WO2021073131A1 - Medicament for efficiently killing drug-resistant disease bacteria and application thereof in inhibiting drug-resistant disease bacteria - Google Patents

Medicament for efficiently killing drug-resistant disease bacteria and application thereof in inhibiting drug-resistant disease bacteria Download PDF

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WO2021073131A1
WO2021073131A1 PCT/CN2020/095683 CN2020095683W WO2021073131A1 WO 2021073131 A1 WO2021073131 A1 WO 2021073131A1 CN 2020095683 W CN2020095683 W CN 2020095683W WO 2021073131 A1 WO2021073131 A1 WO 2021073131A1
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tpad
active ingredient
tpi
bacteria
composition
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PCT/CN2020/095683
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French (fr)
Chinese (zh)
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于日磊
王岩
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中国海洋大学
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Priority claimed from CN201910983456.7A external-priority patent/CN110680904A/en
Application filed by 中国海洋大学 filed Critical 中国海洋大学
Priority to CN202080072646.4A priority Critical patent/CN114650835A/en
Publication of WO2021073131A1 publication Critical patent/WO2021073131A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/04—Derivatives of thiourea
    • C07C335/16—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • 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

  • the invention belongs to the technical field of antibacterial drugs, and specifically relates to a drug for efficiently killing drug-resistant disease bacteria and its application in inhibiting drug-resistant disease bacteria.
  • TPI Tachyplesin I
  • TPI has broad-spectrum antibacterial activity against Gram-positive bacteria and Gram-negative bacteria.
  • follow-up studies have shown that as a polypeptide, TPI has poor stability in plasma and is easily degraded. TPI can easily cause mammalian red blood cell membranes to rupture and hemolysis during the antibacterial process.
  • the antibacterial activity of TPI is still not strong enough, and the antibacterial activity is lower than current clinical drugs. This shortcoming greatly limits its clinical application.
  • TPI is not strong enough in the antibacterial process, has poor stability, is easily degraded, and has strong hemolysis.
  • TPI/TPAD has lower activity.
  • modification of TPI activity based on sequence scanning screening method can mainly improve the activity to a certain extent, the extent of improvement is very limited.
  • the purpose of the present invention is to provide a new drug with high antibacterial activity, especially against drug-resistant bacteria.
  • compositions for killing or inhibiting disease bacteria wherein the composition includes an active combination consisting of a first active ingredient and a second active ingredient; wherein,
  • the TPAD refers to a D-type amino acid analog of TPI, and the TPI is a polypeptide as shown in SEQ ID NO: 3;
  • the second active ingredient is a QseC/B signaling pathway inhibitor
  • the QseC/B signaling pathway inhibitor refers to an active substance that can inhibit QseC, QseB, or a combination thereof.
  • the composition further includes: (b) a pharmaceutically acceptable carrier.
  • the D-type amino acid analog of TPI refers to correspondingly replacing the L-type amino acid in the TPI polypeptide sequence with the D-type amino acid and optionally replacing and/or modifying the amino acid in the polypeptide sequence The analogue obtained later.
  • TPAD is a polypeptide represented by the following structure:
  • X is an unnatural amino acid analog of Leu, Ile, Val, Ala or Leu;
  • each amino acid is D-type amino acid.
  • X is Leu, Ile, Val, or Ala.
  • Cys at position 3 and Cys at position 16 of TPAD form a disulfide bond
  • Cys at position 7 and Cys at position 12 form a disulfide bond
  • TPAD is a polypeptide as shown in SEQ ID NO: 2, and each amino acid in it is a D-type amino acid;
  • the QseC/B signaling pathway inhibitor is LED209 as shown in formula I, or a pharmaceutically acceptable salt thereof;
  • the dosage ratio (mg/pmol) of the first active ingredient and the second active ingredient is 1:100-100:1.
  • the dosage ratio (mg/pmol) of the first active ingredient and the second active ingredient is 1:10 to 1:0.5.
  • the dosage ratio of the first active ingredient and the second active ingredient is 2-500 ⁇ g/mL:5-1000 pM.
  • the total content of the first active ingredient and the second active ingredient is 0.1 to 99.9% by weight, based on the total mass of the composition.
  • the composition includes an active combination composed of TPAD and LED209; wherein the dosage ratio (mg/pmol) of TPAD and LED209 is 2:5.
  • the dosage of the TPAD is ⁇ 2 ⁇ g/mL; and/or the dosage of the LED209 is ⁇ 5pM.
  • the dosage of the TPAD is 2-500 ⁇ g/mL; and/or the dosage of the LED209 is ⁇ 5-1000 pM.
  • the dosage of the TPAD is 2 ⁇ g/mLTPAD; the dosage of the LED209 is 5 pM LED209.
  • composition as described in the first aspect in the preparation of a medicament for treating and/or preventing diseases caused by disease bacteria.
  • the dosage form of the drug is an oral dosage form or a non-oral dosage form.
  • the oral administration dosage form is selected from the group consisting of tablets, powders, granules or capsules, or emulsions or syrups.
  • the non-oral administration dosage form is selected from the group consisting of injections and injections.
  • the diseased bacteria are bacteria with a QseC/B two-component system.
  • the disease bacteria are resistant bacteria.
  • the disease bacteria are selected from the group consisting of Escherichia coli, Bacillus subtilis, Enterobacter cloacae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecium, Lysobacterium (preferably, Enzyme-producing Lysobacterium), Shigella flexneri, Pseudoalteromonas, Stenotrophomonas maltophilia, or a combination thereof.
  • the disease bacteria are selected from the following group: Escherichia coli K-12, Escherichia coli BAA 2469, Escherichia coli ATCC 25923, Bacillus subtilis 168, Enterobacter cloacae BAA 1143, Staphylococcus aureus ATCC 29213, Staphylococcus aureus BAA 41, Staphylococcus aureus BAA 44, Klebsiella pneumoniae BAA 1144, Klebsiella pneumoniae BAA 2470, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa BAA 2108, Bauman no Kinetobacter ATCC 19606, Enterococcus faecium ATCC 29212, Enzyme-producing Lysobacterium YC36, Shigella flexneri ATCC 29903, or a combination thereof.
  • the drug can treat and/or prevent diseases caused by pathogenic bacteria by inhibiting and/or killing disease bacteria.
  • the disease caused by diseased bacteria is a bacterial infection.
  • the diseases caused by disease bacteria include: respiratory tract infections, lung infections (such as pneumonia), and skin infections.
  • a drug combination for killing or inhibiting disease bacteria includes:
  • composition or medicine containing a first active ingredient (i) A composition or medicine containing a first active ingredient; and (ii) A composition or medicine containing a second active ingredient;
  • first active ingredient and the second active ingredient are as defined in the first aspect.
  • a method for inhibiting or killing disease bacteria which includes the steps of: contacting the disease bacteria with the composition as described in the first aspect; or bringing the disease bacteria with the first active ingredient and the first active ingredient. Two active ingredients contact, thereby inhibiting or killing disease bacteria;
  • first active ingredient and the second active ingredient are as defined in the first aspect.
  • the method is non-therapeutic in vitro.
  • the method is therapeutic or preventive.
  • the diseased bacteria are brought into contact with the first active ingredient and the second active ingredient at the same time.
  • the diseased bacteria are contacted with the first active ingredient and the second active ingredient respectively.
  • the diseased bacteria are sequentially contacted with the second active ingredient and the first active ingredient.
  • the amount of the first active ingredient is ⁇ 2 ⁇ g/mL.
  • the amount of the first active ingredient is 2-500 ⁇ g/mL.
  • the amount of the second active ingredient is ⁇ 5pM.
  • the amount of the second active ingredient is 5-1000 pM.
  • the subject includes humans or non-human mammals.
  • the first active ingredient or the composition or medicine containing the first active ingredient and the second active ingredient or the composition or medicine containing the first active ingredient are simultaneously administered to a subject in need.
  • the first active ingredient or the composition or medicine containing the first active ingredient and the second active ingredient or the composition or medicine containing the first active ingredient are administered to a subject in need at intervals.
  • Figure 1 is a schematic diagram of the antibacterial activity of the combination of TPAD and LED209 provided by an embodiment of the present invention on wild-type or qseC or qseB gene knockout bacteria, which shows the inherently drug-resistant bacteria provided by the embodiment of the present invention, the enzyme-producing Lysobacterium YC36 The genome-wide transcription profile of (LeYC36) and the resistance mechanism of bacteria to TPAD at sublethal concentrations; in each figure: WT represents the wild-type strain, ⁇ qseB represents the qseB gene knockout strain, and ⁇ qseC represents the qseC gene knockout strain.
  • the heat map shows the relative transcription level of the drug efflux pump gene, and the ratio below the heat map represents the fold change of the relative expression level
  • Fig. 2 is a schematic diagram of the NMR structure of TPAD provided by an embodiment of the present invention.
  • Fig. 3 is a schematic diagram of molecular dynamics simulation study of peptide conformational stability provided by an embodiment of the present invention.
  • FIG. 4 is a schematic diagram of the orientation of TPAD on the film surface after 600 ns MD simulation provided by an embodiment of the present invention.
  • Figure 5 is a schematic diagram of the hemolytic activity of TPAD on human erythrocytes provided by an embodiment of the present invention.
  • Fig. 6 is a schematic diagram of the lysis activity of TPAD on liposomes composed of E. coli lipid extract provided by an embodiment of the present invention.
  • Fig. 7 is a schematic diagram of the influence of TPAD and TPI on the survival of liver cancer cells provided by an embodiment of the present invention.
  • Fig. 8 is a schematic diagram of Western blotting analysis of the expression of the key protein QseB after L. enzymogenes YC36 is treated by TPAD according to an embodiment of the present invention.
  • FIG. 9 is a schematic diagram of the structure and sequence of TPI and TPAD provided by an embodiment of the present invention; in the figure: (A) and (B) are the structure (PDB ID: 1wo0) and sequence of TPI, respectively; (C) the sequence of TPAD.
  • Fig. 10 is a schematic diagram of the structure and stability of TPI and TPAD provided by an embodiment of the present invention; in the figure: (A) a comparison of H ⁇ secondary chemical shifts between limulin I (TPI) and TPAD.
  • the chemical shift data of TPI comes from BMRB accession number 21044; (B) TPI (PDB ID: 1wo0); (C) NMR solution structure of TPAD (this invention); (D) 600ns molecular dynamics simulation from the initial conformation During the process, the RMSD evolution of the peptide backbone; (E) the stability of TPI (black, lower curve) and TPAD (dark gray, upper curve) in human serum within 6 hours, three parallel experiments were performed, the error bar is the average The standard error of.
  • Figure 11 is a schematic diagram of the hemolytic activity of TPI and TPAD on human erythrocytes and the cytotoxicity of human normal human erythrocytes L02 provided by an embodiment of the present invention
  • Figure 12 is a schematic diagram of the atomic force microscope observation of cell morphology of Lysobacter enzymogenes YC36 treated with TPAD according to an embodiment of the present invention; in the figure: (A) the morphology of LeYC36 cells without TPAD treatment; (B) TPAD The morphology of LeYC36 cells after treatment; (C) A cross-sectional analysis image of the surface morphology of LeYC36 cells untreated or treated with TPAD.
  • Figure 13 is a schematic diagram of the continuous subculture of cells provided by the embodiment of the present invention; the evolution of the LeYC36 minimum inhibitory concentration (MIC) induced by (A) TPAD and (B) TPI for continuous passage; each passage of bacterial strains, TPAD/TPI Increase the concentration of 2 ⁇ g/mL.
  • MIC LeYC36 minimum inhibitory concentration
  • Figure 14 shows that the LED209 provided by the embodiment of the present invention enables TPAD to more effectively kill the maltophilic cells of Pseudomonas and Maltophilia.
  • Figure 15 shows the resistance mechanism of bacteria to TPAD at the sublethal dose provided in the embodiment of the present invention.
  • TPI analogs such as TPAD
  • TPI analogs when used in combination with QsecB/QsecC inhibitors, they also show similar activities to non-mutant strains as TPI analogs have similar activities to ⁇ qseB and ⁇ qseC mutants, TPI analogs and QsecB/QsecC
  • the combination of inhibitors can synergistically promote the antibacterial or bactericidal activity of TPI analogs. Based on this, the inventor completed the present invention.
  • QseC/B signaling pathway inhibitor As used herein, the terms “QseC/B signaling pathway inhibitor”, “QseC/QseB inhibitor” or “QseC/QseB two-component inhibitor” can be used interchangeably and refer to active substances that can inhibit QseC and/or QseB (For example, small molecule compounds or polypeptides, etc.).
  • QseC inhibitor refers to an active substance (small molecule compound or polypeptide, etc.) capable of inhibiting QseC.
  • QseB inhibitor refers to an active substance (small molecule compound or polypeptide, etc.) capable of inhibiting QseB.
  • LED209 is a compound represented by formula I, and LED209 can be commercially available or synthesized according to the prior art.
  • the active polypeptide refers to the D-type amino acid analog (TPAD) of an antibacterial peptide (ie TPI) with broad-spectrum antibacterial activity isolated from stem cells of Tachypleus tridentatus; preferred in the present invention
  • TPI D-type amino acid analog
  • the active polypeptide refers to a full D-type amino acid analog such as TPI.
  • TPI refers to the wild-type polypeptide shown in SEQ ID NO: 3 (KWCFRVCYRGICY RRCR*).
  • TPAD TPAD bacteriostatic peptide
  • TPAD bacteriostatic peptide refers to all D-amino acid analogue of TPI, that is, a derivative or analog obtained after all L-type amino acids of TPI are changed to D-type amino acids. It should be understood that the term also includes analogs with similar bacteriostatic activity formed by replacing 1-3 amino acids with amino acids with similar activities.
  • a preferred TPAD is the polypeptide KWCFRVCY RGLCYRRCR* obtained after the isoleucine at position 11 in TPI is replaced by leucine (type D), as shown in (SEQ ID NO: 2).
  • TPI analogue or "TPI-derived polypeptide” (for example, “TPI D-type amino acid analogue of TPI”) includes SEQ ID NO: 1 or 2 having antibacterial activity similar to TPI or TPAD
  • the variant form of the sequence include (but are not limited to): 1-3 (usually 1-2, preferably 1) amino acid deletions, insertions and/or substitutions, and additions or additions at the C-terminus and/or N-terminus
  • One or several (usually 3 or less, preferably 2 or less, more preferably 1 or less) amino acids are deleted.
  • amino acids with similar or similar properties are substituted, the function of the protein is usually not changed.
  • adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein.
  • the term also includes the polypeptide of the present invention in monomeric and multimeric forms.
  • the term also includes linear and non-linear polypeptides (such as cyclic peptides).
  • a preferred type of active derivative means that compared with the amino acid sequence of the TPI analog or TPAD of the present invention, there are at most 3, preferably at most 2, and most preferably 1 amino acid is similar in nature. Or similar amino acids are replaced to form polypeptides. These conservative variant polypeptides are best produced by amino acid substitutions according to the following table.
  • the D-type amino acid analogs of TPAD or TPI of the present invention also include analogs thereof.
  • the difference between these analogs and the active polypeptide of the present invention may be the difference in amino acid sequence, the difference in modification form that does not affect the sequence, or both.
  • Analogs also include analogs with non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides listed above.
  • Modified (usually without changing the primary structure or sequence) forms include: chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides produced by glycosylation modifications during the synthesis and processing of the polypeptide or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine). It also includes polypeptides that have been modified to improve their resistance to proteolysis or optimize their solubility.
  • the active polypeptide of the present invention can also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base.
  • These salts include (but are not limited to) salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid Acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid or isethionic acid.
  • Other salts include: salts with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium), and in the form of esters, carbamates or other conventional "prodrugs".
  • the active polypeptide of the present invention can be a natural polypeptide (or a wild-type polypeptide), a recombinant polypeptide or a synthetic polypeptide.
  • the polypeptide of the present invention can be isolated, chemically synthesized, or recombinant.
  • the polypeptide of the present invention can be produced by conventional separation and extraction methods, or can be artificially synthesized by conventional methods, or can be produced by recombinant methods.
  • the active polypeptide of the present invention can be synthesized by solid-phase Fmoc chemistry.
  • the TPAD of the present invention can be synthesized by the method shown in flow 1;
  • the method includes the steps:
  • the sulfhydryl protecting group for cysteine at position 3 and 16 is trityl
  • the sulfhydryl protecting group for cysteine at position 7 and 12 is acetamide. methyl.
  • step (1) the resin is activated with 1:1 dichloromethane and N,N-dimethylformamide.
  • step (1) Furthermore, all 17 amino acids in step (1) adopt D-type amino acids.
  • step (3) the disulfide bond at position 3 and position 16 is constructed: air oxidation is used to take 50 mg of solid powder and dissolve it in 150 mL 0.2 M ammonium bicarbonate aqueous solution at a concentration of 0.2 mg/mL. 250mL eggplant-shaped bottle, electromagnetic stirring, react for 48h at room temperature, and use a freeze dryer to obtain a white solid powder.
  • QseC/QseB two-component system and “QseC/B two-component system” can be used interchangeably and refer to a two-component adjustment system composed of QseB and/or QseC.
  • a two-component system usually starts from To the role that foreign signal molecules are recognized by cells, they have the function of signal receiving and signal transmission.
  • QseC is a membrane-bound sensor protein with histidine kinase activity
  • QseB is a cytoplasmic response regulator.
  • pathogenic bacteria or “diseased bacteria” are used interchangeably and refer to microorganisms that can cause disease.
  • pathogenic bacteria or “diseased bacteria” refers to pathogens or diseased bacteria in the QseC/B two-component system.
  • pathogenic bacteria with the QseC/B two-component system refers to bacteria or pathogens that include the QseC/B two-component system. These bacteria or pathogens can sense and respond to environmental conditions through the QseC/B two-component system.
  • these bacteria or pathogenic bacteria include (but are not limited to): Escherichia coli, Bacillus subtilis, Enterobacter cloacae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecium , Lysobacteria (such as enzyme-producing lysobacterium), Shigella flexneri, Pseudoalteromonas, Stenotrophomonas maltophilia, or other pathogens with the QseC/B two-component system.
  • the present invention provides a combination of TPAD and QseC/QseB two-component inhibitor (such as LED209).
  • the combination provided by the present invention can greatly improve the antibacterial effect of TPAD, thereby effectively overcoming the defect of low activity of TPI or TPAD, and significantly improving their clinical application value.
  • the present invention provides a drug for efficiently killing drug-resistant disease bacteria and the application of the drug in inhibiting drug-resistant disease bacteria.
  • the drug for effectively killing drug-resistant disease bacteria includes an active combination consisting of a first active ingredient and a second active ingredient (as described in the first aspect).
  • the drug for efficiently killing drug-resistant disease bacteria provided by the present invention is composed of TPAD and LED209.
  • the drug for efficiently killing drug-resistant disease bacteria provided by the present invention is composed of TPAD and LED209;
  • the dosage of the TPAD is 2 ⁇ g/mL TPAD; the dosage of the LED209 is 5 pM LED209.
  • the present invention also provides the application of the drug for effectively killing drug-resistant disease bacteria in the inhibition of Pseudoalteromonas having resistance to multiple antibiotics.
  • the present invention also provides the application of the drug for efficiently killing drug-resistant disease bacteria in the inhibition of Stenotrophomonas maltophilia resistant to multiple antibiotics.
  • the present invention also provides the application of the drug for efficiently killing drug-resistant disease bacteria in the inhibition of Pseudomonas aeruginosa that is resistant to multiple antibiotics.
  • the present invention also provides the application of the drug for effectively killing drug-resistant disease bacteria in inhibiting microbial diseases.
  • the present invention provides an anti-wild lysobacterium drug, the anti-wild lysobacterium drug is composed of TPAD and LED209;
  • the dosage of the TPAD is 2 ⁇ g/mL TPAD; the dosage of the LED209 is 5 pM LED209.
  • the present invention also provides the application of the anti-wild lysobacterium drug in the inhibition of Pseudoalteromonas which is resistant to multiple antibiotics.
  • the present invention also provides the application of the anti-wild Rongibacillus drug in the inhibition of Stenotrophomonas maltophilia resistant to multiple antibiotics.
  • the present invention also provides a composition, which contains (a) a safe and effective amount of the active combination of the present invention; and (b) a pharmaceutically acceptable carrier or excipient.
  • the dosage of the polypeptide as the first active ingredient is usually 10 micrograms to 100 mg/dose, preferably 100 to 1000 micrograms/dose; QseC/dose as the second active ingredient
  • the dosage of the QseB inhibitor is usually 0.025 pmol-250 pmol/dose, preferably 0.25-2.5 micrograms/dose.
  • an effective dose is about 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg body weight of the active polypeptide of the present invention and an amount corresponding to the amount of active polypeptide administered to an individual per day QseC/QseB inhibitor.
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent.
  • pharmaceutical carriers that do not themselves induce the production of antibodies that are harmful to the individual receiving the composition, and do not have excessive toxicity after administration.
  • Such vectors are well known to those of ordinary skill in the art.
  • Such carriers include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, adjuvants and combinations thereof.
  • the pharmaceutically acceptable carrier in the pharmaceutical composition may contain liquids such as water, saline, glycerol and ethanol.
  • these carriers may also contain auxiliary substances, such as wetting or emulsifying agents, and pH buffering substances.
  • the therapeutic composition can be made into an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.
  • an injectable such as a liquid solution or suspension
  • it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.
  • composition of the invention can be administered by conventional routes, including (but not limited to): intramuscular, intravenous, subcutaneous, intradermal or topical administration.
  • routes including (but not limited to): intramuscular, intravenous, subcutaneous, intradermal or topical administration.
  • the objects to be prevented or treated can be animals; especially humans.
  • various dosage forms of the pharmaceutical composition can be used according to the use situation.
  • tablets, granules, capsules, pills, injections, or oral liquids can be exemplified.
  • compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a customary manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a customary manner according to the dosage form.
  • the preparation can be carried out as follows: the polypeptide of the present invention or a pharmaceutically acceptable salt thereof is dissolved in sterile water (surfactant is dissolved in sterile water) together with the basic substance, and the osmotic pressure and pH are adjusted to a physiological state. , And can optionally add appropriate pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants and thickeners, and then make them completely dissolved.
  • the pharmaceutical composition of the present invention can also be administered in the form of a sustained-release dosage form.
  • the polypeptide of the present invention or its salt can be incorporated into a pill or microcapsule with a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated.
  • the polypeptide or its salt of the present invention can also be applied by inserting an intraocular lens coated with a drug in advance.
  • sustained-release polymers ethylene-vinyl acetate copolymers, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the dosage of the polypeptide of the present invention or its pharmaceutically acceptable salt as the active ingredient can be based on the weight, age, sex, and degree of symptoms of each patient to be treated. And reasonably be determined.
  • the low-concentration TPAD of the present invention can activate the QseC/B two-component system.
  • TPAD can exert a stronger antibacterial effect (compared with drug-resistant bacteria). Therefore, the present invention can significantly enhance the antibacterial effect of TPAD by means of combination medication.
  • the combined administration of TPAD and a QseC/QsecB inhibitor can greatly improve the antibacterial activity against three multi-drug resistant bacteria. It is applied to pathogens in the QseC/B two-component system.
  • very low concentration such as 2 ⁇ g/mL or higher, such as 2-500 ⁇ g/mL
  • very low concentration such as 5pM or higher, such as 5-1000pM
  • QseC/QsecB inhibitor can completely kill drug-resistant bacteria.
  • the combination of TPAD and QseC/QsecB inhibitors can inhibit the production of pathogens such as Pseudoalteromonas and Stenotrophomonas maltophilia.
  • the composition of the present invention has lower hemolytic activity, because the combined drug has higher activity, and produces the same antibacterial effect as compared with TPAD alone.
  • the drugs used in the examples to effectively kill drug-resistant disease bacteria are 2 ⁇ g/mL TPAD and 5 pM LED209.
  • TPI-MD conformational stability
  • TPAD-MD TPAD-MD
  • 10 constellations are extracted from the last 500 ns MD trajectory at the same time interval.
  • TPAD is essentially regarded as a mirror image of TPI.
  • the structure of the TPAD extracted from the MD track (TPADM membrane-MD) in the presence of the membrane is basically the same as the structure of the TPAD in the absence of the membrane.
  • TPAD lytic activity of TPAD on liposomes composed of E. coli lipid extracts.
  • the EC50 of TPAD to 100 ⁇ M large unilamellar vesicles is 1.71 ⁇ 0.32 ⁇ M.
  • P/L is the ratio of peptide to lipid (mol/mol).
  • Tachyplesin I is a cationic ⁇ -hairpin antimicrobial peptide with broad spectrum and effective antimicrobial activity.
  • the present invention synthesizes all D-amino acid analogs of TPI, replaces Ile 11 with D-type Leu (TPAD), and determines its structure and activity.
  • TPAD has antibacterial activity equivalent to TPI on 14 bacterial strains (including 4 resistant bacteria).
  • TPAD has significantly improved stability against enzymatic degradation and reduced hemolytic activity (as shown in Figure 5), indicating that it has better therapeutic potential.
  • Limulus is a class of antimicrobial peptides found in horse crab lymphocyte granular cells.
  • Limulus I was first isolated from human red blood cells of Tachypleus tridentatus. It is an amphiphilic peptide composed of 17 residues (Figure 9A) and two disulfide bonds. The disulfide bond constrains it into an anti-parallel ⁇ -hairpin structure ( Figure 9B).
  • TPI has broad-spectrum antibacterial activity against gram-positive bacteria, gram-negative bacteria and fungi, and the MIC value is usually in the range of 3-6 ⁇ g/mL.
  • a number of studies have shown that TPI acts on both cell membranes and intracellular targets, and the cell membrane is the main target.
  • TPI binds to the membrane by interacting with the negative charge and lipopolysaccharide (LPS) distributed on the membrane surface. After binding, TPI can be translocated across the membrane through pore formation. In addition to directly interacting with the membrane, TPI also inhibits target proteins, such as intracellular esterase and 3-ketoacyl carrier protein reductase FabG, which may affect the composition and biophysical properties of the membrane.
  • LPS lipopolysaccharide
  • TPAD is a D-amino acid analog of TPI, which is formed by replacing all L-amino acids with D-amino acids and replacing Ile11 with D-Leu amino acids (blue).
  • AMP antimicrobial peptide
  • TPI is highly hemolytic to mammalian red blood cells, with a minimum hemolytic concentration (MHC) of 0.25 ⁇ g/mL, which reduces its potential therapeutic application as an antibacterial agent.
  • MHC minimum hemolytic concentration
  • the present invention designed a D-amino acid analog of TPI (called TPAD) by replacing all L amino acids with D-amino acids and replacing Ile- with D-Leu amino acids (Figure 9C). And evaluated its antibacterial activity, stability and hemolytic activity. In addition, an attempt was made to determine the mode of action by investigating changes in the expression of related proteins in bacteria that developed resistance to TPAD through analysis of the genome-wide transcription profile of L. enzymogenes YC36.
  • TPAD D-amino acid analog of TPI
  • the three-dimensional (3D) structure of TPAD was determined using NMR spectroscopy ( Figure 2, Table 3).
  • the large positive value in the secondary ⁇ H chemical shift diagram of TPAD indicates the ⁇ chain secondary structure, which is very similar to TPI ( Figure 10A).
  • the 20 lowest energy level structures are well covered, and the root mean square deviation (RMSD) of the entire skeleton is The root mean square deviation of the ⁇ chain region is (Residuals 3-16).
  • the final structure is comparable to that published by TPI.
  • both TPI and TPAD have a ⁇ -hairpin secondary structure, and TPAD is the mirror image of TPI, except for the side chain difference at position 11 ( Figure 10).
  • the present invention performs MD simulation on TPAD in the presence of the membrane.
  • TPAD 2.3 TPAD is placed parallel to the membrane surface in the starting structure. Although the RMSD of TPAD with membrane is larger, it is still smaller than There are no large fluctuations (Figure 10D), and the ⁇ -chain secondary structure is well maintained ( Figure 3). After MD, a shallow pocket is induced on the membrane surface, and TPAD is tilted to the membrane, and one end of the ⁇ chain is partially embedded in the membrane (A, B in Figure 4). The time scale of MD simulation may be too short to observe the transport process of the peptide completely embedded in the membrane, but the MD of the present invention reveals the interaction between the peptide and the membrane. L- to D-amino acid substitution is a well-known strategy to improve the stability of peptides against enzymatic degradation.
  • TPAD antibacterial activity against 14 kinds of bacteria and the clinical peptide drug Colistin (Colistin) were simultaneously evaluated as a positive control.
  • TPAD and TPI have comparable antibacterial efficacy against most bacteria (including gram-negative bacteria and gram-positive bacteria) (see Table 7 for MIC results).
  • TPAD has the same amount of cationic charge and structure as TPI, which indicates that it has a similar membrane decomposition mechanism and similar antibacterial activity as TPI.
  • the covalent element has 2 to 8 times stronger antibacterial activity than TPAD and TPI, while TPAD and TPI have a broader antibacterial activity, especially against Gram-positive Staphylococcus aureus Higher potency, more than 16 times. Enterococcus faecium ATCC 29212 and Bacillus subtilis 168 are more than 4 times higher.
  • the present invention also tested the minimum bactericidal concentration (MBC) of TPI and TPAD against selected ESKAPE pathogens. The MBC values of TPAD and TPI of all detected strains were similar, ranging from 16 to 64 ⁇ g/mL (see Table 4 for the results).
  • TPAD and TPI have comparable hemolytic activity when the concentration is ⁇ 50 ⁇ g/mL, but it is different when the concentration is >100 ⁇ g/mL (Figure 11A).
  • the hemolytic activity of TPAD reached 100 ⁇ g/mL and reached the maximum, while the hemolytic activity of TPI continued to increase with the increase in concentration ( Figure 11A). Therefore, at a concentration of >100 ⁇ g/mL, the hemolytic activity of TPAD is lower than that of TPI.
  • the MIC of TPAD to the tested bacteria does not exceed 16 ⁇ g/mL. At this concentration, TPAD only causes 10% hemolysis of human red blood cells (Figure 5).
  • TPAD hemolysis of TPAD is acceptable in vitro.
  • the present invention also tested the cytotoxicity of TPI and TPAD to normal human red blood cells. As shown in Figure 11B, TPAD is comparable to TPI, and shows negligible cytotoxicity at a concentration of ⁇ 32 ⁇ g/mL (that is, the concentration on most of the tested bacteria is significantly higher than its MIC). In contrast, both have cytotoxicity to human red blood cells >64 ⁇ g/mL.
  • TPAD 2.5 TPAD induces bacterial membrane leakage.
  • Previous microscopic observations have shown that TPI kills bacteria by acting on cell membranes and intracellular targets, of which the cell membrane is the main target.
  • the present invention uses atomic force microscope to characterize the surface morphology of LeYC36 cell membrane after TPAD is applied. As shown in Figure 12 ( Figure A and Figure B), after treatment with TPAD, the cell surface changed from smooth and intact to severe depression, and intracellular lysate leakage occurred around the bacterial cells. Further slice analysis of the cell surface morphology showed that the surface depression of LeYC36 cells treated with TPAD was significantly lower than that of the control group ( Figure 12C).
  • TPAD can destroy the cell membrane of the enzyme-producing lysobacterium LeYC36, leading to cell death, and its mechanism of action is similar to that of TPI.
  • liposomes that mimic bacterial cell membranes were used to evaluate the membrane decomposing activity of TPAD.
  • TPAD caused the leakage of unilamellar vesicles (LUV) composed of lipid extracts of Escherichia coli in a dose-dependent manner (see Figure 6).
  • LUV unilamellar vesicles
  • TPAD promotes the leakage of contents from the model membrane that mimics the bacterial membrane.
  • the research results of the present invention support the hypothesis that TPAD can induce cell membrane leakage and cause bacterial death.
  • TPAD and TPI showed a concentration-dependent activity on LeYC36.
  • concentration is less than 8 ⁇ g/mL
  • TPAD is more effective on LeYC36 than TPI.
  • concentration of TPAD reaches 4 ⁇ g/mL, it shows strong bactericidal activity, while TPI has little effect on cell survival. Then, the present invention monitored the evolution of resistance to TPAD and TPI through serial passage analysis (Figure 13).
  • the MIC of TPAD on LeYC36 increased from 4 ⁇ g/mL to 16 ⁇ g/mL (a 4-fold increase), and after the same passage, the MIC value of TPI on LeYC36 increased from 8 ⁇ g/mL to 16 ⁇ g/mL ( Increase 2 times). Therefore, compared with the enzymes induced by antibiotics in previous studies, LeYC36 only developed a low level of resistance to TPAD and TPI.
  • the results of the present invention are consistent with studies showing that TPI and TPII (limulus II) have no resistance to various bacteria or only produce low-level resistance.
  • TPAD and TPI basically have the same evolutionary characteristics of MIC. Only after the first generation of TPAD occurred, the MIC increased by 2 times. Such a small difference may be due to the higher sensitivity of LeYC36 to TPAD than TPI at low peptide concentrations.
  • the CpxR/CpxA two-component system caused Salmonella to increase the protamine ⁇ -helical peptide bombesin 2 and melittin by up-regulating the transcription of amiA and amiC.
  • the present invention found 184 genes affected by exogenous TPAD (p value ⁇ 0.005), of which 156 were up-regulated and 28 were down-regulated (PRJNA542247).
  • TPAD exogenous TPAD
  • 156 were up-regulated and 28 were down-regulated
  • Several drug efflux pumps were upregulated (Figure 1A, Figure 1B), including czcB, nodT, yceL and ftsX.
  • TPAD did not activate the drug efflux pump in the ⁇ qseB mutant strain, and the expression level of the efflux pump related protein was similar to that of the untreated wild-type strain.
  • TPAD has a stronger bactericidal effect on ⁇ qseB and ⁇ qseC mutants, which suggests that the intracellular concentration of TPAD can be effectively maintained or increased, resulting in better sterilization or inhibition. Bacterial activity.
  • the cytotoxicity of TPAD to wild-type LeYC36 is not very high, at 10 ⁇ g/mL; unexpectedly, TPAD has high cytotoxicity to the ⁇ qseB mutant LeYC36, and it has a high cytotoxicity to the ⁇ qseC mutant.
  • the cytotoxicity of type LeYC36 is also very high. At 2 ⁇ g/mL, TPAD can kill almost all ⁇ qseB and ⁇ qseC mutant strains.
  • LED209 is a recognized inhibitor of QseC.
  • TPAD and 5pM LED209 are used in combination, wild-type LeYC36 can be completely killed ( Figure 1C, Figure 1D).
  • LED209 In order to exclude the possible sterilization effect of LED209 alone, only LED209 was used as a control experiment to treat LeYC36. LED209 has no bactericidal effect ( Figure 1C, Figure 1D), indicating that the combined application can synergistically promote the bactericidal activity of TPAD.
  • TPAD inactivation/low-level expression of the drug efflux pump on the ⁇ qseB and ⁇ qseC mutants may help increase the intracellular concentration of TPAD and improve its bactericidal activity.
  • TPAD is also believed to have an additional mode of action involving direct interaction with intracellular targets. For example, it has previously been shown that TPI can inactivate intracellular esterases.
  • the intracellular target of TPAD is still unclear, and it will be interesting to determine the specific intracellular target of TPAD in the future.
  • the present invention studies whether the synergy between TPAD and LED2019 can be extended to other pathogenic bacteria with the QseC/B two-component system.
  • the combination of TPAD and LED209 can also effectively resist Pseudomonadaceae, a pathogenic bacteria that is inherently resistant to multiple antibiotics ( Figure 14).
  • the present invention also found that when used in combination with LED209, the activity of TPAD also greatly improves the efficacy against Stenotrophomonas maltophilia, which is also resistant to multiple antibiotics ( Figure 14). Therefore, through the QseC/B two-component system, the combined application strategy of TPAD and LED209 can be extended to other pathogenic bacteria.
  • TPAD may have multiple mechanisms of action.
  • TPAD can destroy the cell membrane of bacteria and cause the death of bacteria, similar to TPI.
  • TPAD triggers changes in the range of gene expression, leading to bacterial resistance to TPAD.
  • some interesting issues such as the mechanism of intracellular transport and the existence of genes that may be regulated, remain to be resolved. The clarification of these issues will help to identify new targets and further improve the bactericidal efficiency of these antimicrobial peptides.
  • TPAD is a full D amino acid analog of TPI, which can maintain the broad-spectrum effective antibacterial activity of natural peptides, but has significantly improved stability and reduced hemolysis at high concentrations. It must be noted that at lower concentrations, the hemolytic activity of TPAD and TPI are equivalent, and it is still necessary to further reduce the hemolytic activity of TPAD analogs in the future. TPAD induces bacterial resistance by activating the QseC/B two-component system, and blocking the two-component system can effectively improve the antibacterial effect of TPAD ( Figure 15).
  • Example 2 The following provides the specific methods of peptide synthesis and activity testing used in Example 2:
  • TPI and TPAD use solid-phase peptide synthesis and neutralization/2 (1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluranium hexafluorophosphate activation procedure, Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry was assembled on rink amide methylbenzylamine resin (Novabiochem).
  • the cleavage is achieved by treating with 88:5:5:2 trifluoroacetic acid, phenol, water and triisopropylsilane as scavengers at room temperature (20-25°C).
  • the trifluoroacetic acid is evaporated at low pressure in a rotary evaporator.
  • the crude peptide is purified by RP-HPLC on a Phenomenex C 18 column and used before being collected and lyophilized for oxidation.
  • the molecular weight was confirmed by electrospray mass spectrometry.
  • the four cystines in the peptide were selectively oxidized in two steps. In the first step, the unprotected cystine was added to 0.1M NH 4 HCO 3 at a concentration of 0.5 mg/mL. (pH 8-8.5), and stirred at room temperature for 24h.
  • the oxidized peptide was separated by RP-HPLC with a detection wavelength of 214nm.
  • the peptide was dissolved in a concentration of 1mg/mL.
  • the cystine protected by Acm was oxidized in the iodine solution and stirred for 30 minutes. Then ascorbic acid was added to stop the oxidation reaction, and the solution was stirred again until no color was visible. After two rounds of oxidation, the peptide was purified by preparative RP-HPLC , And use RP-HPLC and electrospray quality to verify its purity and quality (the results are shown in Table 2).
  • the minimum inhibitory concentration (MIC) is the lowest concentration of a chemical that inhibits the visible growth of bacteria.
  • the MIC of peptides against gram-negative bacteria including E. coli K-12, E. coli BAA 2469, E. coli ATCC 25923, B. subtilis 168 and E. cloacae ) BAA 1143, Gram-positive Staphylococcus aureus (S.aureus) ATCC 29213, S. Staphylococcus aureus BAA 41 and Staphylococcus aureus BAA 44, and K. pneumoniae BAA 1144, pneumonia Klebsiella BAA 2470, P. aeruginosa (P. aeruginosa) ATCC 27853, P.
  • aeruginosa BAA 2108 Acinetobacter baumannii (A. baumannii) ATCC 19606 and Enterococcus faecium (E. faecium) ATCC 29212 measurement method.
  • the experiment was carried out in 96-well plates with serial dilutions of antimicrobial peptides in the wells. Each dilution was done in duplicate. Each well contains 80 ⁇ L of medium, 10 ⁇ L of peptides and 10 ⁇ L of bacterial culture (final bacterial concentration is about 5 ⁇ 10 6 CFU/mL). Only the control of bacteria and culture medium is included to ensure the viability of bacteria and the sterility of culture medium. After incubating at 37°C for 18 hours, the absorbance was measured (see Table 7 for the results).
  • the hemolysis assay was performed using a method similar to that described previously.
  • Human red blood cells were purchased from HaemoScan (Netherlands). An aliquot of the human blood sample was washed twice with 5 mL washing buffer and centrifuged at 2500 rpm for 10 minutes at 4°C. Repeat this step twice with 5 mL dilution buffer, and resuspend the final pellet in 5 mL dilution buffer to a final concentration of 5% erythrocytes. Add 100 ⁇ L of the tested peptide to 100 ⁇ L of the diluted red blood cell suspension.
  • the hemolysis of the peptide was tested at seven concentrations (maximum concentration is 500 ⁇ g/mL, two consecutive dilutions). 100 ⁇ L of MilliQ water was added to 100 ⁇ L of red blood cell suspension as a negative control (0% hemolysis), while 100 ⁇ L of 2% Triton X-100 was added to 100 ⁇ L of red blood cell suspension as a positive control (100% hemolysis). The assay mixture was then incubated at 37°C for 1 h under slow rotation (100 rpm).
  • the sample was centrifuged at 5,000 rpm for 1 minute, and the absorbance of the supernatant was measured by spectrophotometry at 415 nm and 450 nm as reference wavelengths to quantify hemolysis (see Figure 5 and Figure 11A for the results).
  • the cytotoxicity test was performed using normal human red blood cells L02. 100 ⁇ LL02 cells at a concentration of 2 ⁇ 104 cells/mL were added to each well of a 96-well plate and cultured in RPMI-1640 medium for 24 hours. Then the cells were treated with TPI and TPAD at a gradient concentration of 2-128 ⁇ g/mL, the peptide was dissolved and diluted with RPMI-1640 medium, and an equal amount of RPMI-1640 medium was added to the control group. After 48 hours, add 20 ⁇ L of resazurin to each well and incubate for 4 hours, and then perform microplate reader detection under 544nm excitation light and 595nm absorption light. Each concentration was repeated six times at the same time (see Figure 11 for the results).
  • peptide stability As mentioned earlier, male AB human serum (Sigma Aldrich) was used for serum stability determination. The serum was centrifuged at 15000g for 15 minutes to remove lipids, and then incubated at 37°C for 10 minutes. Triplicate samples were prepared with a 1:10 peptide dilution: serum with a working peptide concentration of 20 mM, 40 ⁇ L of 20% TFA was added, and serum proteins were precipitated at 4°C.
  • the spectrum is based on the internal standard, which is 0 ppm 2,2-dimethyl-2-silylpentane-5-sulfonate (DSS).
  • DSS 2,2-dimethyl-2-silylpentane-5-sulfonate
  • the system in the NVT integrated system is gradually heated from 50K to 300K, and used The harmonic potential confines the solute atoms to their initial positions.
  • the simulation was switched to the NPT set, and the solute constraint was changed from within 100ps Gradually decreases.
  • the operation of the production MD is carried out in the NPT ensemble after a simulation time of 100ns, the pressure coupling is 1atm, and the constant temperature is 300K.
  • the MD simulation uses a time step of 2fs, and all bonds involving hydrogen atoms are maintained as using the particle grid Ewald (PME) method to handle all long-distance electrostatic interactions in the MD simulation.
  • PME particle grid Ewald
  • TPAD is located in the presence of POPE (1-palmitoyl-2-oleoyl-sn-glycerol 3-phosphoethanolamine): POPG (1-palmitoyl-2-oleoyl-sn- Glycerol-3-phosphate) in the double layer of the 3:2 mixture-(1'-rac-glycerol)), used to simulate bacterial membranes, the size is And the system uses TIP3P water molecules and Na+ and Clions to dissolve, so the total concentration of the system is 0.15M in the neutral CHARMM-GUI (http://www.charmm-gui.org).
  • the temperature of the system was gradually increased to 310K, and 500ps was balanced in the integration of NVT and NPT respectively, in which protein and lipid were affected by Force constraints.
  • Longman thermostat is used for initial heating.
  • an anisotropic Berendsen weakly coupled barostat is also used to balance the pressure. Then, cancel the restriction on the membrane and simulate the entire system for 20 ns in NPT to properly balance the membrane system.
  • the restriction on protein was gradually removed in 10 steps. After that, a production run of 600ns was carried out.
  • a Langevin thermostat is used to control the temperature, while an anisotropic Berendsen barostat is used to control the pressure. All simulations were performed using Lipid14 force field for lipids and AMBER14SB force field for proteins.
  • the MD simulation uses a time step of 2fs and uses the SHAKE algorithm to keep all bonds involving hydrogen atoms at their standard length.
  • the cut-off point of particle grid Ewald (PME) for non-bonded atom interaction is And the neighbor list is updated every 10 steps.
  • Bacterial strains, plasmids and general methods Wild-type lysobacterium and related mutants (qseC and qseB mutants) were grown in 40% strength TSB medium. E. coli strains DH5 ⁇ and S17-1 were used for bacterial mutation. Table 5 describes the detailed information of bacterial strains and plasmids used in the present invention. Perform molecular manipulations according to the previously described method. Restriction enzymes and molecular biology reagents were purchased from Takara (TaKaRa Bio Group, Japan). The PCR primers were synthesized by Zero2IPO Biotechnology Company (see Table 6 for details).
  • Bioinformatics analysis Use Primer Premier 5. Design primers for real-time PCR and genetic manipulation assays. BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) analyzed the gene sequence. Annotation and bioinformatics analysis were performed by genome sequencing and EMBOSS (European Molecular Biology Open Software Suite) (http://emboss.open-bio.org/).
  • RNA extraction, reverse transcription PCR and real-time PCR LeYC36 was cultured under different conditions (with or without 2 ⁇ g/mL TPAD treatment), and then RNA was extracted using an RNA extraction kit (OMEGA) according to the manufacturer's instructions. The RNA sample was reverse transcribed into cDNA, and real-time PCR was performed in a real-time reaction with a total reaction volume of 20 ⁇ L, containing 250 nM primers, 10 ⁇ LEva Green 2x qPCR Master Mix, 0.5 ⁇ L 10-fold diluted cDNA template, and 8.5 ⁇ L RNase-free water. 16S rRNA was used as the reference gene. Real-time PCR was performed with the StepOne real-time PCR system (AB Applied Biosystems). Design the program as described earlier.
  • RNA of L. enzymogenes YC36 was extracted with TRIzol reagent (Invitrogen) (absent or 2 ⁇ g/mL TPAD).
  • RNA transcription library was constructed using Illumina (San Diego, California) TruSeq RNA Preparation Kit. RiboZero rRNA removal kit (Epicenter) was used to remove the residual rRNA of L. enzymogenes YC36. The original paired end reads have been trimmed using SeqPrep and quality controlled by Sickle (https://github.com/jstjohn/SeqPrep and https://github.com/najoshi/sickle). Use Rockhopper (http://cs.wellesley.edu/ ⁇ btjaden/Rockhopper/) to align the clean readings.
  • the membrane was inoculated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5000, Sangon Biotech) at 37°C for 2 hours.
  • HRP horseradish peroxidase
  • a chemiluminescent substrate was added to the membrane and observed through a CCD imaging system (Bio-Rad) (see Figure 8 for the results).
  • Vesicle leakage assay prepare large unilamellar vesicles (LUV) composed of E. coli lipid extract (Avanti Polar Lipids, Inc.) filled with 5-carboxyfluorescein (Sigma-Aldrich).
  • LUV large unilamellar vesicles
  • E. coli lipid extract Alvanti Polar Lipids, Inc.
  • 5-carboxyfluorescein Sigma-Aldrich
  • 10 mM HEPES buffer 107 mM NaCl, 1 mM EDTA, pH 7.4
  • a 5-carboxyfluorescein-encapsulated LUV with a self-quenching concentration (50 mM) with a diameter of 100 nm was prepared.
  • the LUV containing 5-carboxyfluorescein was separated from the free dye on a Sephadex G75 column, and the lipid concentration of LUV was determined using Stewart analysis.

Abstract

Provided in the present invention are a medicament for effectively killing drug-resistant disease bacteria and an application thereof in inhibiting drug-resistant disease bacteria. Specifically, provided in the present invention is a composition for killing or inhibiting disease bacteria, which comprises an active combination that consists of a first active ingredient TPAD and a second active ingredient QseC/B signaling pathway inhibitor.

Description

高效杀灭耐药病害菌的药物及在抑制耐药病害菌中的应用Drugs for efficiently killing drug-resistant bacteria and their application in inhibiting drug-resistant bacteria 技术领域Technical field
本发明属于抗菌药物技术领域，具体涉及一种高效杀灭耐药病害菌的药物及在抑制耐药病害菌中的应用。The invention belongs to the technical field of antibacterial drugs, and specifically relates to a drug for efficiently killing drug-resistant disease bacteria and its application in inhibiting drug-resistant disease bacteria.
背景技术Background technique
鲎素是存在于马蹄蟹淋巴颗粒细胞中一类抗菌肽。其中的鲎素(Tachyplesin)I(TPI)首次是从三刺鲎(Tachypleus tridentatus)的干细胞中分离得到的。TPI对革兰氏阳性菌和革兰氏阴性菌等菌类具有广谱的抗菌活性。然而，后续的研究表明：TPI作为一种多肽，在血浆中的稳定性差，容易被降解，TPI在抗菌过程中很容易导致哺乳动物红细胞细胞膜破裂发生溶血现象。另外，TPI抗菌活性依然不够强，抗菌活性低于目前临床药物。这种缺点大大限制了其在临床上的应用。Limulus is a type of antimicrobial peptide that exists in horseshoe crab lymph granule cells. Among them, Tachyplesin I (TPI) was first isolated from stem cells of Tachypleus tridentatus. TPI has broad-spectrum antibacterial activity against Gram-positive bacteria and Gram-negative bacteria. However, follow-up studies have shown that as a polypeptide, TPI has poor stability in plasma and is easily degraded. TPI can easily cause mammalian red blood cell membranes to rupture and hemolysis during the antibacterial process. In addition, the antibacterial activity of TPI is still not strong enough, and the antibacterial activity is lower than current clinical drugs. This shortcoming greatly limits its clinical application.
综上所述，现有技术存在的问题是：(1)TPI在抗菌过程中活性不够强、稳定性差，很容易被降解，且溶血性强。(2)TPI/TPAD相比临床药物活性较低。此外，基于序列扫描筛选方法对TPI活性的改造主要虽能一定程度上提高活性，但提高的幅度却非常有限。To sum up, the problems in the prior art are: (1) TPI is not strong enough in the antibacterial process, has poor stability, is easily degraded, and has strong hemolysis. (2) Compared with clinical drugs, TPI/TPAD has lower activity. In addition, although the modification of TPI activity based on sequence scanning screening method can mainly improve the activity to a certain extent, the extent of improvement is very limited.
综上所述，本领域迫切需要开发一种新的具有高抗菌活性的药物。In summary, the field urgently needs to develop a new drug with high antibacterial activity.
发明内容Summary of the invention
本发明的目的就是提供一种新的具有高抗菌活性尤其是针对耐药菌也具有高抗菌活性的药物。The purpose of the present invention is to provide a new drug with high antibacterial activity, especially against drug-resistant bacteria.
在本发明的第一方面，提供了一种用于杀灭或抑制病害菌的组合物，其中，所述的组合物包括由第一活性成分和第二活性成分组成的活性组合；其中，In the first aspect of the present invention, a composition for killing or inhibiting disease bacteria is provided, wherein the composition includes an active combination consisting of a first active ingredient and a second active ingredient; wherein,
(a1)所述第一活性分为TPAD；(a1) The first activity is classified as TPAD;
其中，所述TPAD是指TPI的D型氨基酸类似物，且所述TPI为如SEQ ID NO:3所示的多肽；Wherein, the TPAD refers to a D-type amino acid analog of TPI, and the TPI is a polypeptide as shown in SEQ ID NO: 3;
Figure PCTCN2020095683-appb-000001
Figure PCTCN2020095683-appb-000001
(a2)所述第二活性成分为QseC/B信号通路抑制剂；(a2) The second active ingredient is a QseC/B signaling pathway inhibitor;
其中，所述QseC/B信号通路抑制剂是指能够抑制QseC、QseB，或其组合的活性物质。Wherein, the QseC/B signaling pathway inhibitor refers to an active substance that can inhibit QseC, QseB, or a combination thereof.
在另一优选例中，所述的组合物还包括：(b)药学上可接受的载体。In another preferred embodiment, the composition further includes: (b) a pharmaceutically acceptable carrier.
在另一优选例中，所述TPI的D型氨基酸类似物是指将TPI多肽序列中的L型氨基酸相应替换为D型氨基酸并任选地对该多肽序列中的氨基酸进行替换和/或修饰后得到的类似物。In another preferred example, the D-type amino acid analog of TPI refers to correspondingly replacing the L-type amino acid in the TPI polypeptide sequence with the D-type amino acid and optionally replacing and/or modifying the amino acid in the polypeptide sequence The analogue obtained later.
在另一优选例中，TPAD为下式结构所示的多肽：In another preferred example, TPAD is a polypeptide represented by the following structure:
Figure PCTCN2020095683-appb-000002
Figure PCTCN2020095683-appb-000002
式中，Where
X为Leu、Ile、Val、Ala或Leu的非天然氨基酸类似物；X is an unnatural amino acid analog of Leu, Ile, Val, Ala or Leu;
并且各氨基酸均为D型氨基酸。And each amino acid is D-type amino acid.
在另一优选例中，X为Leu、Ile、Val、或Ala。In another preferred example, X is Leu, Ile, Val, or Ala.
在另一优选例中，TPAD第3位的Cys和第16位的Cys形成二硫键，且第7位的Cys和第12位的Cys形成二硫键。In another preferred example, Cys at position 3 and Cys at position 16 of TPAD form a disulfide bond, and Cys at position 7 and Cys at position 12 form a disulfide bond.
在另一优选例中，TPAD为如SEQ ID NO:2所示的多肽，且其中各氨基酸均为D型氨基酸；In another preferred embodiment, TPAD is a polypeptide as shown in SEQ ID NO: 2, and each amino acid in it is a D-type amino acid;
Figure PCTCN2020095683-appb-000003
Figure PCTCN2020095683-appb-000003
在另一优选例中，所述QseC/B信号通路抑制剂为如式I所示的LED209，或其药学上可接受的盐；In another preferred embodiment, the QseC/B signaling pathway inhibitor is LED209 as shown in formula I, or a pharmaceutically acceptable salt thereof;
Figure PCTCN2020095683-appb-000004
Figure PCTCN2020095683-appb-000004
在另一优选例中，在所述组合物中，第一活性成分和第二活性成分的用量比(mg/pmol)为1:100～100:1。In another preferred example, in the composition, the dosage ratio (mg/pmol) of the first active ingredient and the second active ingredient is 1:100-100:1.
在另一优选例中，在所述组合物中，第一活性成分和第二活性成分的用量比(mg/pmol)为1:10～1:0.5。In another preferred example, in the composition, the dosage ratio (mg/pmol) of the first active ingredient and the second active ingredient is 1:10 to 1:0.5.
在另一优选例中，在所述组合物中，第一活性成分和第二活性成分的用量比为2-500μg/mL:5-1000pM。In another preferred example, in the composition, the dosage ratio of the first active ingredient and the second active ingredient is 2-500 μg/mL:5-1000 pM.
在另一优选例中，第一活性成分和第二活性成分的总含量为0.1～99.9wt％，以组合物的总质量计。In another preferred example, the total content of the first active ingredient and the second active ingredient is 0.1 to 99.9% by weight, based on the total mass of the composition.
在另一优选例中，所述组合物包括由TPAD和LED209组成的活性组合；其中，TPAD和LED209的用量比(mg/pmol)为2:5。In another preferred embodiment, the composition includes an active combination composed of TPAD and LED209; wherein the dosage ratio (mg/pmol) of TPAD and LED209 is 2:5.
在另一优选例中，所述TPAD的用量为≥2μg/mL；和/或所述LED209的用量为≥5pM。In another preferred embodiment, the dosage of the TPAD is ≥2μg/mL; and/or the dosage of the LED209 is ≥5pM.
在另一优选例中，所述TPAD的用量为2-500μg/mL；和/或所述LED209的用量为≥5-1000pM。In another preferred embodiment, the dosage of the TPAD is 2-500 μg/mL; and/or the dosage of the LED209 is ≥ 5-1000 pM.
在另一优选例中，所述TPAD的用量为2μg/mLTPAD；所述LED209的用量为5pM LED209。In another preferred embodiment, the dosage of the TPAD is 2 μg/mLTPAD; the dosage of the LED209 is 5 pM LED209.
在本发明的第二方面，提供了一种如第一方面所述的组合物在制备用于治疗和/或预防由病害菌引起的疾病的药物中的用途。In the second aspect of the present invention, there is provided a use of the composition as described in the first aspect in the preparation of a medicament for treating and/or preventing diseases caused by disease bacteria.
在另一优选例中，所述药物的剂型为口服给药剂型或非口服给药剂型。In another preferred embodiment, the dosage form of the drug is an oral dosage form or a non-oral dosage form.
在另一优选例中，所述的口服给药剂型选自下组：片剂、散剂、颗粒剂或胶囊剂，或乳剂或糖浆剂。In another preferred embodiment, the oral administration dosage form is selected from the group consisting of tablets, powders, granules or capsules, or emulsions or syrups.
在另一优选例中，所述的非口服给药剂型选自下组：注射剂、针剂。In another preferred embodiment, the non-oral administration dosage form is selected from the group consisting of injections and injections.
在另一优选例中，所述病害菌为具有QseC/B双组分***的细菌。In another preferred example, the diseased bacteria are bacteria with a QseC/B two-component system.
在另一优选例中，所述病害菌为耐药菌。In another preferred embodiment, the disease bacteria are resistant bacteria.
在另一优选例中，所述病害菌选自下组：大肠杆菌、枯草芽孢杆菌、阴沟肠杆菌、金黄色葡萄球菌、肺炎克雷伯菌、铜绿假单胞菌、鲍曼不动杆菌、屎肠球菌、溶杆菌(较佳地，产酶溶杆菌)、福氏志贺菌、假交替单胞菌、嗜麦芽寡养单胞菌，或其组合。In another preferred embodiment, the disease bacteria are selected from the group consisting of Escherichia coli, Bacillus subtilis, Enterobacter cloacae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecium, Lysobacterium (preferably, Enzyme-producing Lysobacterium), Shigella flexneri, Pseudoalteromonas, Stenotrophomonas maltophilia, or a combination thereof.
在另一优选例中，所述病害菌选自下组：大肠杆菌K-12、大肠杆菌BAA 2469、大肠杆菌ATCC 25923、枯草芽孢杆菌168、阴沟肠杆菌BAA 1143、金黄色葡萄球菌ATCC 29213、金黄色葡萄球菌BAA 41、金黄色葡萄球菌BAA 44、肺炎克雷伯菌BAA 1144、肺炎克雷伯氏菌BAA 2470、铜绿假单胞菌ATCC 27853、铜绿假单胞菌BAA 2108、鲍曼不动杆菌ATCC 19606、屎肠球菌ATCC 29212、产酶溶杆菌YC36、福氏志贺菌ATCC 29903，或其组合。In another preferred embodiment, the disease bacteria are selected from the following group: Escherichia coli K-12, Escherichia coli BAA 2469, Escherichia coli ATCC 25923, Bacillus subtilis 168, Enterobacter cloacae BAA 1143, Staphylococcus aureus ATCC 29213, Staphylococcus aureus BAA 41, Staphylococcus aureus BAA 44, Klebsiella pneumoniae BAA 1144, Klebsiella pneumoniae BAA 2470, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa BAA 2108, Bauman no Kinetobacter ATCC 19606, Enterococcus faecium ATCC 29212, Enzyme-producing Lysobacterium YC36, Shigella flexneri ATCC 29903, or a combination thereof.
在另一优选例中，所述药物通过抑制和/或杀灭病害菌从而治疗和/或预防 由致病菌引起的疾病。In another preferred example, the drug can treat and/or prevent diseases caused by pathogenic bacteria by inhibiting and/or killing disease bacteria.
在另一优选例中，所述由病害菌引起的疾病为细菌感染。In another preferred embodiment, the disease caused by diseased bacteria is a bacterial infection.
在另一优选例中，所述由病害菌引起的疾病包括：呼吸道感染、肺部感染(如肺炎)、皮肤感染。In another preferred example, the diseases caused by disease bacteria include: respiratory tract infections, lung infections (such as pneumonia), and skin infections.
在本发明的第三方面，提供了一种用于杀灭或抑制病害菌的药物组合，所述的药物组合包括：In the third aspect of the present invention, a drug combination for killing or inhibiting disease bacteria is provided, and the drug combination includes:
(i)包含第一活性成分的组合物或药物；和(ii)包含第二活性成分的组合物或药物；(i) A composition or medicine containing a first active ingredient; and (ii) A composition or medicine containing a second active ingredient;
其中，所述第一活性成分和第二活性成分如第一方面中定义。Wherein, the first active ingredient and the second active ingredient are as defined in the first aspect.
在本发明的第四方面，提供了一种抑制或杀灭病害菌的方法，包括步骤：使病害菌与如第一方面所述的组合物接触；或者使病害菌与第一活性成分和第二活性成分接触，从而抑制或杀灭病害菌；In the fourth aspect of the present invention, a method for inhibiting or killing disease bacteria is provided, which includes the steps of: contacting the disease bacteria with the composition as described in the first aspect; or bringing the disease bacteria with the first active ingredient and the first active ingredient. Two active ingredients contact, thereby inhibiting or killing disease bacteria;
其中，所述第一活性成分和第二活性成分如第一方面中定义。Wherein, the first active ingredient and the second active ingredient are as defined in the first aspect.
在另一优选例中，所述的方法是体外非治疗性的。In another preferred embodiment, the method is non-therapeutic in vitro.
在另一优选例中，所述的方法是治疗性的或预防性的。In another preferred embodiment, the method is therapeutic or preventive.
在另一优选例中，使病害菌同时与第一活性成分和第二活性成分接触。In another preferred embodiment, the diseased bacteria are brought into contact with the first active ingredient and the second active ingredient at the same time.
在另一优选例中，使病害菌分别与第一活性成分和第二活性成分接触。In another preferred embodiment, the diseased bacteria are contacted with the first active ingredient and the second active ingredient respectively.
在另一优选例中，使病害菌依次与第二活性成分接触和第一活性成分接触。In another preferred embodiment, the diseased bacteria are sequentially contacted with the second active ingredient and the first active ingredient.
在另一优选例中，第一活性成分的用量为≥2μg/mL。In another preferred embodiment, the amount of the first active ingredient is ≥2 μg/mL.
在另一优选例中，第一活性成分的用量为2-500μg/mL。In another preferred embodiment, the amount of the first active ingredient is 2-500 μg/mL.
在另一优选例中，第二活性成分的用量为≥5pM。In another preferred embodiment, the amount of the second active ingredient is ≥5pM.
在另一优选例中，第二活性成分的用量为5-1000pM。In another preferred embodiment, the amount of the second active ingredient is 5-1000 pM.
在本发明的第五方面，提供了一种治疗和/或预防由细菌引起的疾病，其中，所述的方法包括步骤：In the fifth aspect of the present invention, there is provided a treatment and/or prevention of diseases caused by bacteria, wherein the method includes the steps:
向需要的对象施用如第一方面所述的组合物；或者向需要的对象施用如第 三方面所述的药物组合；或者向需要的对象施用第一活性成分或包含第一活性成分的组合物或药物和第二活性成分或包含第一活性成分的组合物或药物。Administer the composition as described in the first aspect to a subject in need; or administer the pharmaceutical combination as described in the third aspect to a subject in need; or administer the first active ingredient or a composition containing the first active ingredient to a subject in need Or a medicine and a second active ingredient or a composition or medicine containing the first active ingredient.
在另一优选例中，所述对象包括人或非人哺乳动物。In another preferred embodiment, the subject includes humans or non-human mammals.
在另一优选例中，向需要的对象同时施用第一活性成分或包含第一活性成分的组合物或药物和第二活性成分或包含第一活性成分的组合物或药物。In another preferred example, the first active ingredient or the composition or medicine containing the first active ingredient and the second active ingredient or the composition or medicine containing the first active ingredient are simultaneously administered to a subject in need.
在另一优选例中，向需要的对象间隔施用第一活性成分或包含第一活性成分的组合物或药物和第二活性成分或包含第一活性成分的组合物或药物。In another preferred example, the first active ingredient or the composition or medicine containing the first active ingredient and the second active ingredient or the composition or medicine containing the first active ingredient are administered to a subject in need at intervals.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.
附图说明Description of the drawings
图1是本发明实施例提供的TPAD与LED209的联合用药对野生型或者qseC或者qseB基因敲除的细菌的抗菌活性示意图，其显示了本发明实施例提供的固有耐药菌产酶溶杆菌YC36(LeYC36)的全基因组转录谱以及细菌在亚致死浓度下对TPAD的抗性机制；各图中：WT表示野生型菌株，ΔqseB表示qseB基因敲除的菌株，ΔqseC表示qseC基因敲除的菌株。其中，(A)热图显示药物外排泵基因的相对转录水平，热图下方的比例表示相对表达水平的倍数变化；(B)LeYC36中药物外排泵基因相对表达水平的实时PCR分析。开始培养LeYC36；在培养开始时，将2μg/mL TPAD添加到浓度为40％的TSB培养基中；(C)和(D)TPAD在qseC/B突变体中的杀菌作用，LED209是公认的QseC抑制剂；所示结果具有生物学重复的代表性；误差线代表三个重复的标准偏差。Figure 1 is a schematic diagram of the antibacterial activity of the combination of TPAD and LED209 provided by an embodiment of the present invention on wild-type or qseC or qseB gene knockout bacteria, which shows the inherently drug-resistant bacteria provided by the embodiment of the present invention, the enzyme-producing Lysobacterium YC36 The genome-wide transcription profile of (LeYC36) and the resistance mechanism of bacteria to TPAD at sublethal concentrations; in each figure: WT represents the wild-type strain, ΔqseB represents the qseB gene knockout strain, and ΔqseC represents the qseC gene knockout strain. Among them, (A) the heat map shows the relative transcription level of the drug efflux pump gene, and the ratio below the heat map represents the fold change of the relative expression level; (B) the real-time PCR analysis of the relative expression level of the drug efflux pump gene in LeYC36. Start culturing LeYC36; at the beginning of the culture, add 2μg/mL TPAD to the TSB medium at a concentration of 40%; (C) and (D) the bactericidal effect of TPAD in qseC/B mutants, LED209 is recognized as QseC Inhibitor; the results shown are representative of biological replicates; error bars represent the standard deviation of three replicates.
图2是本发明实施例提供的TPAD的NMR结构示意图。Fig. 2 is a schematic diagram of the NMR structure of TPAD provided by an embodiment of the present invention.
图3是本发明实施例提供的肽构象稳定性的分子动力学模拟研究示意图。Fig. 3 is a schematic diagram of molecular dynamics simulation study of peptide conformational stability provided by an embodiment of the present invention.
图4是本发明实施例提供的在600ns MD模拟后，TPAD在膜表面的取向示意图。4 is a schematic diagram of the orientation of TPAD on the film surface after 600 ns MD simulation provided by an embodiment of the present invention.
图5是本发明实施例提供的TPAD对人红细胞的溶血活性示意图。Figure 5 is a schematic diagram of the hemolytic activity of TPAD on human erythrocytes provided by an embodiment of the present invention.
图6是本发明实施例提供的TPAD对由大肠杆菌脂质提取物组成的脂质体的裂解活性示意图。Fig. 6 is a schematic diagram of the lysis activity of TPAD on liposomes composed of E. coli lipid extract provided by an embodiment of the present invention.
图7是本发明实施例提供的TPAD和TPI对肝癌细胞存活的影响示意图。Fig. 7 is a schematic diagram of the influence of TPAD and TPI on the survival of liver cancer cells provided by an embodiment of the present invention.
图8是本发明实施例提供的TPAD处理L.enzymogenes YC36后，关键蛋白QseB表达的Western blotting分析示意图。Fig. 8 is a schematic diagram of Western blotting analysis of the expression of the key protein QseB after L. enzymogenes YC36 is treated by TPAD according to an embodiment of the present invention.
图9是本发明实施例提供的TPI和TPAD的结构和序列示意图；图中：(A)和(B)分别为TPI的结构(PDB ID：1wo0)和序列；(C)TPAD的序列。9 is a schematic diagram of the structure and sequence of TPI and TPAD provided by an embodiment of the present invention; in the figure: (A) and (B) are the structure (PDB ID: 1wo0) and sequence of TPI, respectively; (C) the sequence of TPAD.
图10是本发明实施例提供的TPI和TPAD的结构和稳定性示意图；图中：(A)鲎素I(TPI)和TPAD之间的Hα二级化学位移比较。TPI的化学位移数据来自BMRB登录号21044；(B)TPI(PDB ID：1wo0)；(C)TPAD的NMR溶液结构(本发明)；(D)在从起始构象开始的600ns分子动力学模拟过程中，肽主链的RMSD演变；(E)TPI(黑色，下方曲线)和TPAD(深灰，上方曲线)在人血清中6小时内的稳定性，进行三次平行实验，误差线为平均值的标准误差。Fig. 10 is a schematic diagram of the structure and stability of TPI and TPAD provided by an embodiment of the present invention; in the figure: (A) a comparison of Hα secondary chemical shifts between limulin I (TPI) and TPAD. The chemical shift data of TPI comes from BMRB accession number 21044; (B) TPI (PDB ID: 1wo0); (C) NMR solution structure of TPAD (this invention); (D) 600ns molecular dynamics simulation from the initial conformation During the process, the RMSD evolution of the peptide backbone; (E) the stability of TPI (black, lower curve) and TPAD (dark gray, upper curve) in human serum within 6 hours, three parallel experiments were performed, the error bar is the average The standard error of.
图11是本发明实施例提供的TPI和TPAD对人红细胞的溶血活性和人正常人红细胞L02的细胞毒性结果示意图；图中：(A)通过在415nm和450nm作为参考波长的光谱法测量上清液的吸光度来获得溶血百分比，重复三次进行测量，误差线为平均值的标准误差；(B)TPI和TPAD对人正常人红细胞L02的细胞毒性。使用对照组作为参考计算细胞存活百分比。假定对照组的细胞存活率为100％。Figure 11 is a schematic diagram of the hemolytic activity of TPI and TPAD on human erythrocytes and the cytotoxicity of human normal human erythrocytes L02 provided by an embodiment of the present invention; Figure: (A) The supernatant was measured by spectroscopy at 415nm and 450nm as reference wavelengths The absorbance of the liquid was used to obtain the percentage of hemolysis, and the measurement was repeated three times. The error bar is the standard error of the mean; (B) Cytotoxicity of TPI and TPAD to normal human erythrocytes L02. Use the control group as a reference to calculate the percentage of cell survival. Assume that the cell survival rate of the control group is 100%.
图12是本发明实施例提供的用TPAD处理的产酶溶杆菌(Lysobacter enzymogenes)YC36的原子力显微镜观察细胞形态示意图；图中：(A)未用TPAD处理的LeYC36细胞的形态；(B)TPAD处理之后的LeYC36细胞的形态；(C)TPAD未处理或处理的LeYC36细胞的表面形态的切面分析图。Figure 12 is a schematic diagram of the atomic force microscope observation of cell morphology of Lysobacter enzymogenes YC36 treated with TPAD according to an embodiment of the present invention; in the figure: (A) the morphology of LeYC36 cells without TPAD treatment; (B) TPAD The morphology of LeYC36 cells after treatment; (C) A cross-sectional analysis image of the surface morphology of LeYC36 cells untreated or treated with TPAD.
图13是本发明实施例提供的的细胞连续传代培养示意图；由(A)TPAD和(B)TPI诱导连续传代的LeYC36最低抑菌浓度(MIC)的演变；细菌菌株每传代一次，TPAD/TPI的浓度增加2μg/mL。Figure 13 is a schematic diagram of the continuous subculture of cells provided by the embodiment of the present invention; the evolution of the LeYC36 minimum inhibitory concentration (MIC) induced by (A) TPAD and (B) TPI for continuous passage; each passage of bacterial strains, TPAD/TPI Increase the concentration of 2μg/mL.
图14显示了本发明实施例提供的LED209使TPAD能够更有效地杀死假单胞菌和嗜麦芽单胞菌的嗜麦芽细胞。Figure 14 shows that the LED209 provided by the embodiment of the present invention enables TPAD to more effectively kill the maltophilic cells of Pseudomonas and Maltophilia.
图15显示了本发明实施例提供的亚致死剂量下细菌对TPAD的耐药机制。Figure 15 shows the resistance mechanism of bacteria to TPAD at the sublethal dose provided in the embodiment of the present invention.
具体实施方式Detailed ways
发明人经过长期而深入地研究，意外地发现TPI的类似物(如TPAD)对ΔqseB突变型和ΔqseC突变型细胞的细胞毒力显著优于对非突变型细胞的细胞毒力，且TPI的类似物在极低浓度能够杀死几乎所有的ΔqseB和ΔqseC突变株。 发明人还意外地发现当TPI的类似物与QsecB/QsecC抑制剂联用时对非突变株同样表现出类似与TPI的类似物对ΔqseB和ΔqseC突变株类似的活性，TPI的类似物和QsecB/QsecC抑制剂联用可协同促进TPI的类似物的抗菌或杀菌活性。基于此，发明人完成了本发明。After long-term and in-depth research, the inventors unexpectedly found that the cytotoxicity of TPI analogs (such as TPAD) to ΔqseB mutant and ΔqseC mutant cells was significantly better than that of non-mutant cells, and TPI was similar The substance can kill almost all ΔqseB and ΔqseC mutant strains at very low concentrations. The inventors also unexpectedly found that when TPI analogs are used in combination with QsecB/QsecC inhibitors, they also show similar activities to non-mutant strains as TPI analogs have similar activities to ΔqseB and ΔqseC mutants, TPI analogs and QsecB/QsecC The combination of inhibitors can synergistically promote the antibacterial or bactericidal activity of TPI analogs. Based on this, the inventor completed the present invention.
术语the term
如本文所用，术语“QseC/B信号通路抑制剂”、“QseC/QseB抑制剂”或“QseC/QseB双组分抑制剂”可以互换使用，是指能够抑制QseC和/或QseB的活性物质(例如，小分子化合物或多肽等)。如本文所用，术语“QseC抑制剂”是指能够抑制QseC的活性物质(小分子化合物或多肽等)。如本文所用，术语“QseB抑制剂”是指能够抑制QseB的活性物质(小分子化合物或多肽等)。As used herein, the terms "QseC/B signaling pathway inhibitor", "QseC/QseB inhibitor" or "QseC/QseB two-component inhibitor" can be used interchangeably and refer to active substances that can inhibit QseC and/or QseB (For example, small molecule compounds or polypeptides, etc.). As used herein, the term "QseC inhibitor" refers to an active substance (small molecule compound or polypeptide, etc.) capable of inhibiting QseC. As used herein, the term "QseB inhibitor" refers to an active substance (small molecule compound or polypeptide, etc.) capable of inhibiting QseB.
在本文中，因此，本文所用的“含有”，“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”；“主要由……构成”、“基本上由……构成”和“由……构成”属于“含有”、“具有”或“包括”的下位概念。In this article, therefore, "containing", "having" or "including" as used herein includes "including", "mainly composed of", "essentially composed of", and "consisting of" ; "Mainly composed of", "basically composed of" and "consisted of" belong to the subordinate concepts of "containing", "having" or "including".
如本文所用，“LED209”为如式I所示的化合物，LED209可市售获的或根据现有技术合成。As used herein, "LED209" is a compound represented by formula I, and LED209 can be commercially available or synthesized according to the prior art.
Figure PCTCN2020095683-appb-000005
Figure PCTCN2020095683-appb-000005
活性多肽Active peptide
在本发明中，活性多肽是指从三刺鲎(Tachypleus tridentatus)的干细胞中分离得到的具有广谱抗菌活性的抗菌肽(即TPI)的D型氨基酸类似物(TPAD)；在本发明的优选实施方式中活性多肽是指如TPI的全D型氨基酸类似物。In the present invention, the active polypeptide refers to the D-type amino acid analog (TPAD) of an antibacterial peptide (ie TPI) with broad-spectrum antibacterial activity isolated from stem cells of Tachypleus tridentatus; preferred in the present invention In the embodiment, the active polypeptide refers to a full D-type amino acid analog such as TPI.
如本文所用，术语“TPI”是指如SEQ ID NO:3所示的野生型多肽(KWCFRVCYRGICY RRCR*)。As used herein, the term "TPI" refers to the wild-type polypeptide shown in SEQ ID NO: 3 (KWCFRVCYRGICY RRCR*).
如本文所用，术语“TPAD”或“TPAD抑菌肽”是指all D-amino acid analogue of TPI，即将TPI的L型氨基酸全部改为D型氨基酸后所获得的衍生物或类似物。应理解，该术语还包括将1-3个氨基酸用活性类似的氨基酸替换所形成的具有类似抑菌活性的类似物。一种优选的TPAD是TPI中第11位的异亮氨酸被亮氨酸(D型)所取代后，获得的多肽KWCFRVCY RGLCYRRCR*，(SEQ ID NO:2)所示的多肽。As used herein, the term "TPAD" or "TPAD bacteriostatic peptide" refers to all D-amino acid analogue of TPI, that is, a derivative or analog obtained after all L-type amino acids of TPI are changed to D-type amino acids. It should be understood that the term also includes analogs with similar bacteriostatic activity formed by replacing 1-3 amino acids with amino acids with similar activities. A preferred TPAD is the polypeptide KWCFRVCY RGLCYRRCR* obtained after the isoleucine at position 11 in TPI is replaced by leucine (type D), as shown in (SEQ ID NO: 2).
如本文所用，术语“TPI类似物”或“TPI衍生多肽”(例如，“TPI的TPI的D型氨基酸类似物”)包括具有类似TPI或TPAD的抗菌活性的SEQ ID NO：1或2所示序列的变异形式。这些变异形式包括(但并不限于)：1-3个(通常为1-2个，较佳地1个)氨基酸的缺失、***和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为3个以内，较佳地为2个以内，更佳地为1个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外，所述术语还包括单体和多聚体形式本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。As used herein, the term "TPI analogue" or "TPI-derived polypeptide" (for example, "TPI D-type amino acid analogue of TPI") includes SEQ ID NO: 1 or 2 having antibacterial activity similar to TPI or TPAD The variant form of the sequence. These variant forms include (but are not limited to): 1-3 (usually 1-2, preferably 1) amino acid deletions, insertions and/or substitutions, and additions or additions at the C-terminus and/or N-terminus One or several (usually 3 or less, preferably 2 or less, more preferably 1 or less) amino acids are deleted. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein. In addition, the term also includes the polypeptide of the present invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).
一类优选的活性衍生物(或衍生多肽)是指与本发明的TPI类似物或TPAD的氨基酸序列相比，有至多3个，较佳地至多2个，最佳地1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据下表进行氨基酸替换而产生。A preferred type of active derivative (or derivative polypeptide) means that compared with the amino acid sequence of the TPI analog or TPAD of the present invention, there are at most 3, preferably at most 2, and most preferably 1 amino acid is similar in nature. Or similar amino acids are replaced to form polypeptides. These conservative variant polypeptides are best produced by amino acid substitutions according to the following table.
最初的残基Initial residue 代表性的取代Representative substitution 优选的取代Preferred substitution
Ala(A)Ala(A) Val；Leu；IleVal; Leu; Ile ValVal
Arg(R)Arg(R) Lys；Gln；AsnLys; Gln; Asn LysLys
Asn(N)Asn(N) Gln；His；Lys；ArgGln; His; Lys; Arg GlnGln
Asp(D)Asp(D) GluGlu GluGlu
Cys(C)Cys(C) SerSer SerSer
Gln(Q)Gln(Q) AsnAsn AsnAsn
Glu(E)Glu(E) AspAsp AspAsp
Gly(G)Gly(G) Pro；AlaPro; Ala AlaAla
His(H)His(H) Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg ArgArg
Ile(I)Ile(I) Leu；Val；Met；Ala；PheLeu; Val; Met; Ala; Phe LeuLeu
Leu(L)Leu(L) Ile；Val；Met；Ala；PheIle; Val; Met; Ala; Phe IleIle
Lys(K)Lys(K) Arg；Gln；AsnArg; Gln; Asn ArgArg
Met(M)Met(M) Leu；Phe；IleLeu; Phe; Ile LeuLeu
Phe(F)Phe(F) Leu；Val；Ile；Ala；TyrLeu; Val; Ile; Ala; Tyr LeuLeu
Pro(P)Pro(P) AlaAla AlaAla
Ser(S)Ser(S) ThrThr ThrThr
Thr(T)Thr(T) SerSer SerSer
Trp(W)Trp(W) Tyr；PheTyr; Phe TyrTyr
Tyr(Y)Tyr(Y) Trp；Phe；Thr；SerTrp; Phe; Thr; Ser PhePhe
Val(V)Val(V) Ile；Leu；Met；Phe；AlaIle; Leu; Met; Phe; Ala LeuLeu
本发明的TPAD或TPI的D型氨基酸类似物还包括其类似物。这些类似物与本发明活性多肽(例如TPAD)的差别可以是氨基酸序列上的差异，也可以是不影 响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述列举的代表性的多肽。The D-type amino acid analogs of TPAD or TPI of the present invention also include analogs thereof. The difference between these analogs and the active polypeptide of the present invention (e.g., TPAD) may be the difference in amino acid sequence, the difference in modification form that does not affect the sequence, or both. Analogs also include analogs with non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides listed above.
修饰(通常不改变一级结构或序列)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。Modified (usually without changing the primary structure or sequence) forms include: chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides produced by glycosylation modifications during the synthesis and processing of the polypeptide or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine). It also includes polypeptides that have been modified to improve their resistance to proteolysis or optimize their solubility.
本发明的活性多肽还可以以由药学上或生理学可接受的酸或碱衍生的盐形式使用。这些盐包括(但不限于)与如下酸形成的盐：氢氯酸、氢溴酸、硫酸、柠檬酸、酒石酸、磷酸、乳酸、丙酮酸、乙酸、琥珀酸、草酸、富马酸、马来酸、草酰乙酸、甲磺酸、乙磺酸、苯磺酸或羟乙磺酸。其他盐包括：与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐，以及以酯、氨基甲酸酯或其他常规的“前体药物”的形式。The active polypeptide of the present invention can also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base. These salts include (but are not limited to) salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid Acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid or isethionic acid. Other salts include: salts with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium), and in the form of esters, carbamates or other conventional "prodrugs".
制备方法Preparation
本发明活性多肽可以是天然多肽(或野生型多肽)、重组多肽或合成多肽。本发明的多肽可以是分离的、化学合成的，或重组的。相应地，本发明多肽可用常规分离提取方法、或可用常规方法人工合成，也可用重组方法生产。优选地，本发明的活性多肽可通过固相Fmoc化学合成。The active polypeptide of the present invention can be a natural polypeptide (or a wild-type polypeptide), a recombinant polypeptide or a synthetic polypeptide. The polypeptide of the present invention can be isolated, chemically synthesized, or recombinant. Correspondingly, the polypeptide of the present invention can be produced by conventional separation and extraction methods, or can be artificially synthesized by conventional methods, or can be produced by recombinant methods. Preferably, the active polypeptide of the present invention can be synthesized by solid-phase Fmoc chemistry.
例如，本发明的TPAD可通过如流程1所示的方法合成；For example, the TPAD of the present invention can be synthesized by the method shown in flow 1;
流程1Process 1
Figure PCTCN2020095683-appb-000006
Figure PCTCN2020095683-appb-000006
Figure PCTCN2020095683-appb-000007
Figure PCTCN2020095683-appb-000007
在一个具体实施例中，所述方法包括步骤：In a specific embodiment, the method includes the steps:
(1)将树脂用二氯甲烷、N，N-二甲基甲酰胺进行活化，后遵循固相合成方法得到直链肽KWCFRVCYRGLCYRRCR*；(1) Activate the resin with dichloromethane and N,N-dimethylformamide, and then follow the solid phase synthesis method to obtain the linear peptide KWCFRVCYRGLCYRRCR*;
(2)使用三氟乙酸、水、苯酚和三异丙基硅烷的混合溶液对树脂进行切割，旋蒸除去三氟乙酸，加入冰***，有白色固体析出，离心后的固体加水溶解，使用冻干机对其冻干，得到固体粉末；(2) Use a mixed solution of trifluoroacetic acid, water, phenol and triisopropylsilane to cut the resin, remove the trifluoroacetic acid by rotary evaporation, add glacial ether, and a white solid precipitates out. The solid after centrifugation is dissolved in water. It is freeze-dried by a dryer to obtain a solid powder;
(3)构建其中的两对二硫键，3位，16位半胱氨酸选用的巯基保护基为三苯甲基，7位和12位的半胱氨酸选用的巯基保护基为乙酰胺甲基。(3) To construct two pairs of disulfide bonds, the sulfhydryl protecting group for cysteine at position 3 and 16 is trityl, and the sulfhydryl protecting group for cysteine at position 7 and 12 is acetamide. methyl.
进一步地，步骤(1)中用1：1的二氯甲烷、N，N-二甲基甲酰胺对树脂进行活化。Further, in step (1), the resin is activated with 1:1 dichloromethane and N,N-dimethylformamide.
进一步地，步骤(1)中17个氨基酸全部采用D型氨基酸。Furthermore, all 17 amino acids in step (1) adopt D-type amino acids.
进一步地，步骤(2)中三氟乙酸：水：苯酚：三异丙基硅烷＝88：5：5：2。Further, in step (2), trifluoroacetic acid: water: phenol: triisopropylsilane=88:5:5:2.
进一步地，步骤(3)中对3位16位的二硫键进行构建：采用空气氧化法，取50mg的固体粉末，以0.2mg/mL的浓度溶解在150mL 0.2M碳酸氢铵水溶液中，于250mL茄形瓶，电磁搅拌，室温条件下反应48h，使用冻干机得到白色固体粉末。Furthermore, in step (3), the disulfide bond at position 3 and position 16 is constructed: air oxidation is used to take 50 mg of solid powder and dissolve it in 150 mL 0.2 M ammonium bicarbonate aqueous solution at a concentration of 0.2 mg/mL. 250mL eggplant-shaped bottle, electromagnetic stirring, react for 48h at room temperature, and use a freeze dryer to obtain a white solid powder.
进一步地，步骤(3)中对7位12位的二硫键进行构建：称取10mg白色固体粉末溶于反应溶剂中，其中水：乙腈：TFA＝5：5：0.01，V/V，反应溶剂中加入3mL碘/乙腈溶液，使溶液保持发黄的状态；于50mL茄形瓶，室温搅拌反应30分钟后，加入抗坏血酸水溶液，混匀，使溶液呈无色澄清，冻干后得到最终产物白色固体粉末TPAD。进一步地，碘/乙腈溶液为5mg/mL，抗坏血酸 水溶液5mg/mL。Further, in step (3), the disulfide bond at position 7 and position 12 is constructed: weigh 10 mg of white solid powder and dissolve it in the reaction solvent, where water: acetonitrile: TFA = 5: 5: 0.01, V/V, react Add 3mL iodine/acetonitrile solution to the solvent to keep the solution yellow; in a 50mL eggplant-shaped flask, stir at room temperature for 30 minutes, add ascorbic acid aqueous solution, mix well, make the solution colorless and clear, and freeze-dry to obtain the final product White solid powder TPAD. Further, the iodine/acetonitrile solution was 5 mg/mL, and the ascorbic acid aqueous solution was 5 mg/mL.
QseC/B双组分***及存在QseC/B双组分***的病原菌QseC/B two-component system and pathogens with QseC/B two-component system
如本文所用，“QseC/QseB双组分***”和“QseC/B双组分***”可以互换使用，是指由QseB和/或QseC组成的双组分调节***，双组分***通常起到外源信号分子被细胞识别的作用，具有信号接收功能和信号传递功能。QseC是具有组氨酸激酶活性的膜结合传感器蛋白，QseB是细胞质反应调节器。As used herein, "QseC/QseB two-component system" and "QseC/B two-component system" can be used interchangeably and refer to a two-component adjustment system composed of QseB and/or QseC. A two-component system usually starts from To the role that foreign signal molecules are recognized by cells, they have the function of signal receiving and signal transmission. QseC is a membrane-bound sensor protein with histidine kinase activity, and QseB is a cytoplasmic response regulator.
如本文所用，术语“病原菌”或“病害菌”可以互换使用，是指能够引起疾病的微生物。在优选实施方案中，“病原菌”或“病害菌”是指存在QseC/B双组分***的病原菌或病害菌。As used herein, the terms "pathogenic bacteria" or "diseased bacteria" are used interchangeably and refer to microorganisms that can cause disease. In a preferred embodiment, "pathogenic bacteria" or "diseased bacteria" refers to pathogens or diseased bacteria in the QseC/B two-component system.
如本文所用，“存在QseC/B双组分***的病原菌”是指包括QseC/B双组分***的细菌或病原菌，这些细菌或病原菌能够通过QseC/B双组分***感知和响应环境条件的变化，这些细菌或病原菌包括(但不限于)：大肠杆菌、枯草芽孢杆菌、阴沟肠杆菌、金黄色葡萄球菌、肺炎克雷伯菌、铜绿假单胞菌、鲍曼不动杆菌、屎肠球菌、溶杆菌(如产酶溶杆菌)、福氏志贺菌、假交替单胞菌、嗜麦芽寡养单胞菌、或其他具有QseC/B双组分***的病原菌。As used herein, "pathogenic bacteria with the QseC/B two-component system" refers to bacteria or pathogens that include the QseC/B two-component system. These bacteria or pathogens can sense and respond to environmental conditions through the QseC/B two-component system. Change, these bacteria or pathogenic bacteria include (but are not limited to): Escherichia coli, Bacillus subtilis, Enterobacter cloacae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecium , Lysobacteria (such as enzyme-producing lysobacterium), Shigella flexneri, Pseudoalteromonas, Stenotrophomonas maltophilia, or other pathogens with the QseC/B two-component system.
TPI类似物(TPAD)与QseC/QseB双组分抑制剂的联用Combination of TPI analog (TPAD) and QseC/QseB two-component inhibitor
TPI或TPAD的抗菌活性相比已有的临床药物依然比较弱，因此，限制了TPI或TPAD的临床应用。本发明针对有技术存在的问题(如活性较低)，提供了一种TPAD与QseC/QseB双组分抑制剂(如LED209)的联用。本发明提供的这种联用可大幅度提高TPAD的抗菌效果，从而有效克服TPI或TPAD的活性较低的缺陷，并显著提高它们的临床应用价值。The antibacterial activity of TPI or TPAD is still relatively weak compared with existing clinical drugs, therefore, the clinical application of TPI or TPAD is limited. In view of the technical problems (such as low activity), the present invention provides a combination of TPAD and QseC/QseB two-component inhibitor (such as LED209). The combination provided by the present invention can greatly improve the antibacterial effect of TPAD, thereby effectively overcoming the defect of low activity of TPI or TPAD, and significantly improving their clinical application value.
典型地，本发明提供了一种高效杀灭耐药病害菌的药物及该药物在抑制耐药病害菌中的应用。Typically, the present invention provides a drug for efficiently killing drug-resistant disease bacteria and the application of the drug in inhibiting drug-resistant disease bacteria.
在一个具体实施方案中，本发明提供的高效杀灭耐药病害菌的药物包括由第一活性成分和第二活性成分组成的活性组合(如第一方面所述)。In a specific embodiment, the drug for effectively killing drug-resistant disease bacteria provided by the present invention includes an active combination consisting of a first active ingredient and a second active ingredient (as described in the first aspect).
在另一个具体实施方案中，本发明提供的高效杀灭耐药病害菌的药物由TPAD和LED209组成。In another specific embodiment, the drug for efficiently killing drug-resistant disease bacteria provided by the present invention is composed of TPAD and LED209.
在另一个具体实施方案中，本发明提供的高效杀灭耐药病害菌的药物由 TPAD和LED209组成；In another specific embodiment, the drug for efficiently killing drug-resistant disease bacteria provided by the present invention is composed of TPAD and LED209;
所述TPAD的用量为2μg/mL TPAD；所述LED209的用量为5pM LED209。The dosage of the TPAD is 2 μg/mL TPAD; the dosage of the LED209 is 5 pM LED209.
在另一个具体实施方案中，本发明还提供了所述高效杀灭耐药病害菌的药物在具有对多种抗生素具有耐药作用的假交替单胞菌抑制中的应用。In another specific embodiment, the present invention also provides the application of the drug for effectively killing drug-resistant disease bacteria in the inhibition of Pseudoalteromonas having resistance to multiple antibiotics.
在另一个具体实施方案中，本发明还提供了所述高效杀灭耐药病害菌的药物在对多种抗生素具有耐药作用的嗜麦芽寡养单胞菌抑制中的应用。In another specific embodiment, the present invention also provides the application of the drug for efficiently killing drug-resistant disease bacteria in the inhibition of Stenotrophomonas maltophilia resistant to multiple antibiotics.
在另一个具体实施方案中，本发明还提供了所述高效杀灭耐药病害菌的药物在对多种抗生素具有耐药作用的铜绿假单胞菌抑制中的应用。In another specific embodiment, the present invention also provides the application of the drug for efficiently killing drug-resistant disease bacteria in the inhibition of Pseudomonas aeruginosa that is resistant to multiple antibiotics.
在另一个具体实施方案中，本发明还提供了所述高效杀灭耐药病害菌的药物在抑制微生物病害中的应用。In another specific embodiment, the present invention also provides the application of the drug for effectively killing drug-resistant disease bacteria in inhibiting microbial diseases.
在一个具体实施方案中，本发明提供了一种抗野生溶杆菌的药物，所述抗野生溶杆菌的药物由TPAD和LED209组成；In a specific embodiment, the present invention provides an anti-wild lysobacterium drug, the anti-wild lysobacterium drug is composed of TPAD and LED209;
所述TPAD的用量为2μg/mL TPAD；所述LED209的用量为5pM LED209。The dosage of the TPAD is 2 μg/mL TPAD; the dosage of the LED209 is 5 pM LED209.
在另一个具体实施方案中，本发明还提供了所述抗野生溶杆菌的药物在具有对多种抗生素具有耐药作用的假交替单胞菌抑制中的应用。In another specific embodiment, the present invention also provides the application of the anti-wild lysobacterium drug in the inhibition of Pseudoalteromonas which is resistant to multiple antibiotics.
在另一个具体实施方案中，本发明还提供了所述抗野生荣杆菌的药物在对多种抗生素具有耐药作用的嗜麦芽寡养单胞菌抑制中的应用。In another specific embodiment, the present invention also provides the application of the anti-wild Rongibacillus drug in the inhibition of Stenotrophomonas maltophilia resistant to multiple antibiotics.
药物组合物和施用方法Pharmaceutical composition and method of administration
另一方面，本发明还提供了一种组合物，它含有(a)安全有效量的本发明的活性组合；以及(b)药学上可接受的载体或赋形剂。在本发明中，作为第一活性成分的多肽(活性多肽，例如TPAD)的用量通常为10微克-100毫克/剂，较佳地为100-1000微克/剂；作为第二活性成分的QseC/QseB抑制剂的用量通常为0.025pmol-250pmol/剂，较佳地为0.25-2.5微克/剂。On the other hand, the present invention also provides a composition, which contains (a) a safe and effective amount of the active combination of the present invention; and (b) a pharmaceutically acceptable carrier or excipient. In the present invention, the dosage of the polypeptide as the first active ingredient (active polypeptide, such as TPAD) is usually 10 micrograms to 100 mg/dose, preferably 100 to 1000 micrograms/dose; QseC/dose as the second active ingredient The dosage of the QseB inhibitor is usually 0.025 pmol-250 pmol/dose, preferably 0.25-2.5 micrograms/dose.
为了本发明的目的，有效的剂量为每天给予个体约0.01毫克/千克至50毫克/千克，较佳地0.05毫克/千克至10毫克/千克体重的本发明的活性多肽以及对应于活性多肽量的QseC/QseB抑制剂。For the purpose of the present invention, an effective dose is about 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg body weight of the active polypeptide of the present invention and an amount corresponding to the amount of active polypeptide administered to an individual per day QseC/QseB inhibitor.
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本 领域普通技术人员所熟知的。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂及其组合。The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent. The term refers to pharmaceutical carriers that do not themselves induce the production of antibodies that are harmful to the individual receiving the composition, and do not have excessive toxicity after administration. These vectors are well known to those of ordinary skill in the art. Such carriers include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, adjuvants and combinations thereof.
药物组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。The pharmaceutically acceptable carrier in the pharmaceutical composition may contain liquids such as water, saline, glycerol and ethanol. In addition, these carriers may also contain auxiliary substances, such as wetting or emulsifying agents, and pH buffering substances.
通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。Generally, the therapeutic composition can be made into an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.
一旦配成本发明的组合物，可将其通过常规途径进行给药，其中包括(但并不限于)：肌内、静脉内、皮下、皮内或局部给药。待预防或治疗的对象可以是动物；尤其是人。Once the composition of the invention is formulated, it can be administered by conventional routes, including (but not limited to): intramuscular, intravenous, subcutaneous, intradermal or topical administration. The objects to be prevented or treated can be animals; especially humans.
当本发明的药物组合物被用于实际治疗时，可根据使用情况而采用各种不同剂型的药物组合物。较佳地，可以例举的有片剂、颗粒剂、胶囊、丸剂、注射剂、或口服液。When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the use situation. Preferably, tablets, granules, capsules, pills, injections, or oral liquids can be exemplified.
这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制，并且偶尔添加合适的药物添加剂，如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型用惯常方式进行。These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a customary manner according to the dosage form.
例如，配制可这样进行：将本发明的多肽或其药学上可接受的盐与基本物质一起溶解于无菌水(在无菌水中溶解有表面活性剂)中，调节渗透压和酸碱度至生理状态，并可任意地加入合适的药物添加剂如防腐剂、稳定剂、缓冲剂、等渗剂、抗氧化剂和增粘剂，然后使其完全溶解。For example, the preparation can be carried out as follows: the polypeptide of the present invention or a pharmaceutically acceptable salt thereof is dissolved in sterile water (surfactant is dissolved in sterile water) together with the basic substance, and the osmotic pressure and pH are adjusted to a physiological state. , And can optionally add appropriate pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants and thickeners, and then make them completely dissolved.
本发明的药物组合物还可以缓释剂形式给药。例如，本发明的多肽或其盐可被掺入以缓释聚合物为载体的药丸或微囊中，然后将该药丸或微囊通过手术植入待治疗的组织。此外，本发明的多肽或其盐还可通过***预先涂有药物的眼内透镜而得以应用。作为缓释聚合物的例子，可例举的有乙烯-乙烯基乙酸酯共聚物、聚羟基甲基丙烯酸酯(polyhydrometaacrylate)、聚丙烯酰胺、聚乙烯吡咯烷酮、甲基纤维素、乳酸聚合物、乳酸-乙醇酸共聚物等，较佳地可例举的是可生物降解的聚合物如乳酸聚合物和乳酸-乙醇酸共聚物。The pharmaceutical composition of the present invention can also be administered in the form of a sustained-release dosage form. For example, the polypeptide of the present invention or its salt can be incorporated into a pill or microcapsule with a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated. In addition, the polypeptide or its salt of the present invention can also be applied by inserting an intraocular lens coated with a drug in advance. As examples of sustained-release polymers, ethylene-vinyl acetate copolymers, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
当本发明的药物组合物被用于实际治疗时，作为活性成分的本发明的多肽或其药学上可接受的盐的剂量，可根据待治疗的每个病人的体重、年龄、性别、症状程度而合理地加以确定。When the pharmaceutical composition of the present invention is used for actual treatment, the dosage of the polypeptide of the present invention or its pharmaceutically acceptable salt as the active ingredient can be based on the weight, age, sex, and degree of symptoms of each patient to be treated. And reasonably be determined.
本发明的主要优点包括：The main advantages of the present invention include:
(1)本发明的低浓度的TPAD能够激活QseC/B双组分***。而在qseC或qseB基因敲除的细菌中，TPAD可以发挥更强的抗菌效果(与耐药病害菌相比)。因此，本发明通过联合用药的方式，可显著增强TPAD的抗菌效果。(1) The low-concentration TPAD of the present invention can activate the QseC/B two-component system. In bacteria knocked out of qseC or qseB gene, TPAD can exert a stronger antibacterial effect (compared with drug-resistant bacteria). Therefore, the present invention can significantly enhance the antibacterial effect of TPAD by means of combination medication.
(2)本发明中，将TPAD与QseC/QsecB抑制剂(如LED209)进行联合给药能够大大提高对三种多重耐药菌的抗菌活性。应用于存在QseC/B双组分***的病原菌。(2) In the present invention, the combined administration of TPAD and a QseC/QsecB inhibitor (such as LED209) can greatly improve the antibacterial activity against three multi-drug resistant bacteria. It is applied to pathogens in the QseC/B two-component system.
(3)本发明的联用有效克服了TPI或TPAD的活性较低的固有缺陷，提高其临床应用价值；联合QseC/QseB双组份抑制剂如LED209用药的技术方案，以大幅度提高TPAD的抗菌效果。(3) The combination of the present invention effectively overcomes the inherent shortcomings of low activity of TPI or TPAD, and improves its clinical application value; combined with the technical solution of QseC/QseB two-component inhibitors such as LED209 medication, to greatly improve TPAD Antibacterial effect.
(4)本发明中，非常低浓度(如2μg/mL或更高，如2-500μg/mL)TPAD与非常低浓度(如5pM或更高，例如5-1000pM)的QseC/QsecB抑制剂(如LED209)的联合用药，能够完全杀死耐药病害菌。TPAD与QseC/QsecB抑制剂(如LED209)的联合用药能够对具有对多种抗生素具有耐药作用的病原菌如假交替单胞菌产生和嗜麦芽寡养单胞菌抑制作用。(4) In the present invention, very low concentration (such as 2μg/mL or higher, such as 2-500μg/mL) TPAD and very low concentration (such as 5pM or higher, such as 5-1000pM) QseC/QsecB inhibitor ( Such as the combination of LED209) can completely kill drug-resistant bacteria. The combination of TPAD and QseC/QsecB inhibitors (such as LED209) can inhibit the production of pathogens such as Pseudoalteromonas and Stenotrophomonas maltophilia.
(5)本发明的组合物具有更低的溶血活性，因为组合用药活性更高，与单独使用TPAD相比，产生相同抗菌效果，使用的TPAD会越少，产生的溶血副作用也会相应变小。(5) The composition of the present invention has lower hemolytic activity, because the combined drug has higher activity, and produces the same antibacterial effect as compared with TPAD alone. The less TPAD used, the hemolytic side effects will be correspondingly smaller. .
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples usually follow the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are percentages by weight and parts by weight.
实施例中所用的高效杀灭耐药病害菌的药物为2μg/mL TPAD和5pM LED209。The drugs used in the examples to effectively kill drug-resistant disease bacteria are 2 μg/mL TPAD and 5 pM LED209.
实施例1Example 1
本实验通过联合应用TPI类似物和QseC/B信号通路抑制剂增强了对多药耐药细菌的活性；通过TPI和TPAD的质谱和RP-HPLC分析，TPAD和TPI的计算分子量与实验值一致。如图2所示，TPAD的NMR结构，显示了20种 最小能量构象。TPAD(A)中的结构旋转180 °导致(B)中的取向。 In this experiment, the combined application of TPI analogs and QseC/B signaling pathway inhibitors enhanced the activity against multidrug-resistant bacteria; through mass spectrometry and RP-HPLC analysis of TPI and TPAD, the calculated molecular weights of TPAD and TPI were consistent with the experimental values. As shown in Figure 2, the NMR structure of TPAD shows 20 minimum energy conformations. Rotation of the structure in TPAD (A) by 180 ° results in the orientation in (B).
如图3所示，肽构象稳定性的分子动力学模拟研究。为了研究TPI(TPI-MD)和TPAD(TPAD-MD)的构象稳定性，进行了600ns的MD模拟。在每个***中，以相同时间间隔从后500ns MD轨迹中提取10帧构象。TPAD本质上被视为TPI的镜像。在存在膜的情况下从MD轨迹(TPADM膜-MD)提取的TPAD的结构与在不存在膜的情况下的TPAD的结构基本相同。As shown in Figure 3, the molecular dynamics simulation study of peptide conformational stability. In order to study the conformational stability of TPI (TPI-MD) and TPAD (TPAD-MD), a 600ns MD simulation was performed. In each system, 10 constellations are extracted from the last 500 ns MD trajectory at the same time interval. TPAD is essentially regarded as a mirror image of TPI. The structure of the TPAD extracted from the MD track (TPADM membrane-MD) in the presence of the membrane is basically the same as the structure of the TPAD in the absence of the membrane.
如图4所示，在600ns MD模拟后，TPAD在膜表面的取向。β链结构倾斜至膜表面，其中一个末端部分嵌入袋中。带正电荷的残基与脂质的磷酸基形成广泛的静电相互作用。As shown in Figure 4, after 600 ns MD simulation, the orientation of TPAD on the film surface. The β chain structure is inclined to the surface of the film, and one of the end portions is embedded in the bag. The positively charged residues form a wide range of electrostatic interactions with the phosphate groups of lipids.
如图5所示，TPAD对人红细胞的溶血活性。As shown in Figure 5, the hemolytic activity of TPAD on human red blood cells.
如图6所示，TPAD对由大肠杆菌脂质提取物组成的脂质体的裂解活性。TPAD对100μM大单层囊泡的EC50为1.71±0.32μM。P/L是肽与脂质的比率(摩尔/摩尔)。As shown in Figure 6, the lytic activity of TPAD on liposomes composed of E. coli lipid extracts. The EC50 of TPAD to 100μM large unilamellar vesicles is 1.71±0.32μM. P/L is the ratio of peptide to lipid (mol/mol).
如图7所示，TPAD和TPI对肝癌细胞存活的影响。显示的结果是生物学重复的代表。As shown in Figure 7, the effects of TPAD and TPI on the survival of liver cancer cells. The results shown are representative of biological duplication.
如图8所示，关键蛋白QseB的Western blotting分析。对添加2μg/ml TPAD处理后和未用TPAD处理的LeYC36菌株进行免疫印迹分析QseB的表达。As shown in Figure 8, Western blotting analysis of the key protein QseB. Western blot analysis of QseB expression was performed on the LeYC36 strain treated with 2μg/ml TPAD and without TPAD.
Western blot结果显示，TPAD处理后，qseB表达上调，与qPCR和转录组结果一致。Western blot results showed that qseB expression was up-regulated after TPAD treatment, which was consistent with qPCR and transcriptome results.
表1(A)制备型RP HPLC和(B)分析性RP HPLC的方案Table 1 (A) Preparative RP HPLC and (B) Analytical RP HPLC scheme
Figure PCTCN2020095683-appb-000008
Figure PCTCN2020095683-appb-000008
表2合成TPI和TPAD的理论质量(Da)和测定分子质量(Da)Table 2 Theoretical mass (Da) and measured molecular mass (Da) of synthetic TPI and TPAD
Figure PCTCN2020095683-appb-000009
Figure PCTCN2020095683-appb-000009
表3 TPAD结构的统计分析Table 3 Statistical analysis of TPAD structure
Figure PCTCN2020095683-appb-000010
Figure PCTCN2020095683-appb-000010
所有统计数据均以平均值±SD给出。All statistical data are given as mean ± SD.
表5本发明中使用的细菌菌株和质粒Table 5 Bacterial strains and plasmids used in the present invention
Figure PCTCN2020095683-appb-000011
Figure PCTCN2020095683-appb-000011
Figure PCTCN2020095683-appb-000012
Figure PCTCN2020095683-appb-000012
表6，本发明中使用的引物Table 6. Primers used in the present invention
Figure PCTCN2020095683-appb-000013
Figure PCTCN2020095683-appb-000013
实施例2Example 2
1、本实验通过联合应用TPI类似物和QseC/B信号通路抑制剂增强了对多药耐药细菌的活性。Tachyplesin I(TPI)是一种阳离子β-发夹式抗菌肽，具有广谱，有效的抗菌活性。本发明合成了TPI的所有D-氨基酸类似物，Ile 11位 以D型Leu替换(TPAD)，并确定了其结构和活性。TPAD在14种细菌菌株(包括4种耐药细菌)上具有与TPI相当的抗菌活性。重要的是，与TPI相比，TPAD具有显著提高的抗酶促降解稳定性和降低的溶血活性(如图5所示)，表明它具有更好的治疗潜力。使用低浓度的TPAD诱导细菌耐药性会激活QseC/B双组分***。删除该***导致TPAD活性至少提高了五倍，并且TPAD与QseC/B抑制剂LED209的组合使用显着增强了对三类多药耐药细菌的杀菌效果。1. In this experiment, the combined application of TPI analogs and QseC/B signaling pathway inhibitors enhanced the activity against multidrug-resistant bacteria. Tachyplesin I (TPI) is a cationic β-hairpin antimicrobial peptide with broad spectrum and effective antimicrobial activity. The present invention synthesizes all D-amino acid analogs of TPI, replaces Ile 11 with D-type Leu (TPAD), and determines its structure and activity. TPAD has antibacterial activity equivalent to TPI on 14 bacterial strains (including 4 resistant bacteria). Importantly, compared with TPI, TPAD has significantly improved stability against enzymatic degradation and reduced hemolytic activity (as shown in Figure 5), indicating that it has better therapeutic potential. Using low concentrations of TPAD to induce bacterial resistance will activate the QseC/B two-component system. The deletion of this system resulted in at least a five-fold increase in TPAD activity, and the combined use of TPAD and QseC/B inhibitor LED209 significantly enhanced the bactericidal effect against three types of multi-drug resistant bacteria.
鲎素是存在于horse蟹淋巴细胞颗粒细胞中的一类抗菌肽。鲎素I(TPI)首先从三刺鲎(Tachypleus tridentatus)人红细胞中分离得到，是一种由17个残基(图9A)和两个二硫键组成的两亲肽。二硫键键约束它成反平行的β-发夹结构(图9B)。TPI对革兰氏阳性菌，革兰氏阴性菌和真菌具有广谱抗菌活性，MIC值通常在3–6μg/mL范围内。多项研究表明，TPI既作用于细胞膜又作用于细胞内靶标，而细胞膜是主要靶标。TPI通过与负电荷以及分布在膜表面的脂多糖(LPS)相互作用而与膜结合。结合后，TPI可通过孔形成而跨膜移位。除了直接与膜相互作用外，TPI还抑制靶蛋白，例如细胞内酯酶和3-酮酰基载体蛋白还原酶FabG，它们可能会影响膜的组成和生物物理特性。Limulus is a class of antimicrobial peptides found in horse crab lymphocyte granular cells. Limulus I (TPI) was first isolated from human red blood cells of Tachypleus tridentatus. It is an amphiphilic peptide composed of 17 residues (Figure 9A) and two disulfide bonds. The disulfide bond constrains it into an anti-parallel β-hairpin structure (Figure 9B). TPI has broad-spectrum antibacterial activity against gram-positive bacteria, gram-negative bacteria and fungi, and the MIC value is usually in the range of 3-6μg/mL. A number of studies have shown that TPI acts on both cell membranes and intracellular targets, and the cell membrane is the main target. TPI binds to the membrane by interacting with the negative charge and lipopolysaccharide (LPS) distributed on the membrane surface. After binding, TPI can be translocated across the membrane through pore formation. In addition to directly interacting with the membrane, TPI also inhibits target proteins, such as intracellular esterase and 3-ketoacyl carrier protein reductase FabG, which may affect the composition and biophysical properties of the membrane.
图9(A)中的“补丁”分别显示了来自带正电荷的残基Lys和Arg的氨基或胍基分布。TPAD是TPI的D-氨基酸类似物，其通过用D-氨基酸替换所有L-氨基酸并用D-Leu氨基酸(蓝色)取代Ile11形成。由于其强大的抗菌活性谱，TPI是一种有吸引力的抗菌肽(AMP)药物。但是，TPI对哺乳动物的红细胞具有高度的溶血性，最低溶血浓度(MHC)为0.25μg/mL，降低了其作为抗菌剂的潜在治疗应用。作为多肽它也容易被酶降解，进一步降低了其临床潜力。在这里，本发明设计了TPI的D-氨基酸类似物(称为TPAD)，方法是将所有L氨基酸替换为D-氨基酸，并用D-Leu氨基酸替换Ile-(图9C)。并评估了其抗菌活性，稳定性和溶血活性。此外，试图通过调查细菌中相关蛋白表达的变化来确定其作用模式，这些细菌通过产酶溶杆菌(L.enzymogenes)YC36的全基因组转录谱分析而对TPAD产生抗性。The "patches" in Figure 9(A) show the distribution of amino groups or guanidine groups from the positively charged residues Lys and Arg, respectively. TPAD is a D-amino acid analog of TPI, which is formed by replacing all L-amino acids with D-amino acids and replacing Ile11 with D-Leu amino acids (blue). Due to its powerful spectrum of antibacterial activity, TPI is an attractive antimicrobial peptide (AMP) drug. However, TPI is highly hemolytic to mammalian red blood cells, with a minimum hemolytic concentration (MHC) of 0.25 μg/mL, which reduces its potential therapeutic application as an antibacterial agent. As a polypeptide, it is also easily degraded by enzymes, further reducing its clinical potential. Here, the present invention designed a D-amino acid analog of TPI (called TPAD) by replacing all L amino acids with D-amino acids and replacing Ile- with D-Leu amino acids (Figure 9C). And evaluated its antibacterial activity, stability and hemolytic activity. In addition, an attempt was made to determine the mode of action by investigating changes in the expression of related proteins in bacteria that developed resistance to TPAD through analysis of the genome-wide transcription profile of L. enzymogenes YC36.
2、结果2. Results
2.1使用固相Fmoc化学合成TPI及其D-氨基酸类似物TPAD，并使用表1(A)中所述的方法通过RP-HPLC纯化，检测波长为214nm。分别使用电喷雾质谱仪和RP-HPLC验证了它们的质量和纯度(≥95％)(结果见表2)。2.1 Use solid-phase Fmoc to chemically synthesize TPI and its D-amino acid analogue TPAD, and use the method described in Table 1(A) to purify by RP-HPLC with a detection wavelength of 214nm. Using electrospray mass spectrometer and RP-HPLC to verify their quality and purity (≥95%) (the results are shown in Table 2).
使用NMR光谱法确定TPAD的三维(3D)结构(图2，表3)。TPAD的二级 αH化学位移图中的大正值表示β链二级结构，与TPI的相似性很高(图10A)。20个最低能级结构很好地覆盖，整个骨架的均方根偏差(RMSD)为
Figure PCTCN2020095683-appb-000014
β链区域的均方根偏差为
Figure PCTCN2020095683-appb-000015
(残差3-16)。最终结构与TPI发布的结构相当。如图10(B和C)所示，TPI和TPAD都具有β-发夹二级结构，并且TPAD是TPI的镜像，除了位置11处的侧链差异(图10)。TPI的Ile-11残基被TPAD中较便宜的D-Leu替代，因为D-Ile和D-Leu具有相似的理化特性。在TPI和TPAD上都进行了分子动力学模拟，以研究它们的构象稳定性。在600ns的分子动力学模拟过程中，两种肽的骨架RMSD在相似的模拟条件下均具有可比性，表明两种肽的构象稳定性均相似(图10D，图3)。 The three-dimensional (3D) structure of TPAD was determined using NMR spectroscopy (Figure 2, Table 3). The large positive value in the secondary αH chemical shift diagram of TPAD indicates the β chain secondary structure, which is very similar to TPI (Figure 10A). The 20 lowest energy level structures are well covered, and the root mean square deviation (RMSD) of the entire skeleton is
Figure PCTCN2020095683-appb-000014
The root mean square deviation of the β chain region is
Figure PCTCN2020095683-appb-000015
(Residuals 3-16). The final structure is comparable to that published by TPI. As shown in Figure 10 (B and C), both TPI and TPAD have a β-hairpin secondary structure, and TPAD is the mirror image of TPI, except for the side chain difference at position 11 (Figure 10). The Ile-11 residue of TPI was replaced by the cheaper D-Leu in TPAD because D-Ile and D-Leu have similar physical and chemical properties. Molecular dynamics simulations were performed on both TPI and TPAD to study their conformational stability. In the 600ns molecular dynamics simulation process, the backbone RMSD of the two peptides were comparable under similar simulated conditions, indicating that the conformational stability of the two peptides were similar (Figure 10D, Figure 3).
此外，本发明在膜存在下对TPAD进行了MD模拟。In addition, the present invention performs MD simulation on TPAD in the presence of the membrane.
2.2 TPAD在起始结构中平行于膜表面放置。尽管带有膜的TPAD的RMSD较大，但在600ns MD后，其仍小于
Figure PCTCN2020095683-appb-000016
没有大的波动(图10D)，并且β链二级结构得到了很好的维护(图3)。MD后，在膜表面诱发一个浅口袋，并且TPAD倾斜至膜，β链的一端部分嵌入膜中(图4的A，B)。MD模拟的时间尺度可能太短，无法观察到肽完全嵌入膜的转运过程，但本发明的MD揭示了肽与膜之间的相互作用。L-至D-氨基酸取代是提高肽抵抗酶促降解的稳定性的众所周知的策略。与野生型TPI相比，TPAD的血浆稳定性确实得到了显着改善(图10E)。2小时后，存在超过80％的TPAD，而只有20％的TPI。6小时后，剩下20％的TPAD，而所有TPI均降解。因此，L-至D-氨基酸取代显着改善了体外肽的稳定性。 2.2 TPAD is placed parallel to the membrane surface in the starting structure. Although the RMSD of TPAD with membrane is larger, it is still smaller than
Figure PCTCN2020095683-appb-000016
There are no large fluctuations (Figure 10D), and the β-chain secondary structure is well maintained (Figure 3). After MD, a shallow pocket is induced on the membrane surface, and TPAD is tilted to the membrane, and one end of the β chain is partially embedded in the membrane (A, B in Figure 4). The time scale of MD simulation may be too short to observe the transport process of the peptide completely embedded in the membrane, but the MD of the present invention reveals the interaction between the peptide and the membrane. L- to D-amino acid substitution is a well-known strategy to improve the stability of peptides against enzymatic degradation. Compared with wild-type TPI, the plasma stability of TPAD has indeed been significantly improved (Figure 10E). After 2 hours, there was more than 80% of TPAD and only 20% of TPI. After 6 hours, 20% of TPAD was left, and all TPI was degraded. Therefore, the L-to D-amino acid substitution significantly improved the stability of the peptide in vitro.
2.3抗菌活性测定。TPAD和TPI对14种细菌的抗菌活性与临床使用的肽类药物大肠粘菌素(Colistin)作为阳性对照同时进行了评估。TPAD和TPI对大多数细菌(包括革兰氏阴性菌和革兰氏阳性菌)具有相当的抗菌效力(MIC结果见表7)。TPAD具有与TPI相同数量的阳离子电荷和结构，这表明它具有与TPI相似的膜分解作用机理和相似的抗菌活性。对于所有测试的革兰氏阴性菌，共价素具有比TPAD和TPI强大的抗菌活性2到8倍，而TPAD和TPI则具有更广泛的抗菌活性，尤其对革兰氏阳性金黄色葡萄球菌具有更高的效力，超过16倍。屎肠球菌ATCC 29212和抗枯草芽孢杆菌168的4倍以上。本发明还测试了TPI和TPAD对所选ESKAPE病原体的最低杀菌浓度(MBC)。所有检测到的菌株的TPAD和TPI的MBC值相似，范围为16至64μg/mL(结果见表4)。2.3 Determination of antibacterial activity. The antibacterial activity of TPAD and TPI against 14 kinds of bacteria and the clinical peptide drug Colistin (Colistin) were simultaneously evaluated as a positive control. TPAD and TPI have comparable antibacterial efficacy against most bacteria (including gram-negative bacteria and gram-positive bacteria) (see Table 7 for MIC results). TPAD has the same amount of cationic charge and structure as TPI, which indicates that it has a similar membrane decomposition mechanism and similar antibacterial activity as TPI. For all gram-negative bacteria tested, the covalent element has 2 to 8 times stronger antibacterial activity than TPAD and TPI, while TPAD and TPI have a broader antibacterial activity, especially against Gram-positive Staphylococcus aureus Higher potency, more than 16 times. Enterococcus faecium ATCC 29212 and Bacillus subtilis 168 are more than 4 times higher. The present invention also tested the minimum bactericidal concentration (MBC) of TPI and TPAD against selected ESKAPE pathogens. The MBC values of TPAD and TPI of all detected strains were similar, ranging from 16 to 64 μg/mL (see Table 4 for the results).
表4 TPI和TPAD对ESKAPE病原体的MBCTable 4 MBC of TPI and TPAD to ESKAPE pathogen
Figure PCTCN2020095683-appb-000017
Figure PCTCN2020095683-appb-000017
表7 TPI和TPAD对ESKAPE病原体的MICTable 7 MIC of TPI and TPAD to ESKAPE pathogen
Figure PCTCN2020095683-appb-000018
Figure PCTCN2020095683-appb-000018
注：MIC进行了两次重复测试。Note: MIC has been tested twice.
2.4溶血活性和细胞毒性测定，TPAD和TPI在浓度<50μg/mL时具有可比的溶血活性，但在浓度>100μg/mL时却有所不同(图11A)。TPAD的溶血活性达到100μg/mL达到最大值，而TPI的溶血活性随着浓度的升高而持续增加(图11A)。因此，在>100μg/mL的浓度下，TPAD的溶血活性低于TPI。TPAD对被测细菌的MIC不超过16μg/mL，在这样的浓度下TPAD仅引起人类红细胞10％的溶血(图5)。因此，当治疗浓度小于16μg/mL时，TPAD的溶血作用在 体外是可以接受的。本发明还测试了TPI和TPAD对正常人人红细胞的细胞毒性。如图11B所示，TPAD与TPI相当，并且在<32μg/mL的浓度下(即在大多数测试细菌上的浓度均显著高于其MIC)都显示出可忽略的细胞毒性。相比之下，两者均对人类人红细胞的细胞毒性>64μg/mL。2.4 Determination of hemolytic activity and cytotoxicity, TPAD and TPI have comparable hemolytic activity when the concentration is <50μg/mL, but it is different when the concentration is >100μg/mL (Figure 11A). The hemolytic activity of TPAD reached 100 μg/mL and reached the maximum, while the hemolytic activity of TPI continued to increase with the increase in concentration (Figure 11A). Therefore, at a concentration of >100μg/mL, the hemolytic activity of TPAD is lower than that of TPI. The MIC of TPAD to the tested bacteria does not exceed 16 μg/mL. At this concentration, TPAD only causes 10% hemolysis of human red blood cells (Figure 5). Therefore, when the therapeutic concentration is less than 16μg/mL, the hemolysis of TPAD is acceptable in vitro. The present invention also tested the cytotoxicity of TPI and TPAD to normal human red blood cells. As shown in Figure 11B, TPAD is comparable to TPI, and shows negligible cytotoxicity at a concentration of <32 μg/mL (that is, the concentration on most of the tested bacteria is significantly higher than its MIC). In contrast, both have cytotoxicity to human red blood cells >64μg/mL.
2.5 TPAD诱导细菌膜渗漏。先前的显微镜观察表明，TPI通过作用于细胞膜以及细胞内靶标杀死细菌，其中细胞膜为主要靶标。在这里，本发明使用原子力显微镜来表征应用TPAD后的LeYC36细胞膜表面形态。如图12所示(图A和图B)，在用TPAD处理后，细胞表面从光滑和完整变化为严重凹陷，细菌细胞周围出现细胞内溶物泄漏。细胞表面形态的进一步切片分析表明，TPAD处理后的LeYC36细胞的表面凹陷明显低于对照组(图12C)。2.5 TPAD induces bacterial membrane leakage. Previous microscopic observations have shown that TPI kills bacteria by acting on cell membranes and intracellular targets, of which the cell membrane is the main target. Here, the present invention uses atomic force microscope to characterize the surface morphology of LeYC36 cell membrane after TPAD is applied. As shown in Figure 12 (Figure A and Figure B), after treatment with TPAD, the cell surface changed from smooth and intact to severe depression, and intracellular lysate leakage occurred around the bacterial cells. Further slice analysis of the cell surface morphology showed that the surface depression of LeYC36 cells treated with TPAD was significantly lower than that of the control group (Figure 12C).
据推测，TPAD可以破坏产酶溶杆菌LeYC36的细胞膜，导致细胞死亡，其作用机制与TPI类似。为了检验该假设，使用模拟细菌细胞膜的脂质体评估了TPAD的膜分解活性。如预期的那样，TPAD以剂量依赖性方式导致大肠埃希氏菌脂质提取物组成的单层囊泡(LUV)泄漏(见图6)。It is speculated that TPAD can destroy the cell membrane of the enzyme-producing lysobacterium LeYC36, leading to cell death, and its mechanism of action is similar to that of TPI. To test this hypothesis, liposomes that mimic bacterial cell membranes were used to evaluate the membrane decomposing activity of TPAD. As expected, TPAD caused the leakage of unilamellar vesicles (LUV) composed of lipid extracts of Escherichia coli in a dose-dependent manner (see Figure 6).
该结果表明TPAD促进了内容物从模拟细菌膜的模型膜中的泄漏。总的来说，本发明的研究结果支持了TPAD可以诱导细胞膜渗漏导致细菌死亡的假说。This result indicates that TPAD promotes the leakage of contents from the model membrane that mimics the bacterial membrane. In general, the research results of the present invention support the hypothesis that TPAD can induce cell membrane leakage and cause bacterial death.
2.6诱导细菌产生抗药性研究，研究了亚致死浓度下细菌对TPAD的抗药性影响，以发现除膜破裂外的其他靶标。产酶溶杆菌是无处不在的环境细菌，已作为一种新的抗生素来源出现，其对多种抗生素具有很高的固有抗药性，因此本发明在TPAD耐药性研究中使用了这些细菌。2.6 Study on the induction of bacterial resistance to TPAD. The effect of bacterial resistance to TPAD at sublethal concentrations was studied to find other targets besides membrane rupture. Enzyme-producing lysobacterium is a ubiquitous environmental bacteria that has emerged as a new source of antibiotics and has high inherent resistance to multiple antibiotics. Therefore, the present invention uses these bacteria in the study of TPAD resistance.
TPAD和TPI对LeYC36表现出浓度依赖性的活性。在浓度<8μg/mL时，TPAD对LeYC36的效力比TPI强。当TPAD的浓度达到4μg/mL时，它显示出很强的杀菌活性，而TPI对细胞存活的影响很小。然后，本发明通过连续传代分析监测了对TPAD和TPI的抗性进化(图13)。在第7代暴露于TPAD后，TPAD对LeYC36的MIC从4μg/mL增加到16μg/mL(增加4倍)，而经过相同的传代TPI对LeYC36的MIC值从8μg/mL增加到16μg/mL(增加2倍)。因此，与先前研究中由抗生素诱导的酶相比，LeYC36仅对TPAD和TPI产生了低水平的抗性。本发明的结果与研究显示TPI和TPII(鲎素II)对多种细菌没有抗药性或仅产生低水平抗药性的研究一致。TPAD和TPI基本上具有相同的MIC进化特征。仅针对TPAD发生的第一代之后，MIC增加了2倍。如此小的差异可能源于低肽浓度下的LeYC36对TPAD的敏感性高于TPI。TPAD and TPI showed a concentration-dependent activity on LeYC36. When the concentration is less than 8μg/mL, TPAD is more effective on LeYC36 than TPI. When the concentration of TPAD reaches 4μg/mL, it shows strong bactericidal activity, while TPI has little effect on cell survival. Then, the present invention monitored the evolution of resistance to TPAD and TPI through serial passage analysis (Figure 13). After exposure to TPAD at the 7th generation, the MIC of TPAD on LeYC36 increased from 4 μg/mL to 16 μg/mL (a 4-fold increase), and after the same passage, the MIC value of TPI on LeYC36 increased from 8 μg/mL to 16 μg/mL ( Increase 2 times). Therefore, compared with the enzymes induced by antibiotics in previous studies, LeYC36 only developed a low level of resistance to TPAD and TPI. The results of the present invention are consistent with studies showing that TPI and TPII (limulus II) have no resistance to various bacteria or only produce low-level resistance. TPAD and TPI basically have the same evolutionary characteristics of MIC. Only after the first generation of TPAD occurred, the MIC increased by 2 times. Such a small difference may be due to the higher sensitivity of LeYC36 to TPAD than TPI at low peptide concentrations.
为了进一步研究细菌对TPAD的抗性机制，对经过或未经过外源TPAD(2μg/mL)处理的LeYC36进行了全基因组转录谱分析。在先前的研究中，已经使用组学技术研究了细菌对抗菌肽的抗性机制。转录组学研究的结果表明，抗菌肽的抗性与多药耐药泵，运动基因和双组分***密切相关。例如，比较转录组学研究表明，蛙皮素(magainin)I下调大肠埃希菌细胞运动性和伴侣相关基因，但上调细胞通讯和多药外排泵相关基因。在另一项研究中，CpxR/CpxA双组分***导致沙门氏菌通过上调amiA和amiC的转录，使对鱼精蛋白α-螺旋肽蛙皮素2和蜂毒肽的抗性增加。在当前研究中，本发明发现184个受外源TPAD影响的基因(p值<0.005)，其中156个被上调，28个下调(PRJNA542247)。上调了几种药物外排泵(图1A，图1B)，包括czcB，nodT，yceL和ftsX。值得注意的是，已知与双组分***QseC和QseB相关的基因簇通过TPAD处理得到上调(先前的研究表明，QseC/B双组分***可以识别外源信号分子。QseC是具有组氨酸激酶活性的膜结合传感器蛋白，QseB是细胞质应答调节剂。In order to further study the resistance mechanism of bacteria to TPAD, the whole genome transcription profile analysis of LeYC36 with or without exogenous TPAD (2μg/mL) treatment was carried out. In previous studies, omics techniques have been used to study the mechanism of bacterial resistance to antimicrobial peptides. The results of transcriptomics studies show that the resistance of antimicrobial peptides is closely related to the multidrug resistance pump, motor genes and the two-component system. For example, comparative transcriptomics studies have shown that magainin I down-regulates E. coli cell motility and partner-related genes, but up-regulates cell communication and multidrug efflux pump-related genes. In another study, the CpxR/CpxA two-component system caused Salmonella to increase the protamine α-helical peptide bombesin 2 and melittin by up-regulating the transcription of amiA and amiC. In the current study, the present invention found 184 genes affected by exogenous TPAD (p value<0.005), of which 156 were up-regulated and 28 were down-regulated (PRJNA542247). Several drug efflux pumps were upregulated (Figure 1A, Figure 1B), including czcB, nodT, yceL and ftsX. It is worth noting that the gene clusters known to be related to the two-component system QseC and QseB are up-regulated by TPAD processing (previous studies have shown that the QseC/B two-component system can recognize foreign signal molecules. QseC has histidine A membrane-bound sensor protein for kinase activity, QseB is a cytoplasmic response modifier.
本发明的结果表明，出乎意料的是，TPAD未激活ΔqseB突变株中的药物外排泵，排泵相关蛋白的表达水平与未处理的野生型菌株相似。与TPAD对野生型LeYC36的杀菌作用相比，TPAD对ΔqseB和ΔqseC突变体具有更强的杀菌作用，这提示TPAD在病菌的胞内浓度得以有效维持或增加，从而导致了更好的杀菌或抑菌活性。The results of the present invention showed that, unexpectedly, TPAD did not activate the drug efflux pump in the ΔqseB mutant strain, and the expression level of the efflux pump related protein was similar to that of the untreated wild-type strain. Compared with the bactericidal effect of TPAD on wild-type LeYC36, TPAD has a stronger bactericidal effect on ΔqseB and ΔqseC mutants, which suggests that the intracellular concentration of TPAD can be effectively maintained or increased, resulting in better sterilization or inhibition. Bacterial activity.
如图1C和图1D所示，TPAD对野生型LeYC36的细胞毒力不是很高，为10μg/mL；出乎意料的是，TPAD对ΔqseB突变型的LeYC36的细胞毒力很高，对ΔqseC突变型的LeYC36的细胞毒力也很高。在2μg/mL时，TPAD可以杀死几乎所有的ΔqseB和ΔqseC突变株。As shown in Figure 1C and Figure 1D, the cytotoxicity of TPAD to wild-type LeYC36 is not very high, at 10 μg/mL; unexpectedly, TPAD has high cytotoxicity to the ΔqseB mutant LeYC36, and it has a high cytotoxicity to the ΔqseC mutant. The cytotoxicity of type LeYC36 is also very high. At 2μg/mL, TPAD can kill almost all ΔqseB and ΔqseC mutant strains.
此外，LED209是QseC的公认抑制剂。当2μg/mL TPAD和5pM LED209的联用时，可以完全杀死野生型LeYC36(图1C，图1D)。In addition, LED209 is a recognized inhibitor of QseC. When 2μg/mL TPAD and 5pM LED209 are used in combination, wild-type LeYC36 can be completely killed (Figure 1C, Figure 1D).
为了排除单独的LED209可能的杀菌作用，仅以LED209作为对照实验处理了LeYC36。LED209没有杀菌作用(图1C，图1D)，表明联合应用可协同促进TPAD的杀菌活性。In order to exclude the possible sterilization effect of LED209 alone, only LED209 was used as a control experiment to treat LeYC36. LED209 has no bactericidal effect (Figure 1C, Figure 1D), indicating that the combined application can synergistically promote the bactericidal activity of TPAD.
据推测，在ΔqseB和ΔqseC突变体上药物外排泵的失活/低水平表达可能有助于增加TPAD的细胞内浓度以及提高其杀菌活性。与TPI一样，TPAD也被认为具有额外的作用方式，涉及与细胞内靶标的直接相互作用。例如，以前 显示TPI可以使细胞内酯酶失活。但是，在当前的研究中，TPAD的细胞内靶标仍然不清楚，将来确定TPAD的特定细胞内靶标将很有趣。It is speculated that the inactivation/low-level expression of the drug efflux pump on the ΔqseB and ΔqseC mutants may help increase the intracellular concentration of TPAD and improve its bactericidal activity. Like TPI, TPAD is also believed to have an additional mode of action involving direct interaction with intracellular targets. For example, it has previously been shown that TPI can inactivate intracellular esterases. However, in the current research, the intracellular target of TPAD is still unclear, and it will be interesting to determine the specific intracellular target of TPAD in the future.
最后，本发明研究了TPAD和LED2019之间的协同作用是否可以扩展到具有QseC/B双组分***的其他病原细菌。TPAD和LED209的组合还可以有效地抵抗假单胞菌(Pseudomonadaceae)，这是一种固有的对多种抗生素具有耐药性的致病细菌(图14)。本发明还发现，与LED209结合使用时，TPAD的活性也大大提高了对嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia)的效用，该菌也对多种抗生素具有抗性(图14)。因此，通过QseC/B双组分***，可以将TPAD和LED209联合应用的策略扩展到其他病原细菌。Finally, the present invention studies whether the synergy between TPAD and LED2019 can be extended to other pathogenic bacteria with the QseC/B two-component system. The combination of TPAD and LED209 can also effectively resist Pseudomonadaceae, a pathogenic bacteria that is inherently resistant to multiple antibiotics (Figure 14). The present invention also found that when used in combination with LED209, the activity of TPAD also greatly improves the efficacy against Stenotrophomonas maltophilia, which is also resistant to multiple antibiotics (Figure 14). Therefore, through the QseC/B two-component system, the combined application strategy of TPAD and LED209 can be extended to other pathogenic bacteria.
2.7通过一系列实验，本发明推测TPAD可能具有多种作用机制。一方面，TPAD可以破坏细菌的细胞膜，并导致细菌死亡，类似于TPI。另外，TPAD触发基因表达范围的变化，导致细菌对TPAD产生抗性。但是，一些有趣的问题，如细胞内转运的机制以及可能受到调控的基因的存在，仍有待解决。这些问题的澄清将有助于鉴定新的靶标并进一步提高这些抗菌肽的杀菌效率。2.7 Through a series of experiments, the present invention speculates that TPAD may have multiple mechanisms of action. On the one hand, TPAD can destroy the cell membrane of bacteria and cause the death of bacteria, similar to TPI. In addition, TPAD triggers changes in the range of gene expression, leading to bacterial resistance to TPAD. However, some interesting issues, such as the mechanism of intracellular transport and the existence of genes that may be regulated, remain to be resolved. The clarification of these issues will help to identify new targets and further improve the bactericidal efficiency of these antimicrobial peptides.
TPAD是TPI的全D氨基酸类似物，可维持天然肽的广谱有效抗菌活性，但在高浓度下具有显着改善的稳定性和降低的溶血作用。必须注意的是，在较低浓度下，TPAD和TPI的溶血活性是相当的，将来进一步降低TPAD类似物的溶血活性仍然是必要的。TPAD通过激活QseC/B双组分***诱导细菌耐药性，而阻断该双组分***可以有效提高TPAD的抗菌作用(图15)。TPAD is a full D amino acid analog of TPI, which can maintain the broad-spectrum effective antibacterial activity of natural peptides, but has significantly improved stability and reduced hemolysis at high concentrations. It must be noted that at lower concentrations, the hemolytic activity of TPAD and TPI are equivalent, and it is still necessary to further reduce the hemolytic activity of TPAD analogs in the future. TPAD induces bacterial resistance by activating the QseC/B two-component system, and blocking the two-component system can effectively improve the antibacterial effect of TPAD (Figure 15).
据本发明所知，这项研究是关于细菌对海洋来源的AMP产生耐药性的机制的第一份报告，它为将来提高AMP的抗菌效率开辟了道路。As far as the present invention knows, this study is the first report on the mechanism by which bacteria develop resistance to marine-derived AMP, and it paves the way for improving the antibacterial efficiency of AMP in the future.
3.肽的合成及活性测试方法3. Peptide synthesis and activity test method
以下提供了实施例2中所用的多肽合成及活性测试的具体方法：The following provides the specific methods of peptide synthesis and activity testing used in Example 2:
3.1实验部分肽合成，TPI和TPAD类似物的固体肽合成步骤与之前所述的步骤类似。简而言之，TPI和TPAD使用固相肽合成与中和/2(1H-苯并***-1-基)-1，1，3，3-四甲基铀六氟磷酸酯活化程序，在rink amide甲基苯甲胺树脂(Novabiochem)上组装Fmoc(N-(9-芴基)甲氧基羰基)化学。 3.1 The experimental part of peptide synthesis , the solid peptide synthesis steps of TPI and TPAD analogs are similar to the steps described previously. In short, TPI and TPAD use solid-phase peptide synthesis and neutralization/2 (1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluranium hexafluorophosphate activation procedure, Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry was assembled on rink amide methylbenzylamine resin (Novabiochem).
通过在室温(20-25℃)下用88：5：5：2的三氟乙酸，苯酚，水和三异丙基硅烷作为清除剂进行处理来实现裂解。在旋转蒸发仪中将三氟乙酸在低压下蒸发。用冰冷的***沉淀肽，过滤，溶解在50％的缓冲液A/B中(缓冲液A由90％的H 2O/10％的CH 3CN/0.05％的三氟乙酸组成，缓冲液B的由90％的 CH 3CN/10％的H 2O/0.045组成，并冻干。通过在Phenomenex C 18柱上的RP-HPLC纯化粗制肽，并在收集和冻干进行氧化之前，使用电喷雾质谱法确认分子量。肽中的四个胱氨酸在两个步骤中被选择性氧化。第一步，将未保护的胱氨酸在浓度为0.5mg/mL的0.1M NH 4HCO 3(pH 8-8.5)中氧化，并在室温下搅拌24h。通过RP-HPLC，用检测波长为214nm的方法分离出氧化的肽。在第二步中，通过将肽溶解在浓度为1mg/mL的碘溶液中来氧化Acm保护的胱氨酸，并搅拌30分钟。然后加入抗坏血酸以终止氧化反应，并再次搅拌溶液直至看不到颜色。经过两轮氧化后，通过制备型RP-HPLC纯化肽，并分别使用RP-HPLC和电喷雾质量验证其纯度和质量(结果见表2)。 The cleavage is achieved by treating with 88:5:5:2 trifluoroacetic acid, phenol, water and triisopropylsilane as scavengers at room temperature (20-25°C). The trifluoroacetic acid is evaporated at low pressure in a rotary evaporator. Precipitate the peptide with ice-cold ether, filter, and dissolve in 50% buffer A/B (buffer A consists of 90% H 2 O/10% CH 3 CN/0.05% trifluoroacetic acid, buffer B It is composed of 90% CH 3 CN/10% H 2 O/0.045 and lyophilized. The crude peptide is purified by RP-HPLC on a Phenomenex C 18 column and used before being collected and lyophilized for oxidation. The molecular weight was confirmed by electrospray mass spectrometry. The four cystines in the peptide were selectively oxidized in two steps. In the first step, the unprotected cystine was added to 0.1M NH 4 HCO 3 at a concentration of 0.5 mg/mL. (pH 8-8.5), and stirred at room temperature for 24h. The oxidized peptide was separated by RP-HPLC with a detection wavelength of 214nm. In the second step, the peptide was dissolved in a concentration of 1mg/mL. The cystine protected by Acm was oxidized in the iodine solution and stirred for 30 minutes. Then ascorbic acid was added to stop the oxidation reaction, and the solution was stirred again until no color was visible. After two rounds of oxidation, the peptide was purified by preparative RP-HPLC , And use RP-HPLC and electrospray quality to verify its purity and quality (the results are shown in Table 2).
3.2生物活性测定。3.2 Determination of biological activity.
最小抑菌浓度(MIC)是抑制细菌可见生长的化学药品的最低浓度。针对革兰氏阴性细菌的肽的MIC，包括大肠杆菌(E.coli)K-12，大肠杆菌BAA 2469，大肠杆菌ATCC 25923，枯草芽孢杆菌(B.subtilis)168和阴沟肠杆菌(E.cloacae)BAA 1143，革兰氏阳性金黄色葡萄球菌(S.aureus)ATCC 29213，S.金黄色葡萄球菌BAA 41和金黄色葡萄球菌BAA 44以及肺炎克雷伯菌(K.pneumoniae)BAA 1144，肺炎克雷伯氏菌BAA 2470，铜绿假单胞菌(P.aeruginosa)ATCC 27853，铜绿假单胞菌BAA 2108，鲍曼不动杆菌(A.baumannii)ATCC 19606和屎肠球菌(E.faecium)ATCC 29212的测定方法。简而言之，实验在96孔板上进行，在孔中连续稀释抗菌肽。每种稀释液一式两份进行。每个孔包含80μL培养基，10μL肽和10μL细菌培养物(最终菌浓度约为5×10 6CFU/mL)。仅包括细菌和培养基的对照，以确保细菌的活性和培养基的无菌性。置于37℃温育18小时后，测定吸光值(结果见表7)。 The minimum inhibitory concentration (MIC) is the lowest concentration of a chemical that inhibits the visible growth of bacteria. The MIC of peptides against gram-negative bacteria, including E. coli K-12, E. coli BAA 2469, E. coli ATCC 25923, B. subtilis 168 and E. cloacae ) BAA 1143, Gram-positive Staphylococcus aureus (S.aureus) ATCC 29213, S. Staphylococcus aureus BAA 41 and Staphylococcus aureus BAA 44, and K. pneumoniae BAA 1144, pneumonia Klebsiella BAA 2470, P. aeruginosa (P. aeruginosa) ATCC 27853, P. aeruginosa BAA 2108, Acinetobacter baumannii (A. baumannii) ATCC 19606 and Enterococcus faecium (E. faecium) ATCC 29212 measurement method. In short, the experiment was carried out in 96-well plates with serial dilutions of antimicrobial peptides in the wells. Each dilution was done in duplicate. Each well contains 80 μL of medium, 10 μL of peptides and 10 μL of bacterial culture (final bacterial concentration is about 5×10 6 CFU/mL). Only the control of bacteria and culture medium is included to ensure the viability of bacteria and the sterility of culture medium. After incubating at 37°C for 18 hours, the absorbance was measured (see Table 7 for the results).
3.3溶血作用测定。溶血测定法使用与先前所述类似的方法进行。人红细胞购自HaemoScan(荷兰)。将人类血液样品的等分试样用5mL洗涤缓冲液洗涤两次，并在4℃下以2500rpm离心10分钟。用5mL稀释缓冲液重复该步骤两次，最后的沉淀物重悬于5mL稀释缓冲液中，最终浓度为5％红细胞。将100μL测试的肽添加到100μL稀释的红细胞悬液中。在七个浓度下测试肽的溶血作用(最大浓度为500μg/mL，连续两次稀释)。将100μL MilliQ水添加到100μL红细胞悬液中作为阴性对照(0％溶血)，同时将100μL 2％Triton X-100添加到100μL红细胞悬液中作为阳性对照(100％溶血)。然后将测定混合物在缓慢旋转(100rpm)下于37℃孵育1h。孵育后，将样品以5，000rpm离心1分钟， 并通过分光光度法在415nm和450nm作为参考波长下测量上清液的吸光度来定量溶血(结果见图5和图11A)。 3.3 Determination of hemolysis. The hemolysis assay was performed using a method similar to that described previously. Human red blood cells were purchased from HaemoScan (Netherlands). An aliquot of the human blood sample was washed twice with 5 mL washing buffer and centrifuged at 2500 rpm for 10 minutes at 4°C. Repeat this step twice with 5 mL dilution buffer, and resuspend the final pellet in 5 mL dilution buffer to a final concentration of 5% erythrocytes. Add 100 μL of the tested peptide to 100 μL of the diluted red blood cell suspension. The hemolysis of the peptide was tested at seven concentrations (maximum concentration is 500 μg/mL, two consecutive dilutions). 100 μL of MilliQ water was added to 100 μL of red blood cell suspension as a negative control (0% hemolysis), while 100 μL of 2% Triton X-100 was added to 100 μL of red blood cell suspension as a positive control (100% hemolysis). The assay mixture was then incubated at 37°C for 1 h under slow rotation (100 rpm). After the incubation, the sample was centrifuged at 5,000 rpm for 1 minute, and the absorbance of the supernatant was measured by spectrophotometry at 415 nm and 450 nm as reference wavelengths to quantify hemolysis (see Figure 5 and Figure 11A for the results).
3.4体外细胞毒性试验。细胞毒性实验是使用人正常人红细胞L02进行的。将浓度为2×104细胞/mL的100μLL02细胞添加到96孔板的每个孔中，并在RPMI-1640培养基中培养24小时。然后以2-128μg/mL的梯度浓度用TPI和TPAD处理细胞，将肽溶解并用RPMI-1640培养基稀释，并向对照组加入等量的RPMI-1640培养基。48小时后，向每个孔中加入20μL刃天青素孵育4小时，然后在544nm激发光和595nm吸收光下进行酶标仪检测。每个浓度同时重复六次(结果见图11)。 3.4 In vitro cytotoxicity test. The cytotoxicity test was performed using normal human red blood cells L02. 100μLL02 cells at a concentration of 2×104 cells/mL were added to each well of a 96-well plate and cultured in RPMI-1640 medium for 24 hours. Then the cells were treated with TPI and TPAD at a gradient concentration of 2-128 μg/mL, the peptide was dissolved and diluted with RPMI-1640 medium, and an equal amount of RPMI-1640 medium was added to the control group. After 48 hours, add 20μL of resazurin to each well and incubate for 4 hours, and then perform microplate reader detection under 544nm excitation light and 595nm absorption light. Each concentration was repeated six times at the same time (see Figure 11 for the results).
3.5肽稳定性测定。如前所述，使用雄性AB人血清(SigmaAldrich)进行血清稳定性测定。将血清以15000g离心15分钟以去除脂质，然后在37℃孵育10分钟。一式三份的样品以1:10的肽稀释液制备：工作肽浓度为20mM的血清，添加40μL的20％TFA，在4℃沉淀血清蛋白。将样品以14000g离心10分钟，将透明的上清液转移至96孔板中，与超纯水混合，并以800rpm的速度摇动10分钟，然后在0.3mL/min的Phenomenex C18色谱柱上使用线性1％梯度的0-50％溶剂B。在每个时间点也将一式三份的PBS中的肽样品作为对照。注入样品的等分试样，并通过在215nm处积分测定完整肽的剩余量(结果见图11)。 3.5 Determination of peptide stability. As mentioned earlier, male AB human serum (Sigma Aldrich) was used for serum stability determination. The serum was centrifuged at 15000g for 15 minutes to remove lipids, and then incubated at 37°C for 10 minutes. Triplicate samples were prepared with a 1:10 peptide dilution: serum with a working peptide concentration of 20 mM, 40 μL of 20% TFA was added, and serum proteins were precipitated at 4°C. Centrifuge the sample at 14000g for 10 minutes, transfer the transparent supernatant to a 96-well plate, mix with ultrapure water, and shake at 800 rpm for 10 minutes, and then apply linearity on a Phenomenex C18 column at 0.3 mL/min. 1% gradient of 0-50% solvent B. Peptide samples in PBS in triplicate were also used as controls at each time point. An aliquot of the sample was injected and the remaining amount of intact peptide was determined by integration at 215 nm (see Figure 11 for the results).
3.6 TPAD的NMR结构测定。在pH值为3.5的90％H2O/10％D2O和75％H2O/25％2，2，2-三氟乙醇(TFE)-d3中的～1mM TPAD上进行NMR分析。二维实验是在Bruker Avance III 600MHz光谱仪上获得的，包括298K的TOCSY，NOESY，1H15N HSQC和1H13C HSQC。还在283-303K的温度下收集TOCSY实验。用TOPSPIN 3.5(Bruker)处理后，使用CCPNMR(版本2.4.4)指定光谱。光谱以内标为基准，即0ppm的2，2-二甲基-2-硅戊烷-5-磺酸盐(DSS)。使用程序CYANA计算初步结构。距离限制是从25％TFE中记录的NOESY光谱中得出的，并使用TALOS-N]程序生成了骨架二面角。然后，利用CNS程序在明确的溶剂中，使用扭转角动力学，优化和能量最小化，生成了最后一组结构。使用MolProbity评估了结构质量。 3.6 NMR structure determination of TPAD. NMR analysis was performed on ~1 mM TPAD in 90% H2O/10% D2O and 75% H2O/25% 2,2,2-trifluoroethanol (TFE)-d3 at pH 3.5. The two-dimensional experiment was obtained on the Bruker Avance III 600MHz spectrometer, including 298K TOCSY, NOESY, 1H15N HSQC and 1H13C HSQC. TOCSY experiments were also collected at a temperature of 283-303K. After processing with TOPSPIN 3.5 (Bruker), specify the spectrum using CCPNMR (version 2.4.4). The spectrum is based on the internal standard, which is 0 ppm 2,2-dimethyl-2-silylpentane-5-sulfonate (DSS). Use the program CYANA to calculate the preliminary structure. The distance limit is derived from the NOESY spectrum recorded in 25% TFE, and the skeleton dihedral angle is generated using the TALOS-N] program. Then, using the CNS program in a clear solvent, using torsion angle dynamics, optimization and energy minimization, the final set of structures was generated. The quality of the structure was evaluated using MolProbity.
3.7分子动力学模拟。通过使用AMBER 16软件包和ff14SB力场的分子动力学(MD)模拟，可以最小化和精炼TPI(pdb代码：1WO0)和TPAD的NMR结构。将肽在截短的八面体TIP3P水箱中溶剂化，并添加抗衡离子(Cl-)以中和溶 剂化肽的净电荷。用3000步最陡下降最小化方案最小化水分子，然后用3000步共轭梯度最小化方案最小化，溶质受
Figure PCTCN2020095683-appb-000019
的谐波力约束。然后使用相同的参数执行第二个最小化步骤，但撤消所有位置限制。然后在100ps的时间内，将NVT集成***中的***逐渐从50K加热到300K，并使用
Figure PCTCN2020095683-appb-000020
Figure PCTCN2020095683-appb-000021
谐波力势将溶质原子限制在其初始位置。之后，将模拟切换到NPT集合，并且对溶质的约束在100ps内从
Figure PCTCN2020095683-appb-000022
逐渐降低。生产MD的运行是在NPT系综中经过100ns的模拟时间进行的，压力耦合为1atm，恒温为300K。MD模拟使用2fs的时间步长，并且所有涉及氢原子的键都保持为使用粒子网格Ewald(PME)方法处理所有MD模拟中的长距离静电相互作用。 3.7 Molecular dynamics simulation. By using AMBER 16 software package and molecular dynamics (MD) simulation of ff14SB force field, the NMR structure of TPI (pdb code: 1WO0) and TPAD can be minimized and refined. The peptide was solvated in a truncated octahedral TIP3P water tank, and a counterion (Cl-) was added to neutralize the net charge of the solvated peptide. Use the 3000-step steepest descent minimization scheme to minimize water molecules, and then use the 3000-step conjugate gradient minimization scheme to minimize the solute.
Figure PCTCN2020095683-appb-000019
Harmonic force constraints. Then perform the second minimization step with the same parameters, but undo all position restrictions. Then in 100ps, the system in the NVT integrated system is gradually heated from 50K to 300K, and used
Figure PCTCN2020095683-appb-000020
Figure PCTCN2020095683-appb-000021
The harmonic potential confines the solute atoms to their initial positions. After that, the simulation was switched to the NPT set, and the solute constraint was changed from within 100ps
Figure PCTCN2020095683-appb-000022
Gradually decreases. The operation of the production MD is carried out in the NPT ensemble after a simulation time of 100ns, the pressure coupling is 1atm, and the constant temperature is 300K. The MD simulation uses a time step of 2fs, and all bonds involving hydrogen atoms are maintained as using the particle grid Ewald (PME) method to handle all long-distance electrostatic interactions in the MD simulation.
使用AMBER 16在膜存在下对TPAD进行MD模拟TPAD位于包含POPE(1-棕榈酰基-2-油酰基-sn-甘油3-磷酸乙醇胺)：POPG(1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸)3：2混合物的双层中-(1’-rac-甘油))，用于模拟细菌膜，尺寸为
Figure PCTCN2020095683-appb-000023
并且该***用TIP3P水分子以及Na+和Clions溶解，因此***在总浓度下为中性CHARMM-GUI(http://www.charmm-gui.org)中的0.15M。***温度逐渐升高至310K，并分别在NVT和NPT集成中平衡500ps，其中蛋白质和脂质受到
Figure PCTCN2020095683-appb-000024
力的约束。朗文温控器用于初始加热。对于第二阶段加热，除了使用Langevin恒温器平衡温度外，还使用各向异性的Berendsen弱耦合恒压器来平衡压力。然后，取消对膜的约束，在NPT中模拟整个***20ns，以适当平衡膜***。在5ns MD模拟中，分10个步骤逐步取消了对蛋白质的限制。之后，进行了600ns的生产运行。在生产过程中，使用Langevin恒温器控制温度，而使用各向异性Berendsen恒压器控制压力。所有模拟均使用脂质的Lipid14力场和蛋白质的AMBER14SB力场进行。MD模拟使用2fs的时间步长，并且使用SHAKE算法将所有涉及氢原子的键保持在其标准长度。粒子网格Ewald(PME)用于非键原子相互作用的截止点为
Figure PCTCN2020095683-appb-000025
并且邻居列表每10步更新一次。 Use AMBER 16 to perform MD simulation of TPAD in the presence of a membrane. TPAD is located in the presence of POPE (1-palmitoyl-2-oleoyl-sn-glycerol 3-phosphoethanolamine): POPG (1-palmitoyl-2-oleoyl-sn- Glycerol-3-phosphate) in the double layer of the 3:2 mixture-(1'-rac-glycerol)), used to simulate bacterial membranes, the size is
Figure PCTCN2020095683-appb-000023
And the system uses TIP3P water molecules and Na+ and Clions to dissolve, so the total concentration of the system is 0.15M in the neutral CHARMM-GUI (http://www.charmm-gui.org). The temperature of the system was gradually increased to 310K, and 500ps was balanced in the integration of NVT and NPT respectively, in which protein and lipid were affected by
Figure PCTCN2020095683-appb-000024
Force constraints. Longman thermostat is used for initial heating. For the second stage heating, in addition to using a Langevin thermostat to balance the temperature, an anisotropic Berendsen weakly coupled barostat is also used to balance the pressure. Then, cancel the restriction on the membrane and simulate the entire system for 20 ns in NPT to properly balance the membrane system. In the 5ns MD simulation, the restriction on protein was gradually removed in 10 steps. After that, a production run of 600ns was carried out. In the production process, a Langevin thermostat is used to control the temperature, while an anisotropic Berendsen barostat is used to control the pressure. All simulations were performed using Lipid14 force field for lipids and AMBER14SB force field for proteins. The MD simulation uses a time step of 2fs and uses the SHAKE algorithm to keep all bonds involving hydrogen atoms at their standard length. The cut-off point of particle grid Ewald (PME) for non-bonded atom interaction is
Figure PCTCN2020095683-appb-000025
And the neighbor list is updated every 10 steps.
3.8细菌菌株，质粒和一般方法。野生型溶杆菌和相关突变体(qseC和qseB突变体)在40％强度的TSB培养基中生长。大肠杆菌菌株DH5α和S17-1用于细菌突变。表5中描述了本发明中使用的细菌菌株和质粒的详细信息。根据先前描述的方法进行分子操作。限制酶和分子生物学试剂购自Takara(TaKaRa Bio Group，日本)。PCR引物由清科生物技术公司合成(具体见表6)。 3.8 Bacterial strains, plasmids and general methods . Wild-type lysobacterium and related mutants (qseC and qseB mutants) were grown in 40% strength TSB medium. E. coli strains DH5α and S17-1 were used for bacterial mutation. Table 5 describes the detailed information of bacterial strains and plasmids used in the present invention. Perform molecular manipulations according to the previously described method. Restriction enzymes and molecular biology reagents were purchased from Takara (TaKaRa Bio Group, Japan). The PCR primers were synthesized by Zero2IPO Biotechnology Company (see Table 6 for details).
3.9生物信息学分析。使用Primer Premier 5.设计用于实时PCR和基因操作 测定的引物。BLAST(http://blast.ncbi.nlm.nih.gov/Blast.cgi)分析了基因序列。注释和生物信息学分析通过基因组测序和EMBOSS(欧洲分子生物学开放软件套件)(http://emboss.open-bio.org/)进行。 3.9 Bioinformatics analysis. Use Primer Premier 5. Design primers for real-time PCR and genetic manipulation assays. BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) analyzed the gene sequence. Annotation and bioinformatics analysis were performed by genome sequencing and EMBOSS (European Molecular Biology Open Software Suite) (http://emboss.open-bio.org/).
3.10 RNA提取，逆转录PCR和实时PCR。LeYC36在不同条件下培养(用或不用2μg/mL TPAD处理)，然后根据制造商的说明使用RNA提取试剂盒(OMEGA)提取RNA。将RNA样品逆转录为cDNA，并在总反应体积为20μL，包含250nM引物，10μLEva Green 2x qPCR Master Mix，0.5μL10倍稀释cDNA模板的实时反应中进行实时PCR，以及8.5μL不含RNase的水。将16S rRNA用作参考基因。实时PCR用StepOne实时PCR***(AB Applied Biosystems)进行。如前所述设计程序。 3.10 RNA extraction, reverse transcription PCR and real-time PCR. LeYC36 was cultured under different conditions (with or without 2μg/mL TPAD treatment), and then RNA was extracted using an RNA extraction kit (OMEGA) according to the manufacturer's instructions. The RNA sample was reverse transcribed into cDNA, and real-time PCR was performed in a real-time reaction with a total reaction volume of 20 μL, containing 250 nM primers, 10 μLEva Green 2x qPCR Master Mix, 0.5 μL 10-fold diluted cDNA template, and 8.5 μL RNase-free water. 16S rRNA was used as the reference gene. Real-time PCR was performed with the StepOne real-time PCR system (AB Applied Biosystems). Design the program as described earlier.
3.11转录分析和分析。3.11 Transcription analysis and analysis.
LeYC36(有和没有TPAD)的转录谱分析由中国上海的Biozeron公司(PRJNA542247)进行。用TRIzol试剂(Invitrogen)提取产酶溶杆菌(L.enzymogenes)YC36的总RNA(不存在或存在2μg/mL TPAD)。Transcription profile analysis of LeYC36 (with and without TPAD) was performed by Biozeron, Shanghai, China (PRJNA542247). The total RNA of L. enzymogenes YC36 was extracted with TRIzol reagent (Invitrogen) (absent or 2μg/mL TPAD).
RNA转录文库用Illumina(加利福尼亚州圣地亚哥)的TruSeq RNA制备试剂盒构建。使用RiboZero rRNA去除试剂盒(Epicenter)去除了L.enzymogenes YC36的残留rRNA。原始的配对末端读段已使用SeqPrep进行了修整，并通过Sickle(https://github.com/jstjohn/SeqPrep和https://github.com/najoshi/sickle)进行了质量控制。使用Rockhopper(http://cs.wellesley.edu/～btjaden/Rockhopper/)对齐干净的读数。分别使用Goatools和KOBAS( https://github.com/tanghaibao/ Goatools和http://kobas.cbi.pku.edu.cn/home.do)进行GO功能富集和KEGG通路分析。EdgeR用于基因表达分析(https://bioconductor.org/packages/release/bioc/html/edgeR.html)。当P值<0.005时，丰度变化大于2倍被认为是显着差异(结果见图1)。 The RNA transcription library was constructed using Illumina (San Diego, California) TruSeq RNA Preparation Kit. RiboZero rRNA removal kit (Epicenter) was used to remove the residual rRNA of L. enzymogenes YC36. The original paired end reads have been trimmed using SeqPrep and quality controlled by Sickle (https://github.com/jstjohn/SeqPrep and https://github.com/najoshi/sickle). Use Rockhopper (http://cs.wellesley.edu/~btjaden/Rockhopper/) to align the clean readings. Goatools and KOBAS ( https://github.com/tanghaibao/ Goatools and http://kobas.cbi.pku.edu.cn/home.do) were used for GO function enrichment and KEGG pathway analysis, respectively. EdgeR is used for gene expression analysis (https://bioconductor.org/packages/release/bioc/html/edgeR.html). When the P value is less than 0.005, a change in abundance greater than 2 times is considered a significant difference (see Figure 1 for the results).
3.12TPAD-LED209组合的杀菌分析。将LeYC36和相关突变菌株培养至OD1.0。将细胞分别在2μg/mL TPAD，5pM LED209和2μg/mL TPAD-5pM LED209组合下处理2-10小时。在连续稀释并在LB琼脂平板上过夜生长后，测量细菌细胞的菌落形成单位(CFU)(结果见图1)。3.12 Sterilization analysis of TPAD-LED209 combination. LeYC36 and related mutant strains were cultured to OD1.0. The cells were treated with a combination of 2μg/mL TPAD, 5pM LED209 and 2μg/mL TPAD-5pM LED209 for 2-10 hours. After serial dilution and overnight growth on LB agar plates, the colony forming units (CFU) of bacterial cells were measured (see Figure 1 for the results).
3.13连续转接传代实验。将细菌接种到含2μg/mL TPAD/TPI的培养基中，并在细菌培养达到固定相时转移至下一代。对于每一代细菌菌株，TPAD/TPI的浓度增加2μg/mL。以初始代LeYC36的MIC值作为对照，研究进化过程中 产酶溶杆菌LeYC36对TPAD/TPI的抗性(结果见图13)。 3.13 Continuous transfer passaging experiment. Bacteria were inoculated into a medium containing 2μg/mL TPAD/TPI, and transferred to the next generation when the bacterial culture reached a stationary phase. For each generation of bacterial strains, the concentration of TPAD/TPI increased by 2 μg/mL. The MIC value of the initial generation LeYC36 was used as a control to study the resistance of the enzyme-producing Lysobacterium LeYC36 to TPAD/TPI during evolution (see Figure 13 for the results).
3.14原子力显微镜。通过在缓冲液(20mM Tris-HCl，pH＝8.0和0.5M NaCl)中超声分解细菌。 3.14 Atomic Force Microscope. The bacteria were decomposed by sonication in a buffer (20mM Tris-HCl, pH=8.0 and 0.5M NaCl).
将裂解物提取物在4x样品缓冲液中稀释并煮沸10分钟。所有样品均经过12％SDS-PAGE凝胶处理，然后转移至0.45μmPVDF膜(Hybond-P，Amersham Biosciences)。在室温下用5％(w/v)脱脂牛奶TBST(20mM Tris-HCl，pH＝7.4，150mM NaCl和0.05％Tween-20)吸干2小时后，将膜与兔多克隆孵育在4℃下抗QseB(1：500，Affinity Biosciences)和GAPDH(1：2000，Sangon Biotech)的抗体16小时。用TBST洗涤3次后，将膜用辣根过氧化物酶(HRP)缀合的山羊抗兔IgG(1：5000，Sangon Biotech)在37℃下接种2小时。再次用TBST洗涤3次后，将化学发光底物(Thermo Fisher Scientific)添加到膜中，并通过CCD成像***(Bio-Rad)进行观察(结果见图8)。The lysate extract was diluted in 4x sample buffer and boiled for 10 minutes. All samples were processed by 12% SDS-PAGE gel, and then transferred to 0.45 μm PVDF membrane (Hybond-P, Amersham Biosciences). After blotting dry with 5% (w/v) skimmed milk TBST (20mM Tris-HCl, pH=7.4, 150mM NaCl and 0.05% Tween-20) at room temperature for 2 hours, incubate the membrane with the rabbit polyclonal at 4℃ Anti-QseB (1:500, Affinity Biosciences) and GAPDH (1:2000, Sangon Biotech) antibodies for 16 hours. After washing 3 times with TBST, the membrane was inoculated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5000, Sangon Biotech) at 37°C for 2 hours. After washing 3 times with TBST again, a chemiluminescent substrate (Thermo Fisher Scientific) was added to the membrane and observed through a CCD imaging system (Bio-Rad) (see Figure 8 for the results).
3.15囊泡泄漏测定，制备由填充有5-羧基荧光素(Sigma-Aldrich)的大肠杆菌脂质提取物(Avanti Polar Lipids，Inc。)组成的大单层囊泡(LUV)。简而言之，在10mM HEPES缓冲液(107mM NaCl，1mM EDTA，pH 7.4)中，制备了直径为100nm的具有自猝灭浓度(50mM)的5-羧基荧光素封装的LUV。在Sephadex G75色谱柱上从游离染料中分离出装有5-羧基荧光素的LUV，并使用Stewart分析法确定了LUV的脂质浓度。对于泄漏分析，将100μM LUV与TPAD以不同浓度孵育(0.78-25在96孔半面积的黑色平底微孔板(Corning)中，在黑暗中放置20分钟，然后使用Tecan Infinite M100Pro多板读数器(λex490nm，λem513nm)测量荧光。通过添加1％Triton X溶液可以完全溶解囊泡，这对应于囊泡内容物100％的泄漏(结果见图6)。 3.15 Vesicle leakage assay, prepare large unilamellar vesicles (LUV) composed of E. coli lipid extract (Avanti Polar Lipids, Inc.) filled with 5-carboxyfluorescein (Sigma-Aldrich). In short, in 10 mM HEPES buffer (107 mM NaCl, 1 mM EDTA, pH 7.4), a 5-carboxyfluorescein-encapsulated LUV with a self-quenching concentration (50 mM) with a diameter of 100 nm was prepared. The LUV containing 5-carboxyfluorescein was separated from the free dye on a Sephadex G75 column, and the lipid concentration of LUV was determined using Stewart analysis. For leak analysis, incubate 100μM LUV and TPAD at different concentrations (0.78-25 in a 96-well half-area black flat-bottom microplate (Corning), place in the dark for 20 minutes, and then use the Tecan Infinite M100Pro multi-plate reader ( λex490nm, λem513nm) were used to measure fluorescence. The vesicles could be completely dissolved by adding 1% Triton X solution, which corresponds to 100% leakage of the vesicle contents (see Figure 6 for the results).
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (13)

  1. 一种用于杀灭或抑制病害菌的组合物，其特征在于，所述的组合物包括由第一活性成分和第二活性成分组成的活性组合；其中，A composition for killing or inhibiting disease bacteria, characterized in that the composition comprises an active combination composed of a first active ingredient and a second active ingredient; wherein,
    (a1)所述第一活性分为TPAD；(a1) The first activity is classified as TPAD;
    其中，所述TPAD是指TPI的D型氨基酸类似物，所述TPI为如SEQ ID NO:3所示的多肽；Wherein, the TPAD refers to a D-type amino acid analog of TPI, and the TPI is a polypeptide shown in SEQ ID NO: 3;
    KWCFRVCYRGICYRRCR (SEQ ID NO:3)；KWCFRVCYRGICYRRCR (SEQ ID NO: 3);
    (a2)所述第二活性成分为QseC/B信号通路抑制剂；(a2) The second active ingredient is a QseC/B signaling pathway inhibitor;
    其中，所述QseC/B信号通路抑制剂是指能够抑制QseC、QseB，或其组合的活性物质。Wherein, the QseC/B signaling pathway inhibitor refers to an active substance that can inhibit QseC, QseB, or a combination thereof.
  2. 如权利要求1所述的组合物，其特征在于，TPAD为下式结构所示的多肽：The composition of claim 1, wherein TPAD is a polypeptide represented by the following structure:
    KWCFRVCYRGXCYRRCR (SEQ ID No.:1)KWCFRVCYRGXCYRRCR (SEQ ID No.: 1)
    式中，Where
    X为Leu、Ile、Val、Ala或Leu的非天然氨基酸类似物；X is an unnatural amino acid analog of Leu, Ile, Val, Ala or Leu;
    并且各氨基酸均为D型氨基酸。And each amino acid is D-type amino acid.
  3. 如权利要求1所述的组合，其特征在于，TPAD为如SEQ ID NO:2所示的多肽，且其中各氨基酸均为D型氨基酸；The combination according to claim 1, wherein TPAD is a polypeptide as shown in SEQ ID NO: 2, and wherein each amino acid is a D-type amino acid;
    KWCFRVCYRGLCYRRCR (SEQ ID NO:2)。KWCFRVCYRGLCYRRCR (SEQ ID NO: 2).
  4. 如权利要求1所述的组合，其特征在于，所述QseC/B信号通路抑制剂为LED209，或其其药学上可接受的盐。The combination of claim 1, wherein the QseC/B signaling pathway inhibitor is LED209, or a pharmaceutically acceptable salt thereof.
  5. 如权利要求1所述的药物组合物，其特征在于，在所述组合物中，第一活性成分和第二活性成分的用量比(mg/pmol)为1:10～1:0.5。The pharmaceutical composition according to claim 1, wherein, in the composition, the dosage ratio (mg/pmol) of the first active ingredient and the second active ingredient is 1:10 to 1:0.5.
  6. 如权利要求1所述的组合物，其特征在于，所述组合物包括由TPAD和LED209组成的活性组合；其中，TPAD和LED209的用量比(mg/pmol)为2:5。The composition of claim 1, wherein the composition comprises an active combination composed of TPAD and LED209; wherein the dosage ratio (mg/pmol) of TPAD and LED209 is 2:5.
  7. 如权利要求1所述的组合物在制备用于治疗和/或预防由病害菌引起的疾病的药物中的用途。The use of the composition according to claim 1 in the preparation of a medicament for the treatment and/or prevention of diseases caused by disease bacteria.
  8. 如权利要求7所述的用途，其特征在于，所述病害菌为具有QseC/B双组分***的细菌。The use according to claim 7, wherein the diseased bacteria are bacteria with a QseC/B two-component system.
  9. 如权利要求7所述的用途，其特征在于，所述病害菌选自下组：大肠杆菌、枯草芽孢杆菌、阴沟肠杆菌、金黄色葡萄球菌、肺炎克雷伯菌、铜绿假单胞菌、鲍曼不动杆菌、屎肠球菌、溶杆菌、福氏志贺菌、假交替单胞菌、嗜麦芽寡养单胞菌，或其组合。The use according to claim 7, wherein the disease bacteria are selected from the group consisting of Escherichia coli, Bacillus subtilis, Enterobacter cloacae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecium, Lysobacterium, Shigella flexneri, Pseudoalteromonas, Stenotrophomonas maltophilia, or a combination thereof.
  10. 如权利要求7所述的用途，其特征在于，所述由病害菌引起的疾病包括：呼吸道感染、肺部感染、皮肤感染。The use according to claim 7, wherein the diseases caused by disease bacteria include: respiratory infections, lung infections, and skin infections.
  11. 一种用于杀灭或抑制病害菌的药物组合，其特征在于，所述的药物组合包括：A drug combination for killing or inhibiting disease bacteria, characterized in that the drug combination includes:
    (i)包含第一活性成分的组合物或药物；和(ii)包含第二活性成分的组合物或药物；(i) A composition or medicine containing a first active ingredient; and (ii) A composition or medicine containing a second active ingredient;
    其中，所述第一活性成分和第二活性成分如权利要求1中定义。Wherein, the first active ingredient and the second active ingredient are as defined in claim 1.
  12. 一种抑制或杀灭病害菌的方法，其特征在于，包括步骤：使病害菌与如权利要求1所述的组合物接触，或者使病害菌与第一活性成分和第二活性成分接触，从而抑制或杀灭病害菌；A method for inhibiting or killing disease bacteria, which is characterized by comprising the steps of: contacting the disease bacteria with the composition according to claim 1, or contacting the disease bacteria with the first active ingredient and the second active ingredient, thereby Inhibit or kill disease bacteria;
    其中，所述第一活性成分和第二活性成分如权利要求1中定义。Wherein, the first active ingredient and the second active ingredient are as defined in claim 1.
  13. 一种治疗和/或预防由细菌引起的疾病，其特征在于，所述的方法包括步骤：A treatment and/or prevention of diseases caused by bacteria, characterized in that the method comprises the steps:
    向需要的对象施用如权利要求1所述的组合物；或者向需要的对象施用如权利要求11所述的药物组合；或者向需要的对象施用第一活性成分或包含第一活性成分的组合物或药物和第二活性成分或包含第一活性成分的组合物或药物。Administer the composition according to claim 1 to a subject in need; or administer the pharmaceutical combination according to claim 11 to a subject in need; or administer the first active ingredient or a composition containing the first active ingredient to a subject in need Or a medicine and a second active ingredient or a composition or medicine containing the first active ingredient.
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