiment 76, wherein the targeting agent broadens the antibiotic spectrum. 79. The peptide of any one of embodiments 1-75, wherein Y2 comprises an amide. Embodiment 80. The peptide of embodiment 76, wherein Y2 is selected from the group consisting of -NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2. Embodiment 81. The peptide of any one of embodiments 1-75, wherein Y2 comprises a water-soluble polymer. Embodiment 82. The peptide of embodiment 81, wherein Y2 comprises PEG (polyethylene glycol). Embodiment 83. The peptide of embodiment 82, wherein PEG has an average molecular weight of 10,000-85,000 daltons. 84. The peptide of any one of embodiments 1-75, wherein Y2 comprises a peptide or protein. Embodiment 85. The peptide of embodiment 84, wherein Y2 comprises XTEN or PAS. Embodiment 86. The peptide of embodiment 85, comprises a polymer having 100-1000 XTEN or PAS repeats. Embodiment 87. The peptide of embodiment 84, wherein Y2 comprises Y or K. Embodiment 88. The peptide of embodiment 84, wherein Y2 comprises YYKK. Embodiment 89. The peptide of embodiment 84, wherein Y2 comprises Y, K, DAB, or DAP. Embodiment 90. The peptide of embodiment 84, wherein Y2 comprises at least one DAP or DAB amino acids. Embodiment 91. The peptide of embodiment 84, wherein Y2 comprises at least one homoamino acid. Embodiment 92. The peptide of embodiment 84, wherein Y2 comprises YY{DAB}{DAB} or YY{DAP}{DAP}. Embodiment 93. The peptide of any one of embodiments 1-75, wherein Y2 comprises an antibody or antibody fragment. Embodiment 94. The peptide of embodiment 93, wherein Y2 comprises an Fc. Embodiment 95. The peptide of embodiment 93, wherein Y2 comprises an IgG. Embodiment 96. The peptide of embodiment 93, wherein Y2 comprises a lipid. Embodiment 97. The peptide of any one of embodiments 1-96, wherein Y1 comprises an acyl group. Embodiment 98. The peptide of embodiment 97, wherein Y1 is -C(=O)( C1-C6 alkyl). Embodiment 99. The peptide of embodiment 97, wherein Y1 is selected from the group consisting of formyl, acetyl, propionyl, butyryl, and isovaleryl. Embodiment 100. The peptide of any one of embodiments 1-96, wherein Y1 comprises a water-soluble polymer. 101. The peptide of embodiment 97, wherein Y1 comprises PEG (polyethylene glycol). Embodiment 102. The peptide of embodiment 101, wherein PEG has an average molecular weight of 10,000-85,000 daltons. Embodiment 103. The peptide of any one of embodiments 1-96, wherein Y1 comprises a peptide or protein. Embodiment 104. The peptide of embodiment 103, wherein Y1 comprises XTEN or PAS. Embodiment 105. The peptide of embodiment 104, comprises a polymer having 100-1000 XTEN or PAS repeats. Embodiment 106. The peptide of any one of embodiments 1-96, wherein Y1 comprises an antibody or antibody fragment. Embodiment 107. The peptide of embodiment 106, wherein Y1 comprises an Fc. Embodiment 108. The peptide of embodiment 106, wherein Y1 comprises an IgG. Embodiment 109. The peptide of any one of embodiments 1-96, wherein Y1 comprises a lipid. Embodiment 110. An isolated peptide, wherein the peptide has at least 70% identity with SEQ ID NO: 1, wherein in the peptide is not identical to SEQ ID NO: 1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. 111. The peptide of embodiment 110, wherein the peptide has at least 75% identity with SEQ ID NO: 1. Embodiment 112. The peptide of embodiment 110, wherein the peptide has at least 85% identity with SEQ ID NO : 1. Embodiment 113. The peptide of embodiment 110, wherein the peptide has at least 90% identity with SEQ ID NO: 1. Embodiment 114. The peptide of embodiment 110, wherein the peptide has at least 95% identity with SEQ ID NO: 1. Embodiment 115. An isolated peptide, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 2-154, wherein in the peptide is not identical to SEQ ID NO: 1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. Embodiment 116. An isolated peptide, wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 2-50, wherein in the peptide is not identical to SEQ ID NO: 1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. Embodiment 117. The peptide of embodiment 116, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 2-50. Embodiment 118. The peptide of embodiment 116, wherein the peptide has at least 90% identity with any one of SEQ ID NOs: 2-50. Embodiment 119. The peptide of embodiment 116, wherein the peptide has at least 95% identity with any one of SEQ ID NOs: 2-50. Embodiment 120. An isolated peptide, wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 51-100, wherein in the peptide is not identical to SEQ ID NO:
1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. Embodiment 121. The peptide of embodiment 120, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 51-100. Embodiment 122. The peptide of embodiment 120, wherein the peptide has at least 90% identity with any one of SEQ ID NOs: 51-100. Embodiment 123. The peptide of embodiment 120, wherein the peptide has at least 95% identity with any one of SEQ ID NOs: 51-100. Embodiment 124. An isolated peptide, wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 101-154, wherein in the peptide is not identical to SEQ ID NO: 1, and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. Embodiment 125. The peptide of embodiment 123, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 101-154. Embodiment 126. The peptide of embodiment 123, wherein the peptide has at least 90% identity with any one of SEQ ID NOs: 101-154. Embodiment 127. The peptide of embodiment 123, wherein the peptide has at least 95% identity with any one of SEQ ID NOs: 101-154. Embodiment 128. An isolated peptide, wherein the peptide has at least 70% identity with SEQ ID NO: 1 over a range of at least 10 amino acids and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M. Embodiment 129. The peptide of embodiment 128, wherein the peptide has at least 80% identity with SEQ ID NO: 1 over a range of at least 13 amino acids. Embodiment 130. The peptide of embodiment 128, wherein the peptide has at least 80% identity with SEQ ID NO: 1 over a range of at least 16 amino acids. Embodiment 131. The peptide of embodiment 128, wherein the peptide has at least 80% identity with SEQ ID NO:l over a range of at least 9-18 amino acids. Embodiment 132. The peptide of any one of embodiments 110-131, wherein the peptide comprises an N- terminal deletion. Embodiment 133. The peptide of embodiment 132, wherein the N-terminal deletion consists of one amino acid. Embodiment 134. The peptide of embodiment 132, wherein the N-terminal deletion consists of two amino acids. Embodiment 135. The peptide of embodiment 132, wherein the N-terminal deletion consists of three amino acids. Embodiment 136. The peptide of embodiment 132, wherein the N-terminal deletion consists of one to five amino acids. Embodiment 137. The peptide of any one of embodiments 110- 136, wherein the peptide comprises at least one non-canonical amino acid. Embodiment 138. The peptide of embodiment 137, wherein the non-canonical amino acid is selected from the group consisting of a D-amino acid, a beta-amino acid, an N-acyl amino acid, or a C-amidyl amino acid. Embodiment 139. The peptide of embodiment any one of embodiments 110-138, wherein the peptide comprises at least one C-terminal or N-terminal modification. Embodiment 140. The peptide of embodiment any one of embodiments 110-138, wherein the at least one C-terminal or N-terminal modification comprises a modification which extends half-life or broadens the antibiotic spectrum. Embodiment 141. The peptide of embodiment any one of embodiments 110-138, wherein the at least one C-terminal or N-terminal modification comprises a water-soluble polymer. Embodiment 142. The peptide of embodiment 141, wherein the at least one C-terminal or N-terminal modification comprises a PEG group. Embodiment 143. The peptide of embodiment any one of embodiments 110-138, wherein the at least one C-terminal or N-terminal modification comprises a lipid. Embodiment 144. The peptide of embodiment any one of embodiments 110-138, wherein the at least one C-terminal or N-terminal modification comprises a protein or half-life extending peptide. Embodiment 145. The peptide of embodiment any one of embodiments 110-139, wherein the peptide comprises at least one intramolecular linkage. Embodiment 146. The peptide of embodiment 145, wherein the intramolecular linkage is formed by two amino acid side chains. Embodiment 147. The peptide of embodiment 146, wherein the peptide comprises at least one disulfide linkage. Embodiment 148. The peptide of embodiment any one of embodiments 110-147, wherein the peptide comprises at least one substitution relative to SEQ ID NO: 1. Embodiment 149. A pharmaceutical composition comprising a peptide of any one of embodiments 1-148 and an excipient, delivery vehicle, second therapeutic agent, or a combination thereof. Embodiment 150. The pharmaceutical composition of embodiment 149, wherein the pharmaceutical composition comprises an excipient. Embodiment 151. The pharmaceutical composition of embodiment 149, wherein the pharmaceutical composition comprises a second therapeutic agent. Embodiment 152. The pharmaceutical composition of embodiment 149, wherein the pharmaceutical composition is formulated for topical administration. Embodiment 153. The pharmaceutical composition of embodiment 149, wherein the pharmaceutical composition is formulated for intravenous administration. Embodiment 154. The pharmaceutical composition of embodiment 149, wherein the pharmaceutical composition is formulated for oral administration. Embodiment 155. The pharmaceutical composition of embodiment 149, wherein the pharmaceutical composition is formulated for intramuscular or subcutaneous administration. Embodiment 156. A method of treating an infection comprising administering to a subject a therapeutically effective amount of a peptide or pharmaceutical composition of any one of embodiments 1-155 or SEQ ID NO: 1. Embodiment 157. The method of embodiment 156, wherein the infection comprises a bacterial infection. Embodiment 158. The method of embodiment 157, wherein the bacterial infection is caused by a Gram-negative bacterium. Embodiment 159. The method of embodiment 157, wherein the bacterial infection is caused by an enterobacterium. Embodiment 160. The method of embodiment 158, wherein the bacterium is Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, or Pseudomonas aeruginosa. Embodiment 161. The method of any one of embodiments 156-159, wherein the bacterium is a drug- resistant or multi-drug resistant bacterium. Embodiment 162. The method of any one of embodiments 156-161, wherein the subject is a mammal. Embodiment 163. The method of embodiment 162, wherein the mammal is a human. Embodiment 164. The method of any one of embodiments 156-163, wherein the peptide is administered orally, enterically, topically, intravenously, intraperitoneally, intramuscularly, endoscopically, percutaneously, subcutaneously, regionally, by inhalation, or by direct injection. Embodiment 165. The method of embodiment 164, wherein the orally administered peptide is a capsule or tablet. Embodiment 166. The method of any one of embodiments 156-165, further comprising administering a second therapeutic agent. Embodiment 167. The method of embodiment 166, wherein the second therapeutic agent is an antibiotic or a protease inhibitor. Embodiment 168. The method of embodiment 167, wherein the antibiotic is a beta-lactam antibiotic. Embodiment 169. The method of embodiment 167, wherein the antibiotic is amoxicillin, bacitracin, chloramphenicol, clindamycin, capreomycin, colistimethate, ciprofloxacin, doxycycline, erythromycin, fusidic acid, fosfomycin, fusidate sodium, gramicidin, gentamycin, lincomycin, minocycline, macrolides, monobactams, nalidixic acid, novobiocin, ofloxcin, rifamycins, tetracyclines, vancomycin, tobramycin, fluoroquinolones, polymyxins, DNA gyrase inhibitors, bacterial polymerase inhibitors, folate synthesis inhibitors, or trimethoprim.
EXAMPLES
[0235] Example 1. Measurement of MICs of Peptides [00236] Minimum inhibitory concentration (MIC) was determined by broth microdilution using Mueller-Hinton broth (MHB) as the test medium. Bacterial strains were cultured at 37°C in a shaking incubator until log phase was reached between 0.5-0.8 at O.D. 600nm. Cell concentration was adjusted to approximately 5 x 105 cells/ml. Peptides were prepared as 10X stock solutions and serially diluted 2-fold in bovine serum albumin (BSA) acetic acid media to prevent binding to the plastic. Using 96 well plates 90 microliters of cells and 10 ul of 10X peptide dilutions were added to each well. Growth control and media control lanes were included in each plate. Each plate was then parafilmed and incubated at 37°C for 16-18 hours. Plates were read with a spectrophotometer at O.D. 600 nm. The MIC was defined as the lowest concentration of peptide with an O.D. of 0.
[00237] Table 2 shows MIC99 data for Compound A (SEQ ID NO: 1) against several drug resistant isolates, including multi-drug resistant E. coli , colistin resistant E. coli , and E. coli with resistant mutations in New Delhi metallo-beta-lactamase 1. Compound A also shows activity against Klebsiella pneumoniae and its New Delhi mutations. In addition, activity was measured against the New Delhi mutant of enterbacter cloacae.
Figure imgf000096_0001
[00238] Table 3 shows sequences and data associated with Compound A (SEQ ID NO: 1) and analogs (SEQ ID NOS: 1-8).
Figure imgf000097_0001
[00239] Example 2. Comparison of Compound A and thanatin.
[00240] Following the general protocol of Example 1, Compound A and thanatin were tested against a panel of Enterobacteriaceae, additional Gram-negative species, and Grampositive strains.
[00241] Table 4 shows MIC data for various bacterial strains. Compound A demonstrated increased potency compared to thanatin against a panel of MDR E. coli strains including multi-drug (MDR), carbapenem (NDM) and colistin resistant (ColR) isolates.
Figure imgf000097_0002
Figure imgf000098_0001
KPC = K. pneumoniae carbapenemase, MDR = multi-drug resistant, CoP = colistin resistant, NDM = New Delhi metallo-fi-lactamase-l .
[00242] Example 3. Compound A antimicrobial activity
[00243] Compound A was tested in both a time-kill assay (Fig. 1A), and beta-lactamase release assay (Fig. IB, 4 hours). Compound A showed rapid bactericidal activity consistent with other bactericidal antibiotics and distinct from membrane lytic agents such as melittin. The D-form of Compound A was not active (Fig. 1A). Compound A did not demonstrate measurable release of periplasmic bacterial β-lactamase from E. coli even well above its MIC (Fig. IB). Colistin was used as a positive control for the beta-lactamase release assay.
[00244] Example 4. In-vitro toxicity study with Compound A in mammalian cells [00245] Compound A, thanatin, and melittin were tested for toxicity in red blood cells and HEK293 cells using standard protocols. Traditional cationic AMPS (anti-microbial peptides, such as cAMPS, e.g., melittin) in some instances cause significant mammalian cell toxicity through membrane lysis. In constrast, Compound A demonstrated less than 3% red blood cell hemolysis at 128 μg/ml (Fig. 2A), and no significant release of LDH from HEK293 cells (Fig. 2B).
[00246] Example 5. In-vivo disease model
[00247] Drug-resistant E. coli (strain 2340) was delivered intraperitoneally (IP) followed by peptide administration IP at 1 hr and 6 hrs post-infection to a mouse model. Peripheral organs were harvested at 24 hrs post infection and bacterial load assessed by CFU.
Compound A showed greater than 2 log reduction in peripheral organ bacterial load, similar to positive control gentamicin.
[00248] Example 6: General procedure for synthesis of antimicrobial peptides
[00249] Solid Phase Peptide Synthesis. The peptide (targeting 10 mg) was synthesized on
0.2 mmol CTC resin using standard Fmoc chemistry. The first amino acid (0.2 mmol, 1.0 eq ) was added to the resin in DCM (4.0 mL), and then DIE A (4.0 eq) was added dropwisely and mixed for 2 hr with N2 bubbling. 0.4 mL MeOH was then added and mixed for an additional 30 min. Then the resin was washed with DMF for 5 times, and proceeded to Fmoc removal and next AA (amino acid) coupling. For Fmoc removal, the resin was added with 20% piperidine/DMF and incubated for 20 min. For AA coupling, the resin was incubated with a mixture of Fmoc-AA-OH: HBTU: DIEA (3 eq: 2.85 eq: 6 eq, in DMF) for 45 min with N2 bubbling. The coupling process was monitored by ninhydrin test, where colorless beads indicated no free amine thus the reaction was complete. Between coupling of amino acids and de-protection of Fmoc groups, the resin was washed exhaustively with DMF.
[00250] Cleavage. The linear peptide was cleaved off from the resin by treatment of (90% TFA/5% 3-mercaptopropionic acid/2.5% TIS/2.5% H2O, 10 mL in total) for 2 hr, and the solution fraction was collected. The liquid fraction was slowly added to cold tert-butyl methyl ether (50 mL), and peptide crushed out. The precipitate was collected after centrifugation (3000 rpm/2 min), and the white crude was washed with cold tert-butyl methy ether for 2 additional times, and then dried in desiccator for 2 hr.
[00251] Cyclization. The crude peptide was dissolved in ACN/ H2O (30/70, ~ 4 mg/mL), and disulfide bond was promoted by adding I2/AcOH (0.1 M) dropwisely at 25°C until the yellow color persisted, and then the mixture was stirred at 20°C for 2 min. The oxidation was quenched by adding sodium thiosulfate (0.1 M in water) dropwisely until yellow color disappeared. The mixture was lyophilized to give the crude cyclic peptide.
[00252] Purification. The crude peptide was purified by prep-HPLC (TFA condition; 30 °C, A: 0.075% TFA in FLO, B: CFLCN, Table 5) to give the pure peptide, then exchanged in to AcOH salt via prep-HPLC (AcOH condition; 50 °C, A:0.5% AcOH in H2O, B:CH3CN, Table 6) to give the purified peptide.
Table 5
Figure imgf000099_0001
Table 6
Figure imgf000099_0002
Figure imgf000100_0001
[00253] Example 7: Synthesis of Compound B (SEQ ID NO: 294)
[00254] Peptide Synthesis. Compound B was synthesized using standard Fmoc chemistry (Table 7): 1. Resin preparation: To the 1-chloro-2-[chloro(diphenyl)methyl]benzene (0.20 mmol, 1.00 eq) was added Fmoc-Met-OH (0.2 mmol, 1.00 eq) and DIEA (0.8 mmol, 4.00 eq) in DCM (4.00 mL). The mixture was agitated with N2 for 2 h at 25°C, then added MeOH (0.40 mL) and agitated with N2 for another 30 min. The resin was washed with DMF (5.00 mL * 3). Then 20% piperidine in DMF (5.00 mL) was added and the mixture was agitated with N2 for 20 min at 25°C. Then the mixture was filtered to get the resin. The resin was washed with DMF (5.00 mL * 5) and filtered to get the resin. 2. Coupling: a solution of DIEA (1.2 mmol, 6.00 eq), Fmoc-Dab(Boc)-OH (0.60 mmol, 3.00 eq) and HBTU (0.57 mmol, 2.85 eq) in DMF (2.50 mL) was added to the resin and agitated with N2 for 45 min at 25°C. The resin was then washed with DMF (4.00 mL * 3). 3. Deprotection: 20% piperidine in DMF (4.00 mL) was added to the resin and the mixture was agitated with N2 for 20 min at 25°C. The resin was washed with DMF (4.00 mL * 5) and filtered to get the resin. 4. Above steps 3 to 4 was repeated for the coupling of following amino acids.
Table 7
Figure imgf000100_0002
Figure imgf000101_0001
20% piperidine in DMF was used for Fmoc deprotection for 20 min. The coupling reaction was monitored by ninhydrin test, and the resin was washed with DMF for 5 times.
[00255] Peptide Cleavage and Purification. 1. Added cleavage buffer (90% TFA/5% 3- mercaptopropionic acid/2.5% TIS/2.5% H2O) to the flask containing the side chain protected peptide at room temperature and stir for 2 hours. 2. The peptide was precipitated with cold tert-butyl methyl ether. 3. Filtered and the filter cake was collected. 4. Tert-butyl methyl ether washes were performed two more times. 5. Crude peptide was dried under vacuum 2 hours.
6. To the crude peptide (0.5 g) in water (100 mL) and CFFCN (40 mL) was added I2 / AcOH (0.1 M) dropwise at 25°C until the yellow color persisted. Then the mixture was stirred at 25°C for 2 min. 7. Then after 2 min, sodium thiosulfate (0.1 M in water) was added dropwise until yellow color disappeared. The mixture was lyophilized to give the crude peptide. 8. The crude peptide was purified by prep-HPLC (TFA condition; 30 °C, A:0.075% TFA in H2O, B:CH3CN) to give the peptide, then the peptide was purified by prep-HPLC (AcOH condition; 50 °C, A:0.5% AcOH in H2O, B:CH3CN) to give Compound B (41.6 mg, 1.66% yield, 99.2% purity, AcOH).
[00256] Purification conditions are shown in Table 8 and Table 9.
Table 8
Figure imgf000101_0002
Table 9
Figure imgf000101_0003
Figure imgf000102_0001
[00257] Example 8: In-vitro antimicrobial activity assay
[00258] Following the general procedure of Example 6, peptides were synthesized. These peptides were then tested for antimicrobial activity following the general procedures of Example 1 (Tables 10 and Table 11).
Table 10
Figure imgf000102_0002
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
[00259] MIC Key: A: <0.5; B: 0.5-1; C: 1.01-10; D: 10.01-50; F: >50. nd: not determined.
Table 11
Figure imgf000107_0002
[00260] MIC Key: A: <0.5; B: 0.5-1; C: 1.01-10; D: 10.01-50; F: >50.
[00261] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS What is claimed is:
1. An isolated peptide, wherein the peptide has a sequence comprising Formula (I):
Y1-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-Y2
Formula (I); wherein
Y1 is absent or an N-terminal modification;
X1 and X2 are independently absent, or any amino acid;
X3 is absent or any amino acid;
X4 is positively charged amino acid;
X5 is any amino acid;
X6 is a non-polar amino acid;
X7 is any amino acid;
X8 is a non-polar amino acid;
X9 is any amino acid;
X10 is any amino acid;
X11 is a bridging amino acid;
X12 is a polar amino acid or a positively charged amino acid;
X13 is any amino acid;
X14 is any amino acid;
X15 is a polar amino acid;
X16 is a non-polar amino acid;
X17 is any amino acid;
X18 is a bridging amino acid;
X19 is a polar amino acid;
X20 is a positively charged amino acid;
X21 is absent or any amino acid;
Y2 is absent or a C-terminal modification, wherein if: X1 and X16 are G; X2 is S, X3, X4, X17 are K; X5 and X7 are P; X6 is V; X8 and X9 are I; X10 is Y; X11 is C; X12 is N; X13, X14, and X20 are R; X15 is T, X16 is G, X18 is C, X19 is Q; then X21 is not M; and the peptide is not identical to SEQ ID NO: 1
2. The peptide of claim 1, wherein at least three of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10,
X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, or X21 are independently a non-canonical amino acid.
3. The peptide of claim 2, wherein X1 and X2 are independently G, S, A, V, L, I, M, F, W, or P.
4. The peptide of claim 1, wherein X3 is K, R, H, DAB (2,4-diaminobutyric acid), DAP (diaminopimelic acid), or absent.
5. The peptide of claim 1, wherein X4 is K, R, H, DAB (2,4-diaminobutyric acid), or DAP (diaminopimelic acid).
6. The peptide of claim 1, wherein X5 and X7 are each independently a constrained amino acid.
7. The peptide of claim 6, wherein X5 is P, X7 is P, and X6 is V, L, or I.
8. The peptide of claim 1, wherein X8 is I or F.
9. The peptide of claim 1, wherein X9 is I, T, N, S, V, E, Y, or F.
10. The peptide of claim 1, wherein X10 is F, W, or Y.
11. The peptide of claim 1, wherein X11 and X18 are taken together to form an intramolecular linkage.
12. The peptide of claim 11, wherein at least one of X11 and X18 is C or penicillamine.
13. The peptide of claim 11, wherein the intramolecular linkage comprises a disulfide linkage.
14. The peptide of claim 11, wherein the intramolecular linkage comprises the structure:
Figure imgf000110_0001
Figure imgf000111_0001
wherein
R is a substituent, Ar is aryl, and HA is heteroaryl.
15. The peptide of claim 14, wherein R is alkyl, aralkyl, or cycloalkyl; Ar is optionally substituted phenyl or naphthyl; and HA is optionally substituted: pyridinyl, imidazolyl, thiophenyl, pyrrolyl, thiazolyl, oxazolyl, or furanyl.
16. The peptide of claim 1, wherein X12 is S, T, C, Y, N, or Q.
17. The peptide of claim 1, wherein X13 is R, Q, L, or F.
18. The peptide of claim 1, wherein X14, X17, and X20 are independently a positively charged amino acid.
19. The peptide of claim 18, wherein X14 and X17, and X20 are independently a K, R, H, DAB (2,4-diaminobutyric acid), or DAP (diaminopimelic acid).
20. The peptide of claim 1, wherein X16 is G, A, V, L, I, M, F, W, or P.
21. The peptide of claim 1, wherein X21 is M, L, V, or F.
22. The peptide of claim 2, wherein the non-canonical amino acid is selected from the group consisting of alanine derivatives; alicyclic amino acids; arginine derivatives; aromatic amino acids; asparagine derivatives; aspartic acid derivatives; beta-amino acids; cysteine derivatives; dab (2,4-diaminobutyric acid); dap (2,3-diaminopropionic acid); glutamic acid derivatives; glutamine derivatives; glycine derivatives; homo-amino acids; isoleucine derivatives; leucine derivatives; linear core amino acids; lysine derivatives; methionine derivatives; n-methyl amino acids; norleucine derivatives; norvaline derivatives; ornithine derivatives; penicillamine derivatives; phenylalanine derivatives; phenylglycine derivatives; proline derivatives; pyroglutamine derivatives; serine derivatives; threonine derivatives; tryptophan derivatives; tyrosine derivatives; and valine derivatives.
23. The peptide of claim 22, wherein at least one of X4, X17, or X20 are DAB (2,4- diaminobutryic acid) or DAP (diaminopimelic acid).
24. The peptide of claim 22, wherein at least one of X3, X4, X17, are DAB (2,4- diaminobutryic acid) or DAP (diaminopimelic acid).
25. The peptide of claim 1, wherein at least one of X1, X2, X3, and Y2 are non-canonical amino acids.
26. The peptide of claim 25, wherein at least one of X20, X21, or Y1 are non-canonical amino acids.
27. The peptide of claim 1, wherein Y1 is absent or an N-terminal modification;
XI is absent, or G;
X2 is absent, or S;
X3 is absent, K, DAB, or DAP;
X4 is K, DAB, or DAP;
X5 is P;
X6 is V, I, L, or F;
X7 is P;
X8 is I or F;
X9 is I, T, N, S, V, E, Y, F;
X10 is Y, F, H, or L;
XII is C or penicillamine;
X12 is N, S, H, or R;
X13 is R, Q, L, F, DAB, or DAP;
X14 is any amino acid;
X15 is T or S;
X16 is G;
X17 is any amino acid;
X18 is C or penicillamine;
X19 is Q or T;
X20 is R, DAB or DAP;
X21 is absent, I, L, V, M, F, or a non-canonical amino acid; and Y2 is absent or a C-terminal modification.
28. The peptide of any one of claims 1-27, wherein the non-canonical amino acid is linked to a half-life extending moiety or a moiety which broadens the antibiotic spectrum.
29. The peptide of any one of claims 1-28, wherein the peptide comprises at least one N- terminal modification or C-terminal modification.
30. The peptide of claim 29, wherein the N-terminal modification or C-terminal modification comprises at least one targeting agent.
31. The peptide of claim 30, wherein the targeting agent increases the local concentration of the peptide.
32. The peptide of claim 30, wherein the targeting agent broadens the antibiotic spectrum.
33. The peptide of any one of claims 1-32, wherein Y2 comprises an amide.
34. The peptide of claim 33, wherein Y2 is selected from the group consisting of -NH2, - NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2.
35. An isolated peptide, wherein the peptide has at least 70% identity with SEQ ID NO: 1, wherein in the peptide is not identical to SEQ ID NO: 1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
36. The peptide of claim 35, wherein the peptide has at least 75% identity with SEQ ID NO:l.
37. The peptide of claim 35, wherein the peptide has at least 85% identity with SEQ ID NO:l.
38. The peptide of claim 35, wherein the peptide has at least 90% identity with SEQ ID NO:l.
39. The peptide of claim 35, wherein the peptide has at least 95% identity with SEQ ID NO:l.
40. An isolated peptide, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 2-313, wherein in the peptide is not identical to SEQ ID NO: 1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R;
T18Q; and F20M.
41. The peptide of claim 40, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 2-50.
42. An isolated peptide, wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 51-100, wherein in the peptide is not identical to SEQ ID NO: 1 and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R;
T18Q; and F20M.
43. The peptide of claim 42, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 51-100.
44. An isolated peptide, wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 101-154, wherein in the peptide is not identical to SEQ ID NO: 1, and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
45. The peptide of claim 44, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 101-150.
46. An isolated peptide, wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 151-200, wherein in the peptide is not identical to SEQ ID NO: 1, and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
47. The peptide of claim 46, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 151-200.
48. An isolated peptide, wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 201-250, wherein in the peptide is not identical to SEQ ID NO: 1, and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
49. The peptide of claim 48, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 201-250.
50. An isolated peptide, wherein the peptide has at least 70% identity with any one of SEQ ID NOs: 251-313, wherein in the peptide is not identical to SEQ ID NO: 1, and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
51. The peptide of claim 50, wherein the peptide has at least 80% identity with any one of SEQ ID NOs: 251-313.
52. An isolated peptide, wherein the peptide has at least 70% identity with SEQ ID NO: 1 over a range of at least 10 amino acids and wherein the peptide is not a variant of SEQ ID NO: 1 comprising mutations S2_K3insK; K13R; T18Q; and F20M.
53. The peptide of claim 52, wherein the peptide has at least 80% identity with SEQ ID NO: 1 over a range of at least 13 amino acids.
54. The peptide of claim 52, wherein the peptide has at least 80% identity with SEQ ID NO: 1 over a range of at least 16 amino acids.
55. The peptide of claim 52, wherein the peptide has at least 80% identity with SEQ ID NO: 1 over a range of at least 9-18 amino acids.
56. The peptide of any one of claims 35-55, wherein the peptide comprises an N-terminal deletion.
57. The peptide of any one of claims 35-55, wherein the peptide comprises at least one non- canonical amino acid.
58. The peptide of claim 57, wherein the non-canonical amino acid is selected from the group consisting of a D-amino acid, a beta-amino acid, an N-acyl amino acid, or a C-amidyl amino acid.
59. The peptide of claim any one of claims 35-58, wherein the peptide comprises at least one C-terminal orN-terminal modification.
60. The peptide of claim any one of claims 35-58, wherein the at least one C-terminal or N- terminal modification comprises a modification which extends half-life or broadens the antibiotic spectrum.
61. The peptide of claim any one of claims 35-60, wherein the peptide comprises at least one intramolecular linkage.
62. The peptide of claim 61, wherein the intramolecular linkage is formed by two amino acid side chains.
63. The peptide of claim 62, wherein the peptide comprises at least one disulfide linkage.
64. The peptide of claim any one of claims 35-63, wherein the peptide comprises at least one substitution relative to SEQ ID NO: 1.
65. A pharmaceutical composition comprising a peptide of any one of claims 1-64 and an excipient, delivery vehicle, second therapeutic agent, or a combination thereof.
66. The pharmaceutical composition of claim 65, wherein the pharmaceutical composition comprises an excipient.
67. The pharmaceutical composition of claim 65, wherein the pharmaceutical composition comprises a second therapeutic agent.
68. The pharmaceutical composition of claim 65, wherein the pharmaceutical composition is formulated for topical administration.
69. The pharmaceutical composition of claim 65, wherein the pharmaceutical composition is formulated for intravenous administration.
70. The pharmaceutical composition of claim 65, wherein the pharmaceutical composition is formulated for oral administration.
71. The pharmaceutical composition of claim 65, wherein the pharmaceutical composition is formulated for intramuscular or subcutaneous administration.
72. A method of treating an infection comprising administering to a subject a therapeutically effective amount of a peptide or pharmaceutical composition of any one of claims 1-71 or a peptide comprising SEQ ID NO: 1.
73. The method of claim 72, wherein the infection comprises a bacterial infection.
74. The method of claim 73, wherein the bacterial infection is caused by a Gram-negative bacterium.
75. The method of claim 73, wherein the bacterial infection is caused by an enterobacterium.
76. The method of claim 74, wherein the bacterium is Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii, or Pseudomonas aeruginosa.
77. The method of any one of claims 72-75, wherein the bacterium is a drug-resistant or multi-drug resistant bacterium.
78. The method of any one of claims 72-77, wherein the subject is a mammal.
79. The method of claim 78, wherein the mammal is a human.
80. The method of any one of claims 72-79, wherein the peptide is administered orally, enterically, topically, intravenously, intraperitoneally, intramuscularly, endoscopically, percutaneously, subcutaneously, regionally, by inhalation, or by direct injection.
81. The method of claim 80, wherein the orally administered peptide is a capsule or tablet.
82. The method of any one of claims 72-81, further comprising administering a second therapeutic agent.
83. The method of claim 82, wherein the second therapeutic agent is an antibiotic or a protease inhibitor.
84. The method of claim 83, wherein the antibiotic is a beta-lactam antibiotic.
85. The method of claim 83, wherein the antibiotic is amoxicillin, bacitracin, chloramphenicol, clindamycin, capreomycin, colistimethate, ciprofloxacin, doxycycline, erythromycin, fusidic acid, fosfomycin, fusidate sodium, gramicidin, gentamycin, lincomycin, minocycline, macrolides, monobactams, nalidixic acid, novobiocin, ofloxcin, rifamycins, tetracyclines, vancomycin, tobramycin, fluoroquinolones, polymyxins, DNA gyrase inhibitors, bacterial polymerase inhibitors, folate synthesis inhibitors, or trimethoprim.
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