WO2024076728A1 - Cyclic nucleotides and uses thereof - Google Patents

Cyclic nucleotides and uses thereof Download PDF

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Publication number
WO2024076728A1
WO2024076728A1 PCT/US2023/034632 US2023034632W WO2024076728A1 WO 2024076728 A1 WO2024076728 A1 WO 2024076728A1 US 2023034632 W US2023034632 W US 2023034632W WO 2024076728 A1 WO2024076728 A1 WO 2024076728A1
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groups
group
analog
composition
derivative
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PCT/US2023/034632
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French (fr)
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Matthew Meyerson
Douglas Wheeler
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Dana-Farber Cancer Institute, Inc.
The Broad Institute, Inc.
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Publication of WO2024076728A1 publication Critical patent/WO2024076728A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • Second messenger molecules released by the cell in response to extracellular signaling molecules can trigger intracellular signal transduction cascades, and include cyclic adenosine monophosphate and cyclic guanosine monophosphate. Novel second messengers generated in human cells in response to challenges to the innate immune system.
  • compositions comprising a cyclic dinucleotide molecule, a cyclic trinucleotide molecule, an analog or derivative thereof, or any combination thereof.
  • a composition comprises a cyclic dinucleotide molecule comprising a cytidine moiety and an adenosine moiety (cCAMP molecule), a cyclic trinucleotide molecule comprising an adenosine moiety and two cytidine moieties (cACCMP molecule), an analog or derivative thereof, or any combination thereof.
  • a composition comprises an n’,n’ cyclic cytidine monophosphate-adenosine monophosphate analog (n’,n’ cCAMP analog), wherein each n’ is 2’ or 3’, an n’,n’,n’ cyclic adenosine monophosphate-cytidine monophosphate cytidine analog (n’,n’,n’ cACCMP analog), wherein each n’ is 2’ or 3’, an analog or derivative thereof, or any combination thereof.
  • a composition is according to:
  • a composition is according to:
  • a composition is an isonucleotide analog of any one of the previous embodiments.
  • an isonucleotide analog of any one of the previous embodiments comprises one or more sugar ring(s), each comprising the adenosine or the cytosine group at the 2’ carbon position versus the 1’ carbon position, and the R1 group at the 1 ’ carbon position versus the 2’ carbon position.
  • composition is a cCAMP isonucleotide analog according to:
  • R6 or R7 is an adenine moiety or an analog or derivative thereof, and, when not an adenine moiety or an analog or derivative thereof, R6 or R7 is selected from hydrogen, hydroxyl, and fluorine groups;
  • R8 or R9 is a cytidine moiety or an analog or derivative thereof, and, when not a cytidine moiety or an analog or a derivative thereof, R8 or R9 is selected from hydrogen, hydroxyl, and fluorine groups; each RIO is independently selected from oxygen and sulfur groups; and each R11 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R11 is optionally further substituted.
  • composition is a cACCMP isonucleotide analog wherein:
  • R6 or R7 is an adenine group or an analog or derivative thereof, and, when not an adenine group or an analog or derivative thereof, R6 or R7 is selected from hydrogen, hydroxyl, and fluorine groups; at each occurrence, R8 or R9 is a cytidine group or an analog or derivative thereof, and, at each occurrence, when not a cytidine group or an analog or a derivative thereof, R8 or R9 is selected from hydrogen, hydroxyl, and fluorine groups; each RIO is independently selected from oxygen and sulfur groups; and each R11 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R11 is optionally further substituted.
  • the adenine moiety of an isonucleotide analog of any one of the previous embodiments is according to: analog or derivative thereof; and/or the cytidine moiety of an isonucleotide analog of any one of the previous embodiments is according to: analog or derivative thereof; wherein each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and a detectable labels, and wherein each R4 can be optionally further substituted.
  • a composition is according to any one of the previous embodiments, wherein Rl, R6 or R7, and/or R8 or R9 is/are a fluorine group; R2 or R10 is a sulfur group; R3 or Rl 1 is selected from a methyl group, a hydroxyl group, and alkoxyl (-OR5) groups, wherein R5 is a C1-C10 alkyl group optionally substituted with a C1-C10 alkyl ester group, optionally wherein R5 is a group; and/or R4 is selected from a hydrogen group, a methyl group, and an alkyne group selected from cycloalkyne groups, optionally wherein the cycloalkyne group is a cyclooctyne group, or a detectable label, optionally selected from biotin, fluorophore, and a radiolabel.
  • a composition is according to any one of the previous embodiments, wherein the composition is a prodrug composition.
  • Illa, Illb, inc, Illd, or IV is according to:
  • R11 of a composition according to Formula III, Illa, Illb, inc, Hid, or IV is selected from a hydroxyl and alkoxyl (-OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group.
  • group, and/or the composition is a prodrug composition.
  • the present disclosure provides pharmaceutical compositions comprising one or more composition(s) of any one of the previous embodiments, or pharmaceutically acceptable salt(s) thereof.
  • the present disclosure provides methods of modulating immune signaling in a cell.
  • a method comprises administering one or more composition(s) of any one of the previous embodiments to the cell.
  • the present disclosure provides methods of modulating an immune signaling in a subject in need thereof.
  • a method of any one of the previous embodiments comprises administering an effective amount of one or more pharmaceutical composition(s) of any one of the previous embodiments to the subject.
  • a method of any one of the previous embodiments administers composition(s) or pharmaceutical composition(s) of any one of the previous embodiments via a delivery vehicle comprising liposomes, lipid particles, or nanoparticles.
  • the present disclosure provides methods of identifying one or more pathways modulated by one or more composition(s) of any one of the previous embodiments.
  • a method comprises delivering the composition(s) to a cell, and evaluating differential expression of one or more genes of the one or more pathways by RNAseq to thereby identify the one or more pathways.
  • FIG. 1 depiction of methodology approach for discovery of example molecules.
  • FIG. 2 - depiction of 2D metabolomics approach reveals a diverse array of dsRNA- induced second messengers.
  • FIG. 3A-3B - (A) schematic showing method of discovery using linkage and basespecific nuclease to enable the diagnosis of molecule structure of unknown molecules; (B) digestion with enzymes enables assignment of structure to example novel metabolites.
  • FIG. 4 depiction of analytical results and predicted structure identifying 3’,3’- cCAMP.
  • FIG. 5A-5B - (A) sensitivity and resistance to enzymes rule out a linear structure for putative cCAMP; (B) sensitivity and resistance to enzymes suggest cCAMP is 3’,3’-cCAMP.
  • FIG. 6 example alternative phosphodiester linkage isomers of cCAMP, circles indicate chemical modification relative to natural 3’,3’-cCAMP.
  • FIG. 7 example fluorinated analogs of cCAMP, circles indicate chemical modification relative to natural 3 ’,3 ’-cCAMP.
  • FIG. 8 example phosphorothioate analogs of cCAMP, circles indicate chemical modification relative to natural 3 ’,3 ’-cCAMP.
  • FIG. 9 example methylphosphonate analogs of cCAMP, circles indicate chemical modification relative to natural 3 ’,3 ’-cCAMP.
  • FIG. 10 example base modified analogs of cCAMP, circles indicate chemical modification relative to natural 3 ’,3 ’-cCAMP.
  • FIG. 11 depiction of analytical results and predicted structure identifying 3’,3’,3’- cACCMP
  • FIG. 12 example alternative phosphodiester linkage isomers of cACCMP, circles indicate chemical modification relative to natural 3’,3’,3’-cCAMP.
  • FIG. 13 example fluorinated analogs of cACCMP, circles indicate chemical modification relative to natural 3 ’,3 ’,3 ’-cACCMP.
  • FIG. 14 example phosphorothioate analogs of cACCMP, circles indicate chemical modification relative to natural 3’, 3’3’, -cACCMP.
  • FIG. 15 example methylphosphonate analogs of cACCMP, circles indicate chemical modification relative to natural 3 ’,3 ’,3’- cACCMP.
  • FIG. 16 example base modified analogs of cACCMP, circles indicate chemical modification relative to natural 3 ’,3 ’,3’- cACCMP.
  • FIG. 17 example cCAMP nucleotides and/or isonucleotides, where: if R1 is an adenine group, then R2 is either H, OH, or F; if R2 is an adenine group, then R1 is H or OH; if R3 is a cytosine group, then R4 is either H, OH or F; if R4 is a cytosine group, then R3 is H or OH; and/or where R5 is either an H or a handle, an acyl group with carbon tail, etc.
  • FIG. 18 example 3’,3’-cCAMP with an isonucleotidic AMP group.
  • FIG. 19 example 3’,3’-cCAMP with an isonucleotidic CMP group.
  • FIG. 20 example cACCMP nucleotides and/or isonucleotides, where: if R1 is an adenine group, then R2 is either H, OH, or F; if R2 is an adenine group, then R1 is H or OH; if R3 is a cytosine group, then R4 is either H, OH or F; if R4 is a cytosine group, then R3 is H or OH; and/or where R5 is either an H or a handle, an acyl group with carbon tail, etc.
  • a “biological sample” can contain whole cells and/or live cells and/or cell debris.
  • the biological sample can contain (or be derived from) a “bodily fluid”.
  • the present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof.
  • Biological samples include cell cultures, bodily fluids,
  • subject refers to a vertebrate, preferably a mammal, more preferably a human.
  • Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • group refers to a chemical entity that is monovalent (i.e., comprises one terminus that can be covalently bonded to other chemical species), divalent, or polyvalent (i.e., comprises two or more termini that can be covalently bonded to other chemical species).
  • group also includes radicals (e.g., monovalent and multivalent, such as, for example, divalent radicals, trivalent radicals, and the like).
  • radicals e.g., monovalent and multivalent, such as, for example, divalent radicals, trivalent radicals, and the like.
  • Illustrative, non-limiting examples of groups include:
  • aliphatic group refers to branched or unbranched hydrocarbon groups that, optionally, contain one or more degree(s) of unsaturation. Degrees of unsaturation include, but are not limited to, carbon-carbon double bonds and carbon-carbon triple bonds. Non-limiting examples, of aliphatic groups with one or more degree(s) of unsaturation include alkenyl groups, alkynyl groups, and aliphatic cyclic groups, and the like. An aliphatic group can be an alkyl group.
  • an aliphatic group is a Ci to C20 aliphatic group, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., Ci, C2, C3, C4, C5, C6, C7, C8, C9, C10, Cll, C12, C13, C14, C15, C16, C17, C18, C19, and C20, Cl to C10. C3 to C10, and C5 to C10).
  • An aliphatic group can be unsubstituted or substituted with one or more substituent(s).
  • substituent groups include, but are not limited to, various substituents such as, for example, halide groups (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups (e.g., a Cl to C30 alkyl groups (e.g., methyl, ethyl, propyl, and the like), monocycloalkyl groups (e.g., cyclohexyl, cyclopentyl, and the like), polycycloalkyl groups (e.g., bicyclic groups, and the like), alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group and the like), aryl groups (e.g., phenyl group and the like), polyaryl groups (e.g., pyrenyl group, and the like), halogenated aryl groups, hydroxyl groups, amine groups,
  • alkyl groups include, but are not limited to, methyl groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert-butyl groups, cyclohexyl groups, and structural analogs thereof.
  • a substituent group of an aliphatic group can be further substituted with one or more substituent group(s) described herein.
  • alkyl group refers to branched or unbranched hydrocarbon groups that include only single bonds between carbon atoms (not including substituent(s), if any).
  • an alkyl group is a saturated group.
  • an alkyl group is a Ci to C20 alkyl group, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., Ci, C2, C3, C4, C5, C6, C7, C8, C9, C10, Cll, C12, C13, C14, C15, C16, C17, C18, C19, and C20, Cl to C10. C3 to C10, and C5 to C10).
  • an alkyl group is a cycloalkyl group, e.g., a monocycloalkyl group or a polycycloalkyl group (e.g. bicyclic and the like). In various examples, an alkyl group is unsubstituted or substituted with one or more substituent group(s).
  • substituent groups include, but are not limited to, various substituents such as, for example, halide groups (-F, -Cl, - Br, and -I), aliphatic groups (e.g., alkyl groups (e.g., a Cl to C20 alkyl groups (e.g., methyl, ethyl, propyl, and the like), monocycloalkyl groups (e.g., cyclohexyl, cyclopentyl, and the like), polycycloalkyl groups (e.g., bicyclic groups, and the like), alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group and the like), aryl groups (e.g., phenyl group and the like), polyaryl groups (e.g., pyrenyl group, and the like), halogenated aryl groups, hydroxyl groups, amine groups, nitro
  • alkyl groups include, but are not limited to, methyl groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert-butyl groups, cyclohexyl groups, and structural analogs thereof.
  • a substituent group of an alkyl group can be further substituted with one or more substituent group(s) described herein.
  • alkenyl group refers to branched or unbranched hydrocarbon groups comprising one or more C-C double bond(s).
  • alkenyl groups include, but are not limited to, an ethenyl (vinyl) group, 1 -propenyl groups, 2- propenyl (allyl) groups, 1-, 2-, and 3-butenyl groups, isopropenyl groups, and the like.
  • an alkenyl group is a Ci to C20 alkenyl group, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., Ci, C2, C3, C4, C5, C6, C7, C8, C$>, C10, Cu, C12, C13, C14, C15, C16, C17, C18, C19, and C20, Cl to C10. C3 to C10, and C5 to C10).
  • An alkenyl group can be unsubstituted or substituted with one or more substituent(s).
  • substituent groups include, but are not limited to, various substituents such as, for example, halide groups (- F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups (e.g., a Cl to C30 alkyl groups (e.g., methyl, ethyl, propyl, and the like), monocycloalkyl groups (e.g., cyclohexyl, cyclopentyl, and the like), polycycloalkyl groups (e.g., bicyclic groups, and the like), alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group and the like), aryl groups (e.g., phenyl group and the like), polyaryl groups (e.g., pyrenyl group, and the like), halogenated aryl groups, hydroxyl groups, amine groups,
  • alkyl groups include, but are not limited to, methyl groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert-butyl groups, cyclohexyl groups, and structural analogs thereof.
  • a substituent group of an alkenyl group can be further substituted with one or more substituent group(s) described herein.
  • alkynyl group refers to branched or unbranched hydrocarbon groups comprising one or more C-C triple bond(s).
  • alkynyl groups include, but are not limited to an ethyne group, 1- and 2-propyne groups, 1-, 2-, and 3-butyne groups, and the like.
  • an alkynyl group is a Ci to C20 alkynyl group, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., Cl, C2, C3, C4, C5, C6, C7, C8, C 9 , C10, Cll, C12, C13, C14, C15, C16, C17, C18, C19, and C20, Cl to C10, C3 to C10, and C5 to C10).
  • An alkynyl group can be unsubstituted or substituted with one or more substituent(s).
  • An alkynyl group can be unsubstituted or substituted with one or more substituent(s).
  • substituent groups include, but are not limited to, various substituents such as, for example, halide groups (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups (e.g., a Cl to C30 alkyl groups (e.g., methyl, ethyl, propyl, and the like), monocycloalkyl groups (e.g., cyclohexyl, cyclopentyl, and the like), poly cycloalkyl groups (e.g., bicyclic groups, and the like), alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group and the like), aryl groups (e.g., phenyl group and the like), polyaryl groups (e.g., pyrenyl group, and the like), halogenated aryl groups, hydroxyl groups, amine groups,
  • alkyl groups include, but are not limited to, methyl groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert-butyl groups, cyclohexyl groups, and structural analogs thereof.
  • a substituent group of an alkynyl group can be further substituted with one or more substituent group(s) described herein.
  • aryl group refers to C5 to C30 aromatic or partially aromatic carbocyclic groups, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., C5, C6, C7, C8, C9, C10, Cll, C12, C13, C14, C15, C16, C17, C18, C19 C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, and C30).
  • Aryl groups can comprise polyaryl groups such as, for example, fused ring groups, biaryl groups, and the like, and any combination thereof.
  • the aryl group is unsubstituted or substituted with one or more substituent group(s).
  • substituent groups include, but are not limited to, substituents such as, for example, halide groups (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group and the like), aryl groups, halogenated aryl groups, hydroxyl groups, amine groups, nitro groups, cyano groups, isocyano groups, silyl groups, alkoxide groups, alcohol groups, ether groups, ketone groups, carboxylate groups, carboxylic acid groups, ester groups, amide groups, thioether groups, structural analogs thereof, and the like, and any combination thereof.
  • Aryl groups can contain hetero atoms, such as, for example, oxygen, nitrogen (e.g., pyridinyl groups and the like), sulfur, and the like, and any combination thereof.
  • aryl groups include, but are not limited to, phenyl groups, biaryl groups (e.g., biphenyl groups and the like), fused ring groups (e.g., naphthyl groups and the like), hydroxybenzyl groups, tolyl groups, xylyl groups, furanyl groups, benzofuranyl groups, indolyl groups, imidazolyl groups, benzimidazolyl groups, pyridinyl groups, structural analogs thereof, and the like.
  • a substituent group of an aryl group can be further substituted with one or more substituent group(s) described herein.
  • analog refers to any compound or group that can be envisioned to arise from an original compound or group, respectively, if one atom or group of atoms, functional groups, or substructures is replaced with another atom or group of atoms, functional groups, substructures, or the like. Examples of analogs include, but are not limited to isomers, homologs, and the like. In various examples, an analog is not a functional analog (e.g., does not exhibit significantly different physical, chemical, biochemical, or pharmacological properties from the original compound or group).
  • the term “derivative” refers to any compound or group that is derived from an original compound or group, respectively, by a chemical reaction, where the compound or group is modified or partially substituted such that at least one structural feature of the original compound or group is retained.
  • “Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The absolute stereochemistry is specified according to the Cahn-Ingold-Prelog R-S system When a compound is an enantiomer, the stereochemistry at each chiral carbon can be specified by either R or S.
  • Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line.
  • Certain of the compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry at each asymmetric atom, as (R)- or (S)-.
  • the present chemical entities, pharmaceutical compositions and methods are meant to include all such possible isomers, including racemic mixtures, optically substantially pure forms and intermediate mixtures.
  • stereocenters can be identified with "wavy" bonds indicating that the stereocenter can be in the R or S configuration, unless otherwise specified. However, stereocenters without a wavy bond (i.e., a "straight" bond) can also be in the (R) or (S) configuration, unless otherwise specified.
  • Compositions comprising compounds can comprise stereocenters which each can independently be in the (R) configuration, the (S) configuration, or racemic mixtures.
  • a bond substitution coming out of a ring means that the substitution can be at any of the available position on the ring.
  • Embodiments disclosed herein provide cyclic nucleotides generated in cells in response to innate immune stimulation with double stranded RNA (dsRNA).
  • the molecules disclosed herein can be useful as chemical tools or therapeutic agents, as cellular receptors for the molecules are likely modulated by the cyclic nucleotides.
  • Cyclic dinucleotide molecules and cyclic trinucleotide molecules are provided.
  • cyclic CMP -AMP molecules cyclic CMP -AMP molecules
  • cACCMP molecules cyclic AMP-CMP-CMP molecules
  • Compositions of the cyclic nucleotide molecules, including analogs or derivatives thereof are also provided.
  • Nonnatural analogs and derivatives of the molecules are also provided.
  • Example nonnatural analogs include isonucleotide analogs.
  • Example nonnatural analogs include isomers with phosphodiester linkages. Such isomers with nonnatural phosphodiester linkages can modulate target specificity or biological activity.
  • Cyclic analogs can comprise additional functional groups, for example, one or more fluorines, to enhance cell permeability.
  • Cyclic nucleotide derivatives and analogs detailed herein are provided with enhanced nuclease resistance relative to the naturally occurring cyclic nucleotides, for example, via phosphorothioation or methylphosphonation.
  • cyclic nucleotides modified via labels, e.g., biotin, or other modification, e.g., methylation are also provided and can be useful for applications including chemical probes.
  • Molecules and compositions can be utilized in methods, including methods of modulating immune signaling in a cell.
  • the molecule or composition is administered via a delivery vehicle.
  • methods of identifying one or more pathways modulated by the cyclic nucleotide molecules and compositions are provided.
  • Cyclic nucleotide compositions comprising a cyclic dinucleotide molecule comprising a cytidine moiety and an adenosine moiety, a cyclic trinucleotide molecule comprising an adenosine moiety and two cytidine moieties, an analog or derivative thereof, or any combination thereof are detailed herein.
  • cCAMP Molecules comprising a cyclic dinucleotide molecule comprising a cytidine moiety and an adenosine moiety, a cyclic trinucleotide molecule comprising an adenosine moiety and two cytidine moieties, an analog or derivative thereof, or any combination thereof are detailed herein.
  • compositions detailed herein comprise a cyclic dinucleotide molecule, an analog or derivative thereof, or any combination thereof.
  • the cyclic dinucleotide molecule comprises one adenosine monophosphate and one cytidine monophosphate (cCAMP molecule).
  • the cCAMP molecule comprises a phosphodiester linkage analog (e.g., a phosphodiester linkage isomer).
  • the cyclic dinucleotide molecule is an n’,n’ cCAMP analog, wherein each n’ is 2’ or 3’ (e.g., where n’,n’ indicates the carbon numbers of the AMP and CMP sugar rings, respectively, linked by a phosphodiester linkage to the 5’ sugar ring carbon of the CMP and AMP sugar rings, respectively).
  • a composition comprising a cCAMP molecule, analog or derivative thereof, or any combination thereof is a pharmaceutically active composition.
  • a composition comprising a cCAMP molecule, analog or derivative thereof, or any combination thereof is a prodrug composition.
  • the cCAMP molecule is a 3’3’ cCAMP analog according to the formula:
  • each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups
  • each R2 is independently selected from oxygen and sulfur groups
  • each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted
  • each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups and detectable labels, wherein each R4 is optionally further substituted.
  • the cCAMP molecule is a 2’, 3’ cCAMP analog according to the formula:
  • each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups
  • each R2 is independently selected from oxygen and sulfur groups
  • each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted
  • each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups and detectable labels, wherein each R4 is optionally further substituted.
  • the cCAMP molecule is a 3 ’,2’ cCAMP analog according to the formula:
  • each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups
  • each R2 is independently selected from oxygen and sulfur groups
  • each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted
  • each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups and detectable labels, wherein each R4 is optionally further substituted.
  • the cCAMP molecule is a 2’, 2’ cCAMP analog according to the formula:
  • each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups
  • each R2 is independently selected from, oxygen and sulfur groups
  • each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted
  • each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups and detectable labels, wherein each R4 is optionally further substituted.
  • compositions according to any one of Formula la, lb, Ic, or Id can have various substituent groups.
  • R1 is a fluorine group.
  • R2 is a sulfur group.
  • R3 is a methyl group.
  • R3 is selected from a hydroxyl group and alkoxyl (-OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group.
  • R5 is a group.
  • the composition is a prodrug composition.
  • R5 is a group and the composition is a prodrug composition.
  • R4 is a hydrogen group.
  • R4 is a methyl group.
  • R4 is an alkyne group selected from cycloalkyne and groups, optionally wherein the cycloalkyne group is a cyclooctyne group.
  • R4 is a detectable label, optionally selected from biotin, fluorophore, and a radiolabel.
  • compositions detailed herein comprise a cyclic trinucleotide molecule, an analog or derivative thereof, or any combination thereof.
  • the cyclic trinucleotide molecule comprises one adenosine monophosphate and two cytidine monophosphates (cACCMP molecule).
  • the cyclic trinucleotide is an n’,n’,n’ cACCMP analog, wherein each n’ is 2’ or 3’ (e.g., where n’,n’,n’ indicates the carbon numbers of the AMP, first CMP, and second CMP sugar rings, respectively, linked by a phosphodiester linkage to the 5’ sugar ring carbon of the first CMP, the second CMP, and the AMP sugar rings, respectively).
  • a composition comprising a cACCMP molecule, analog or derivative thereof, or any combination thereof is a pharmaceutical composition.
  • a composition comprising a cACCMP molecule, analog or derivative thereof, or any combination thereof is a prodrug composition.
  • the cACCMP molecule is a 3 ’,3 ’,3 ’-cACCMP analog according to the formula:
  • each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups
  • each R2 is independently selected from oxygen and sulfur groups
  • each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1-C10 alkyl group optionally substituted with a C1-C10 alkyl ester group, and wherein each R3 is optionally further substituted
  • each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 is optionally further substituted.
  • the cACCMP molecule is a 2’, 3’, 3’-cACCMP analog according to the formula:
  • each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups
  • each R2 is independently selected from, oxygen and sulfur groups
  • each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted
  • each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 is optionally further substituted.
  • the cACCMP molecule is a 2’,2’,3’-cACCMP analog according to the formula:
  • each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups
  • each R2 is independently selected from oxygen and sulfur groups
  • each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 can be optionally further substituted
  • each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 can be optionally further substituted.
  • the cACCMP molecule is a 2’,2’,2’-cACCMP analog according to the formula:
  • each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups
  • each R2 is independently selected from oxygen and sulfur groups
  • each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 can be optionally further substituted
  • each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 can be optionally further substituted.
  • the cACCMP molecule is a 2’,3’,2’-cACCMP analog according to the formula:
  • each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups
  • each R2 is independently selected from oxygen and sulfur groups
  • each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted
  • each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 is optionally further substituted.
  • the cACCMP molecule is a 3’, 2’, 2’-cACCMP analog according to the formula:
  • each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups
  • each R2 is independently selected from oxygen and sulfur groups
  • each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted
  • each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 is optionally further substituted.
  • the cACCMP molecule is a 3’, 2’, 3’- cACCMP analog according to the formula:
  • each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups
  • each R2 is independently selected from oxygen and sulfur groups
  • each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted
  • each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 is optionally further substituted.
  • the cACCMP molecule is a 3 ’,3 ’,2’ -cACCMP analog according to the formula:
  • each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups
  • each R2 is independently selected from oxygen and sulfur groups
  • each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1-C10 alkyl group optionally substituted with a C1-C10 alkyl ester group, and wherein each R3 is optionally further substituted
  • each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 is optionally further substituted.
  • compositions according to any one of Formula Ila, lib, lie, lid, lie, Ilf, Ilg, or Ilh can have various substituent groups.
  • R1 is a fluorine group.
  • R2 is a sulfur group.
  • R3 is a methyl group.
  • R3 is selected from a hydroxyl group and alkoxyl (-OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group.
  • R5 is a group.
  • the composition is a prodrug composition.
  • R5 is a group and the composition is a prodrug composition.
  • R4 is a hydrogen group.
  • R4 is a hydrogen group.
  • R4 is a methyl group.
  • R4 is an alkyne group selected from a cycloalkyne and groups, optionally wherein the cycloalkyne group is a cyclooctyne group.
  • R4 is a detectable label, optionally selected from biotin, fluorophore, and a radiolabel.
  • analogs and derivatives of the cyclic nucleotides include modifications or substitutions at one or more positions of the purine or pyrimidine moieties, one or more positions of the sugar ring, including sugar substitutions, and modifications and/or replacements of the phosphodiester moiety or linkage. Isomeric configurations are also contemplated for use. Each of the modifications described herein can be used in any combination independently with each of the other modifications described herein.
  • compositions detailed herein comprise a nucleotide analog of a cyclic dinucleotide molecule, a cyclic trinucleotide molecule, an analog or derivative thereof, or any combination thereof.
  • a composition comprises an isonucleotide analog of a cCAMP, a cACCMP, an analog or derivative thereof, or any combination thereof.
  • the composition comprises an isonucleotide analog of a cyclic nucleotide molecule according to any one of Formula la, lb, Ic, Id, Ila, lib, lie, lid, lie, Ilf, Ilg, or Ilh.
  • the isonucleotide analog comprises one or more sugar ring(s) each comprising the adenosine group or the cytosine group at the 2’ carbon position and the R1 group at the 1’ carbon position.
  • the R3 of the isonucleotide analog, an analog or derivative thereof, or any combination thereof is selected from a hydroxyl group and alkoxyl (-OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group.
  • R5 is a group, and/or the composition is a prodrug composition.
  • composition is a cCAMP isonucleotide analog according to:
  • R6 or R7 is an adenine moiety or an analog or derivative thereof, and, when not an adenine moiety or an analog or derivative thereof, R6 or R7 is selected from hydrogen, hydroxyl, and fluorine groups;
  • R8 or R9 is a cytidine moiety or an analog or derivative thereof, and, when not a cytidine moiety or an analog or a derivative thereof, R8 or R9 is selected from hydrogen, hydroxyl, and fluorine groups; each RIO is independently selected from oxygen and sulfur groups; and each R11 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R11 is optionally further substituted.
  • a adenine moiety is according to: wherein each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and a detectable labels, wherein each R4 can be optionally further substituted.
  • R4 is a hydrogen group.
  • R4 is a methyl group.
  • R4 is an alkyne group selected from cycloalkyne and groups, optionally wherein the cycloalkyne group is a cyclooctyne group.
  • R4 is a detectable label, optionally selected from biotin, fluorophore, and a radiolabel.
  • R11 is selected from a hydroxyl group and alkoxyl (-OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group.
  • R5 is a group.
  • the composition is a prodrug composition.
  • R5 is a o group and the composition is a prodrug composition.
  • the composition is a cACCMP isonucleotide analog of
  • R6 or R7 is an adenine group or an analog or derivative thereof, and, when not an adenine group or an analog or derivative thereof, R6 or R7 is selected from hydrogen, hydroxyl, and fluorine groups; at each occurrence, R8 or R9 is a cytidine group or an analog or derivative thereof, and, at each occurrence, when not a cytidine group or an analog or a derivative thereof, R8 or R9 is selected from hydrogen, hydroxyl, and fluorine groups; each RIO is independently selected from oxygen and sulfur groups; and each R11 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1-C10 alkyl group optionally substituted with a C1-C10 alkyl ester group, and wherein each R11 can be optional
  • a adenine moiety is according to: wherein each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and a detectable labels, and wherein each R4 can be optionally further substituted.
  • R4 is a hydrogen group.
  • R4 is a methyl group.
  • R4 is an alkyne group selected from cycloalkyne and groups, optionally wherein the cycloalkyne group is a cyclooctyne group.
  • R4 is a detectable label, optionally selected from biotin, fluorophore, and a radiolabel.
  • R3 is selected from a hydroxyl and alkoxyl (-OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group. In one example embodiment, R5 is a group. In one example embodiment, the composition is a prodrug composition. In one example embodiment, R5 is a group and the composition is a prodrug composition.
  • the 3’,3’,3’-cACCMP isonucleotide analog comprises an isonucleotidic AMP, one or more isonucleotidic CMP(s), or any combination thereof.
  • the 3’,3’,3’-cACCMP isonucleotide analog is according to:
  • a cyclic nucleotide molecule analog or derivative comprises a cyclic n’,n’ CMP -AMP analog (e.g., isomer) or a cyclic n’,n’,n’ cyclic AMP-CMP- CMP (cACCMP) analog (e.g., isomer).
  • the cCAMP molecule is a n’,n’ cCAMP analog, where n’,n’ indicates the carbon numbers of the AMP and CMP sugar rings, respectively, linked by a phosphodiester linkage to the 5’ sugar ring carbon of the CMP and AMP sugar rings, respectively.
  • the cACCMP molecule is a n’,n’,n’ cACCMP analog, where n’,n’,n’ indicates the carbon numbers of AMP, first CMP, and second CMP sugar rings, respectively, linked by a phosphodiester linkage to the 5’ sugar ring carbon of the first CMP, second CMP, and AMP sugar rings, respectively.
  • the compositions comprise a 2’, 3’ cCAMP, a 2’, 2’ cCAMP, a 3’2’ cCAMP, a 2’, 3 ’,3 ’-cACCMP, a 2’, 2’, 3 ’-cACCMP, a 2’,2’,2’-cACCMP, a 2’,3’,2’-cACCMP, a 3’,2’,2’- cACCMP a 3’,2’,3’-cACCMP, a 3’,3’,2’-cACCMP, an analog (e.g., an isonucleotide analog, a phosphodiester linkage analog, a fluorinated analog, a phosphodiester analog, a modified nucleotide base analog, or the like) or derivative thereof, or any combination thereof Fluorinated Analogs
  • an analog e.g., an isonucleotide analog, a phosphodiester linkage analog, a flu
  • a cyclic nucleotide analog or derivative comprises one or more fluorine modifications.
  • the cyclic nucleotide analog or derivative can be a mono-fluorinated or di-fluorinated cCAMP molecule, a mono-fluorinated, di-fluorinated, or tri-fluorinated cACCMP molecule, analog (e.g., an isonucleotide analog, a phosphodiester linkage analog, a phosphodiester analog, a modified nucleotide base analog, or the like) or derivative thereof, or any combination thereof.
  • the fluorinated cyclic nucleotide molecule, analog or derivative thereof, or any combination thereof has increased cell permeability relative to a cyclic nucleotide that lacks fluorination. Additional modifications at the Ri can comprise amino, methoxy, and azido groups.
  • a cyclic nucleotide analog or derivative comprises one or more phosphorothioate modifications.
  • the cyclic nucleotide analog or derivative can be a mono-phosphorothioate or di- phosphorothioate cCAMP molecule, or a mono-phosphorothioate di-phosphorothioate, or tri-phosphorothioate cACCMP molecule, analog (e.g., an isonucleotide analog, a phosphodiester linkage analog, a fluorinated analog, a methylphosphonate modified analog, a modified nucleotide base analog, or the like) or derivative thereof, or any combination thereof.
  • analog e.g., an isonucleotide analog, a phosphodiester linkage analog, a fluorinated analog, a methylphosphonate modified analog, a modified nucleotide base analog, or the like
  • the phosphorothioate cyclic nucleotide molecule has increased resistance to cellular nucleases relative to a cyclic nucleotide molecule that lacks the phosphorothioate modification.
  • a cyclic nucleotide molecule comprises one or more methylphosphonate modifications.
  • the cyclic nucleotide can be a mono-methylphosphonate or di-methylphosphonate cCAMP molecule, a mono- methylphosphonate, di-methylphosphonate, or tri-methylphosphonate cACCMP, an analog (e.g., an isonucleotide analog, a phosphodiester linkage analog, a fluorinated analog, a phosphorothioate modified analog, a modified nucleotide base analog, or the like) or derivative thereof, or any combination thereof.
  • an analog e.g., an isonucleotide analog, a phosphodiester linkage analog, a fluorinated analog, a phosphorothioate modified analog, a modified nucleotide base analog, or the like
  • the methylphosphonate cyclic nucleotide molecule has increased resistance to cellular nucleases relative to a cyclic nucleotide molecule that lacks the methylphosphonate modifications.
  • a cyclic nucleotide molecule comprises one or more nucleotide base modifications.
  • the cyclic nucleotide can be a mono-, or di-, or tri -nucleotide base-substituted cCAMP molecule, a mono-, di-, tri-, tetra- or penta-nucleotide base-substituted cACCMP molecule, an analog (e.g., an isonucleotide analog, a phosphodiester linkage analog, a fluorinated analog, a phosphodiester analog, or the like) or derivative thereof, or any combination thereof.
  • an analog e.g., an isonucleotide analog, a phosphodiester linkage analog, a fluorinated analog, a phosphodiester analog, or the like
  • the cyclic nucleotide molecule is substituted with a chemical handle.
  • a cyclic nucleotide can comprise a handle via a click chemistry substitution at R4 of any one of Formula la, lb, Ic, Id, Ila, lib, lie, lid, lie, Ilf, Ilg, Ilh, III, IIIa-IIID, or IV, III, IIIa- IIID, or IV.
  • exemplary click chemistry molecules can comprise trans-cyclooctene, cyclooctyne, or terminal alkyne, [e.g.
  • BCN amine N-(lR,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl- l,8-diamino-3,6-di oxaoctane
  • BCN amine reagent N-(lR,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl- l,8-diamino-3,6-di oxaoctane
  • one or more of R4 is an alkyne selected from a cycloalkyne and .
  • the cycloalkyne is a cyclooctyne.
  • a detectable label can comprise radiolabels, biotin labels, fluorophores or other optical tags, including amine-reactive dyes.
  • the cyclic nucleotide molecules can comprise one or more non-naturally occurring nucleotide or nucleotide analog such as a nucleotide with phosphorothioate linkage, boranophosphate linkage, a locked nucleic acid (LNA) nucleotides comprising a methylene bridge between the 2’ and 4’ carbons of the ribose ring, an amino-LNA or thio-LNA, peptide nucleic acids (PNA), or bridged nucleic acids (BNA).
  • LNA locked nucleic acid
  • modified nucleotides include 2'- O-methyl analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, or 2'- fluoro analogs.
  • Further examples of modification to one or more nucleotides in the cyclic nucleotides disclosed herein include further linkage of chemical moieties at the 2' position, including but not limited to peptides, nuclear localization sequence (NLS), peptide nucleic acid (PNA), polyethylene glycol (PEG), triethylene glycol, or tetraethyleneglycol (TEG).
  • Synthetic nucleic acid analogies comprising a different sugar backbone, e.g.
  • xeno nucleic acid are also included as analogs that can be incorporated in the compositions detailed herein., and can include 1,5-anhydrohexitol nucleic acid (HNA), Cyclohexene nucleic acid (CeNA), Threose nucleic acid (TNA), and glycol nucleoic acid (GNA)., and 2'-Fluoro-arabinonucleic Acid (FANA).
  • HNA 1,5-anhydrohexitol nucleic acid
  • CeNA Cyclohexene nucleic acid
  • TPA Threose nucleic acid
  • GNA glycol nucleoic acid
  • FANA 2'-Fluoro-arabinonucleic Acid
  • XNAs can be chosen for particular applications, including, e.g., probing biomolecular interactions.
  • Methods of use of the compositions include use of the cyclic nucleotides in modulating immune signaling in the cell, which can comprise administering any of the compositions as disclosed herein to a cell. Methods of modulating immune signaling in a subject in need thereof can comprise administration of an effective amount a pharmaceutical composition as detailed herein. Further methods of identifying novel cyclic nucleotide molecules, pathways modulated by the molecules, and cell receptors of the molecules are also provided.
  • the cyclic nucleotides, their analogs and derivatives can be utilized to module immune signaling in a cell.
  • modulate broadly denotes a qualitative and/or quantitative alteration, change or variation in that which is being modulated. Where modulation can be assessed quantitatively - for example, where modulation comprises or consists of a change in a quantifiable variable such as a quantifiable property of a cell or where a quantifiable variable provides a suitable surrogate for the modulation - modulation specifically encompasses both increase (e g., activation) or decrease (e.g., inhibition) in the measured variable.
  • modulation can encompass an increase in the value of the measured variable by at least about 10%, e.g., by at least about 20%, preferably by at least about 30%, e.g., by at least about 40%, more preferably by at least about 50%, e.g., by at least about 75%, even more preferably by at least about 100%, e.g., by at least about 150%, 200%, 250%, 300%, 400% or by at least about 500%, compared to a reference situation without said modulation; or modulation can encompass a decrease or reduction in the value of the measured variable by at least about 10%, e.g., by at least about 20%, by at least about 30%, e.g., by at least about 40%, by at least about 50%, e.g., by at least about 60%, by at least about 70%, e.
  • modulating or “to modulate” generally means either reducing or inhibiting the expression or activity of, or alternatively increasing the expression or activity of a target or antigen.
  • modulating or “to modulate” can mean either reducing or inhibiting the activity of, or alternatively increasing a (relevant or intended) biological activity of, a target or antigen as measured using a suitable in vitro, cellular or in vivo assay (which will usually depend on the target involved), by at least 5%, at least 10%, at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more, compared to activity of the target in the same assay under the same conditions but without the presence of an agent.
  • an “increase” or “decrease” refers to a statistically significant increase or decrease respectively.
  • an increase or decrease will be at least 10% relative to a reference, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, a t least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or more, up to and including at least 100% or more, in the case of an increase, for example, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, or more.
  • Modulating can also involve effecting a change (which can either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of a target or antigen. “Modulating” can also mean effecting a change with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the target or antigen (or in which its substrate(s), ligand(s) or pathway(s) are involved, such as its signaling pathway or metabolic pathway and their associated biological or physiological effects) is involved.
  • such an action as an agonist or an antagonist can be determined in any suitable manner and/or using any suitable assay known or described herein (e.g., in vitro or cellular assay), depending on the target or antigen involved.
  • Modulating can, for example, also involve allosteric modulation of the target and/or reducing or inhibiting the binding of the target to one of its substrates or ligands and/or competing with a natural ligand, substrate for binding to the target. Modulating can also involve activating the target or the mechanism or pathway in which it is involved. Modulating can for example also involve effecting a change in respect of the folding or confirmation of the target, or in respect of the ability of the target to fold, to change its conformation (for example, upon binding of a ligand), to associate with other (sub)units, or to disassociate. Modulating can for example also involve effecting a change in the ability of the target to signal, phosphorylate, dephosphorylate, and the like.
  • agent broadly encompasses any condition, substance or agent capable of modulating one or more phenotypic aspects of a cell or cell population as disclosed herein. Such conditions, substances or agents can be of physical, chemical, biochemical and/or biological nature.
  • the agent is a cyclic nucleotide, derivative, analog, or any combination thereof, as described herein.
  • candidate agent refers to any condition, substance or agent that is being examined for the ability to modulate one or more phenotypic aspects of a cell or cell population as disclosed herein in a method comprising applying the candidate agent to the cell or cell population (e.g., exposing the cell or cell population to the candidate agent or contacting the cell or cell population with the candidate agent) and observing whether the desired modulation takes place.
  • Agents can include any potential class of biologically active conditions, substances or agents, such as for instance antibodies, proteins, peptides, nucleic acids, oligonucleotides, small molecules, or combinations thereof, as described herein.
  • the methods of phenotypic analysis can be utilized for evaluating environmental stress and/or state, for screening of chemical libraries, and to screen or identify structural, syntenic, genomic, and/or organism and species variations.
  • a culture of cells can be exposed to an environmental stress, such as but not limited to heat shock, osmolarity, hypoxia, cold, oxidative stress, radiation, starvation, a chemical (for example a therapeutic agent or potential therapeutic agent) and the like.
  • a representative sample can be subjected to analysis, for example at various time points, and compared to a control, such as a sample from an organism or cell, for example a cell from an organism, or a standard value.
  • a further aspect of the invention relates to a method for identifying the modulating of one or more phenotypic aspects of a cell or cell population as disclosed herein, comprising: a) applying a candidate agent to the cell or cell population; b) detecting modulation of one or more phenotypic aspects of the cell or cell population by the candidate agent, thereby identifying the agent.
  • the phenotypic aspects of the cell or cell population that is modulated can be a gene signature or biological program specific to a cell type or cell phenotype or phenotype specific to a population of cells (e.g., an inflammatory phenotype or suppressive immune phenotype).
  • steps can include administering candidate modulating agents to cells, detecting identified cell (sub)populations for changes in signatures, or identifying relative changes in cell (sub) populations which can comprise detecting relative abundance of particular gene signatures.
  • aspects of the present disclosure relate to the correlation of an agent with the spatial proximity and/or epigenetic profile of the nucleic acids in a sample of cells.
  • the disclosed methods can be used to screen the compositions for modulation of chromatin architecture epigenetic profiles, and/or relationships thereof.
  • screening of test agents involves testing a combinatorial library containing a large number of potential modulator compounds.
  • a combinatorial chemical library can be a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library, is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (for example the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • the present invention provides for gene signature screening.
  • signature screening was introduced by Stegmaier et al. (Gene express! on -based high-throughput screening (GE-HTS) and application to leukemia differentiation. Nature Genet. 36, 257-263 (2004)), who realized that if a gene-expression signature was the proxy for a phenotype of interest, it could be used to find small molecules that effect that phenotype without knowledge of a validated drug target.
  • the signatures or biological programs of the present invention can be used to screen for drugs that reduce the signature or biological program in cells as described herein.
  • the signature or biological program can be used for GE-HTS.
  • pharmacological screens can be used to identify drugs that are selectively toxic to cells having a signature.
  • the Connectivity Map is a collection of genome-wide transcriptional expression data from cultured human cells treated with bioactive small molecules and simple pattern-matching algorithms that together enable the discovery of functional connections between drugs, genes and diseases through the transitory feature of common gene-expression changes (see, Lamb et al., The Connectivity Map: Using Gene-Expression Signatures to Connect Small Molecules, Genes, and Disease. Science 29 Sep 2006: Vol. 313, Issue 5795, pp. 1929-1935, DOI: 10.1126/science.1132939; and Lamb, J., The Connectivity Map: a new tool for biomedical research. Nature Reviews Cancer January 2007: Vol. 7, pp. 54-60).
  • Cmap can be used to screen for small molecules capable of modulating a signature or biological program of the present invention in silica.
  • Detection of differential expression of one or more genes for identification of one or more pathways modulated by the compositions is provided. It is to be understood that also when referring to proteins (e.g. differentially expressed proteins), such can fall within the definition of “gene” signature. Levels of expression or activity or prevalence can be compared between different cells in order to characterize or identify for instance signatures specific for cell (sub)populations. Increased or decreased expression or activity of signature genes can be compared between different cells in order to characterize or identify for instance specific cell (sub)populations. The detection of a signature in single cells can be used to identify and quantitate for instance specific cell (sub)populations.
  • proteins e.g. differentially expressed proteins
  • a signature can include a gene or genes, protein or proteins, or epigenetic element(s) whose expression or occurrence is specific to a cell (sub)population, such that expression or occurrence is exclusive to the cell (sub)population.
  • a gene signature as used herein can thus refer to any set of up- and down-regulated genes that are representative of a cell type or subtype.
  • a gene signature as used herein can also refer to any set of up- and down-regulated genes between different cells or cell (sub)populations derived from a gene-expression profile.
  • a gene signature can comprise a list of genes differentially expressed in a distinction of interest.
  • the signature as defined herein can be used to indicate the presence of a cell type, a subtype of the cell type, the state of the microenvironment of a population of cells, a particular cell type population or subpopulation, and/or the overall status of the entire cell (sub)population. Furthermore, the signature can be indicative of cells within a population of cells in vivo.
  • the signature according to certain embodiments of the present invention can comprise or consist of one or more genes, proteins and/or epigenetic elements, such as for instance 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.
  • the signature can comprise or consist of two or more genes, proteins and/or epigenetic elements, such as for instance 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.
  • the signature can comprise or consist of three or more genes, proteins and/or epigenetic elements, such as for instance 3, 4, 5, 6, 7, 8, 9, 10 or more.
  • the signature can comprise or consist of four or more genes, proteins and/or epigenetic elements, such as for instance 4, 5, 6, 7, 8, 9, 10 or more.
  • the signature can comprise or consist of five or more genes, proteins and/or epigenetic elements, such as for instance 5, 6, 7, 8, 9, 10 or more. In certain embodiments, the signature can comprise or consist of six or more genes, proteins and/or epigenetic elements, such as for instance 6, 7, 8, 9, 10 or more. In certain embodiments, the signature can comprise or consist of seven or more genes, proteins and/or epigenetic elements, such as for instance 7, 8, 9, 10 or more. In certain embodiments, the signature can comprise or consist of eight or more genes, proteins and/or epigenetic elements, such as for instance 8, 9, 10 or more. In certain embodiments, the signature can comprise or consist of nine or more genes, proteins and/or epigenetic elements, such as for instance 9, 10 or more.
  • the signature can comprise or consist of ten or more genes, proteins and/or epigenetic elements, such as for instance 10, 11, 12, 13, 14, 15, or more. It is to be understood that a signature according to the invention can for instance also include genes or proteins as well as epigenetic elements combined.
  • a signature is characterized as being specific for a particular target cell or target cell (sub)population if it is upregulated or only present, detected or detectable in that particular target cell or target cell (sub)population, or alternatively is downregulated or only absent, or undetectable in that particular target cell or target cell (sub)population.
  • a signature consists of one or more differentially expressed genes/proteins or differential epigenetic elements when comparing different cells or cell (sub)populations, including comparing different target cell or target cell (sub)populations, as well as comparing target cell or target cell (sub)populations with non-target cell or non-target cell (sub)populations.
  • genes/proteins include genes/proteins which are up- or down-regulated as well as genes/proteins which are turned on or off.
  • up- or down-regulation in certain embodiments, such up- or down-regulation is preferably at least two-fold, such as two-fold, three-fold, four-fold, five-fold, or more, such as for instance at least ten-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or more.
  • differential expression can be determined based on common statistical tests, as is known in the art.
  • differentially expressed genes/proteins, or differential epigenetic elements can be differentially expressed on a single cell level, or can be differentially expressed on a cell population level.
  • the differentially expressed genes/ proteins or epigenetic elements as discussed herein, such as constituting the gene signatures as discussed herein, when as to the cell population or subpopulation level refer to genes that are differentially expressed in all or substantially all cells of the population or subpopulation (such as at least 80%, preferably at least 90%, such as at least 95% of the individual cells). This allows one to define a particular subpopulation of target cells.
  • a “subpopulation” of cells preferably refers to a particular subset of cells of a particular cell type which can be distinguished or are uniquely identifiable and set apart from other cells of this cell type.
  • the cell subpopulation can be phenotypically characterized and is preferably characterized by the signature as discussed herein.
  • a cell (sub)population as referred to herein can constitute of a (sub)population of cells of a particular cell type characterized by a specific cell state.
  • induction or alternatively suppression of a particular signature preferable is meant induction or alternatively suppression (or upregulation or downregulation) of at least one gene/protein and/or epigenetic element of the signature, such as for instance at least two, at least three, at least four, at least five, at least six, or all genes/proteins and/or epigenetic elements of the signature.
  • Detection of differential expression can comprise single cell RNA sequencing.
  • the invention involves single cell RNA sequencing (see, e.g., Kalisky, T., Blainey, P. & Quake, S. R. Genomic Analysis at the Single-Cell Level. Annual review of genetics 45, 431-445, (2011); Kalisky, T. & Quake, S. R. Single-cell genomics. Nature Methods 8, 311- 314 (2011); Islam, S. et al. Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Research, (2011); Tang, F. et al. RNA-Seq analysis to capture the transcriptome landscape of a single cell.
  • the invention involves plate based single cell RNA sequencing (see, e.g., Picelli, S. et al., 2014, “Full-length RNA-seq from single cells using Smart-seq2” Nature protocols 9, 171-181, doi:10.1038/nprot.2014.006).
  • the invention involves high-throughput single-cell RNA-seq.
  • Macosko et al. 2015, “Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets” Cell 161, 1202-1214; International patent application number PCT/US2015/049178, published as WQ2016/040476 on March 17, 2016; Klein et al., 2015, “Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells” Cell 161, 1187-1201; International patent application number PCT/US2016/027734, published as WO2016168584A1 on October 20, 2016; Zheng, et al., 2016, “Haplotyping germline and cancer genomes with high-throughput linked-read sequencing” Nature Biotechnology 34, 303-311; Zheng, et al., 2017, “Massively parallel digital transcriptional profiling of single cells” Nat.
  • the invention involves single nucleus RNA sequencing.
  • Swiech et al., 2014 “In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9” Nature Biotechnology Vol. 33, pp. 102-106; Habib et al., 2016, “Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons” Science, Vol. 353, Issue 6302, pp. 925-928; Habib et al., 2017, “Massively parallel single-nucleus RNA-seq with DroNc-seq” Nat Methods.
  • the invention involves the Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq) as described, (see, e.g., Buenrostro, et al., Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature methods 2013; 10 (12): 1213-1218; Buenrostro et al., Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-490 (2015); Cusanovich, D. A., Daza, R., Adey, A., Pliner, H., Christiansen, L., Gunderson, K.
  • TPP thermal proteome profiling
  • Exemplary methods of detection and identification of novel cyclic nucleotides can comprise stimulating innate immune signaling of a population of cells, extracting polar metabolites from the cells, fractionating extract via reversed-phase high pressure liquid chromatography, and analyzing the fractions by liquid chromatography-tandem mass spectrometry (LC-MS/MS) thereby detecting novel second messenger small molecules, e.g., cyclic nucleotides.
  • Stimulating the cells typically comprises exposing the cells to double stranded RNA or double stranded DNA.
  • Elucidating the chemical structures of the small molecules identified comprises digestion with one or more enzymes to assign structure to novel metabolites, which can comprise one or more of rsAP, RNAseA, RNaseTl, RNAse T2, Nuclease SI and/or PDEII, as described in more detail in the examples.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LipofectinTM), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax.
  • vesicles such as LipofectinTM
  • DNA conjugates such as LipofectinTM
  • anhydrous absorption pastes oil-in- water and water-in-oil emulsions
  • emulsions carbowax polyethylene glycols of various molecular weights
  • semi-solid gels and semi-solid mixtures containing carbowax.
  • the medicaments of the invention are prepared in a manner known to those skilled in the art, for example, by means of conventional dissolving, lyophilizing, mixing, granulating or confectioning processes. Methods well known in the art for making formulations are found, for example, in Remington: The Science and Practice of Pharmacy, 20th ed., ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York.
  • Administration of medicaments of the invention can be by any suitable means that results in a compound concentration that is effective for treating or inhibiting (e.g., by delaying) the development of a disease.
  • the compound is admixed with a suitable carrier substance, e.g., a pharmaceutically acceptable excipient that preserves the therapeutic properties of the compound with which it is administered.
  • a suitable carrier substance e.g., a pharmaceutically acceptable excipient that preserves the therapeutic properties of the compound with which it is administered.
  • One exemplary pharmaceutically acceptable excipient is physiological saline.
  • the suitable carrier substance is generally present in an amount of 1-95% by weight of the total weight of the medicament.
  • the medicament can be provided in a dosage form that is suitable for administration.
  • the medicament can be in form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, delivery devices, injectables, implants, sprays, or aerosols.
  • Pharmaceutical Compositions including hydrogels, pastes, ointments, creams, plasters, drenches, delivery devices, injectables, implants, sprays, or aerosols.
  • compositions of the present disclosure include pharmaceutical composition comprising one or more molecules or compositions as detailed herein, or pharmaceutically acceptable salts thereof.
  • the agents disclosed herein e.g., cyclic nucleotides
  • Such compositions comprise a therapeutically effective amount of the agent and a pharmaceutically acceptable carrier.
  • Such a composition can also further comprise (in addition to an agent and a carrier) diluents, fdlers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • Compositions comprising the agent can be administered in the form of salts provided the salts are pharmaceutically acceptable. Salts can be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry.
  • salts refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids.
  • Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.
  • basic ion exchange resins such
  • pharmaceutically acceptable salt further includes all acceptable salts such as acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide, bromide, methylnitrate, calcium edetate, methylsulfate, camsylate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate, N- methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, edisylate, pamoate (embonate), estolate, palmitate, esylate, pantothenate, fumarate, phosphate/di phosphate, gluceptate, polygalacturonate, gluconate, salicylate, glutamate, stearate, glycosulfonate
  • composition of the invention can also advantageously be formulated in order to release 2’ -ATP mimetics or derivatives, and/or agonist in the subject in a timely controlled fashion.
  • composition of the invention is formulated for controlled release.
  • the agents of the present invention can be modified, such that they acquire advantageous properties for therapeutic use (e.g., stability and specificity), but maintain their biological activity.
  • the agents include a protecting group covalently joined to the N-terminal amino group.
  • a protecting group covalently joined to the N-terminal amino group of the agonists reduces the reactivity of the amino terminus under in vivo conditions.
  • Amino protecting groups include — Cl-10 alkyl, — Cl- 10 substituted alkyl, — C2-10 alkenyl, — C2-10 substituted alkenyl, aryl, — Cl-6 alkyl aryl, — C(O)— (CH2)l-6— COOH, — C(O)— Cl-6 alkyl, — C(O)-aryl, — C(O)— O— Cl-6 alkyl, or — C(O) — O-aryl.
  • the amino terminus protecting group is selected from the group consisting of acetyl, propyl, succinyl, benzyl, benzyloxycarbonyl, and t- butyloxy carbonyl.
  • deamination of the N-terminal amino acid is another modification that can be used for reducing the reactivity of the amino terminus under in vivo conditions.
  • compositions of the agents are also included within the scope of the present invention.
  • the polymer selected is usually modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be controlled.
  • Included within the scope of polymers is a mixture of polymers.
  • the polymer will be pharmaceutically acceptable.
  • the polymer or mixture thereof can include but is not limited to polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (for example, glycerol), and polyvinyl alcohol.
  • PEG polyethylene glycol
  • monomethoxy-polyethylene glycol dextran, cellulose, or other carbohydrate based polymers
  • poly-(N-vinyl pyrrolidone) polyethylene glycol propylene glycol homopolymers
  • a polypropylene oxide/ethylene oxide co-polymer for example, glycerol
  • polyoxyethylated polyols for example, glycerol
  • the present invention provides for one or more therapeutic agents.
  • the one or more agents comprises a small molecule inhibitor, small molecule degrader (e.g., PROTAC), genetic modifying agent, antibody, antibody fragment, antibody-like protein scaffold, aptamer, protein, or any combination thereof.
  • small molecule inhibitor e.g., PROTAC
  • PROTAC small molecule degrader
  • genetic modifying agent e.g., antibody, antibody fragment, antibody-like protein scaffold, aptamer, protein, or any combination thereof.
  • therapeutic agent refers to a molecule or compound that confers some beneficial effect upon administration to a subject.
  • the beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
  • treatment or “treating,” or “palliating” or “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.
  • the compositions can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom can not have yet been manifested.
  • treating includes ameliorating, curing, preventing it from becoming worse, slowing the rate of progression, or preventing the disorder from re-occurring (i.e., to prevent a relapse).
  • the present invention provides for one or more therapeutic agents against combinations of targets identified. Targeting the identified combinations can provide for enhanced or otherwise previously unknown activity in the treatment of disease.
  • PROTAC Proteolysis Targeting Chimera
  • combinations of targets are modulated (e.g., one or more targets related to cyclic nucleotide generation or modulation).
  • an agent against one of the targets in a combination can already be known or used clinically.
  • targeting the combination can require less of the agent as compared to the current standard of care and provide for less toxicity and improved treatment.
  • Methods of administrating the pharmacological compositions, including agonists, antagonists, antibodies or fragments thereof, to an individual include, but are not limited to, intradermal, intrathecal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, by inhalation, and oral routes.
  • the compositions can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (for example, oral mucosa, rectal and intestinal mucosa, and the like), ocular, and the like and can be administered together with other biologically-active agents. Administration can be systemic or local.
  • compositions into the central nervous system can be advantageous to administer by any suitable route, including intraventricular and intrathecal injection.
  • Pulmonary administration can also be employed by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • the agent can be delivered in a vesicle, in particular a liposome.
  • a liposome the agent is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. No. 4,837,028 and U.S. Pat. No. 4,737,323.
  • the pharmacological compositions can be delivered in a controlled release system including, but not limited to: a delivery pump (See, for example, Saudek, et al., New Engl. J. Med.
  • the controlled release system can be placed in proximity of the therapeutic target (e g., a tumor), thus requiring only a fraction of the systemic dose. See, for example, Goodson, In: Medical Applications of Controlled Release, 1984. (CRC Press, Boca Raton, Fla.).
  • Anderson et al. provides a modified dendrimer nanoparticle for the delivery of therapeutic, prophylactic and/or diagnostic agents to a subject, comprising: one or more zero to seven generation alkylated dendrimers; one or more amphiphilic polymers; and one or more therapeutic, prophylactic and/or diagnostic agents encapsulated therein.
  • One alkylated dendrimer can be selected from the group consisting of poly(ethyleneimine), poly(polyproylenimine), diaminobutane amine polypropylenimine tetramine and poly(amido amine).
  • Anderson et al. (US20050123596) provides examples of microparticles that are designed to release their payload when exposed to acidic conditions, wherein the microparticles comprise at least one agent to be delivered, a pH triggering agent, and a polymer, wherein the polymer is selected from the group of polymethacrylates and polyacrylates.
  • Anderson et al (US 20020150626) providing lipid-protein-sugar particles for delivery of nucleic acids, wherein the polynucleotide is encapsulated in a lipid-protein-sugar matrix by contacting the polynucleotide with a lipid, a protein, and a sugar; and spray drying mixture of the polynucleotide, the lipid, the protein, and the sugar to make microparticles.
  • Nanoparticles with one half hydrophilic and the other half hydrophobic are termed Janus particles and are particularly effective for stabilizing emulsions. They can self-assemble at water/oil interfaces and act as solid surfactants.
  • a nanolipid delivery system in particular a nano-particle concentrate, comprising: a composition comprising a lipid, oil or solvent, the composition having a viscosity of less than 100 cP at 25°C and a Kauri Butanol solvency of greater than 25 Kb; and at least one amphipathic compound selected from the group consisting of an alkoxylated lipid, an alkoxylated fatty acid, an alkoxylated alcohol, a heteroatomic hydrophilic lipid, a heteroatomic hydrophilic fatty acid, a heteroatomic hydrophilic alcohol, a diluent, and combinations thereof, wherein the compound is derived from a starting compound having a viscosity of less than 1000 cP at 50° C, wherein the concentrate is configured to provide a stable nano emulsion having a D50 and a mean average particle size distribution of less than 100 nm when diluted.
  • Liu et al. provides a protocell nanostructure comprising: a porous particle core comprising a plurality of pores; and at least one lipid bilayer surrounding the porous particle core to form a protocell, wherein the protocell is capable of loading one or more cargo components to the plurality of pores of the porous particle core and releasing the one or more cargo components from the porous particle core across the surrounding lipid bilayer.
  • Bader et al. provides a method for producing a lipid particle comprising the following: i) providing a first solution comprising denatured apolipoprotein, ii) adding the first solution to a second solution comprising at least two lipids and a detergent but no apolipoprotein, and iii) removing the detergent from the solution obtained in ii) and thereby producing a lipid particle.
  • the delivery system can be an administration device.
  • an administration device can be any pharmaceutically acceptable device adapted to deliver a composition of the invention (e.g., to a subject's nose).
  • a nasal administration device can be a metered administration device (metered volume, metered dose, or metered-weight) or a continuous (or substantially continuous) aerosol -producing device. Suitable nasal administration devices also include devices that can be adapted or modified for nasal administration. In some embodiments, the nasally administered dose can be absorbed into the bloodstream of a subject.
  • a metered nasal administration device delivers a fixed (metered) volume or amount (dose) of a nasal composition upon each actuation.
  • Exemplary metered dose devices for nasal administration include, by way of example and without limitation, an atomizer, sprayer, dropper, squeeze tube, squeeze-type spray bottle, pipette, ampule, nasal cannula, metered dose device, nasal spray inhaler, breath actuated bi-directional delivery device, pump spray, pre-compression metered dose spray pump, monospray pump, bispray pump, and pressurized metered dose device.
  • the administration device can be a single-dose disposable device, single-dose reusable device, multidose disposable device or multi-dose reusable device.
  • the compositions of the invention can be used with any known metered administration device.
  • a continuous aerosol-producing device delivers a mist or aerosol comprising droplet of a nasal composition dispersed in a continuous gas phase (such as air).
  • a nebulizer, pulsating aerosol nebulizer, and a nasal continuous positive air pressure device are exemplary of such a device.
  • Suitable nebulizers include, by way of example and without limitation, an air driven jet nebulizer, ultrasonic nebulizer, capillary nebulizer, electromagnetic nebulizer, pulsating membrane nebulizer, pulsating plate (disc) nebulizer, pulsating/vibrating mesh nebulizer, vibrating plate nebulizer, a nebulizer comprising a vibration generator and an aqueous chamber, a nebulizer comprising a nozzle array, and nebulizers that extrude a liquid formulation through a self- contained nozzle array.
  • the device can be any commercially available administration devices that are used or can be adapted for nasal administration of a composition of the invention (see, e.g., US patent publication US20090312724A1).
  • the amount of the agents (e.g., cyclic nucleotides, analogs and derivatives) which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques by those of skill within the art. In addition, in vitro assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the overall seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Ultimately, the attending physician will decide the amount of the agent with which to treat each individual patient. In certain embodiments, the attending physician will administer low doses of the agent and observe the patient's response.
  • the agents e.g., cyclic nucleotides, analogs and derivatives
  • suitable dosage ranges for intravenous administration of the agent are generally about 5-500 micrograms (pg) of active compound per kilogram (Kg) body weight.
  • suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight.
  • a composition containing an agent of the present invention is subcutaneously injected in adult patients with dose ranges of approximately 5 to 5000 pg/human and preferably approximately 5 to 500 pg/human as a single dose. It is desirable to administer this dosage 1 to 3 times daily. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient. Ultimately the attending physician will decide on the appropriate duration of therapy using compositions of the present invention. Dosage will also vary according to the age, weight and response of the individual patient.
  • small particle aerosols of antibodies or fragments thereof can be administered (see e.g., Piazza et al., J. Infect. Dis., Vol. 166, pp. 1422-1424, 1992; and Brown, Aerosol Science and Technology, Vol. 24, pp. 45-56, 1996).
  • antibodies are used as agonists to depress inflammatory diseases.
  • antibodies can be administered in liposomes, i.e., immunoliposomes (see, e.g., Maruyama et al., Biochim. Biophys. Acta, Vol. 1234, pp. 74-80, 1995).
  • immunoconjugates, immunoliposomes or immunomicrospheres containing an agent of the present invention is administered by inhalation.
  • antibodies can be topically administered to mucosa, such as the oropharynx, nasal cavity, respiratory tract, gastrointestinal tract, eye such as the conjunctival mucosa, vagina, urogenital mucosa, or for dermal application.
  • mucosa such as the oropharynx
  • nasal cavity such as the oropharynx
  • respiratory tract such as the conjunctival mucosa, vagina, urogenital mucosa, or for dermal application.
  • antibodies are administered to the nasal, bronchial or pulmonary mucosa.
  • a surfactant such as a phosphoglyceride, e.g. phosphatidylcholine, and/or a hydrophilic or hydrophobic complex of a positively or negatively charged excipient and a charged antibody of the opposite charge.
  • excipients suitable for pharmaceutical compositions intended for delivery of antibodies to the respiratory tract mucosa can be a) carbohydrates, e.g., monosaccharides such as fructose, galactose, glucose. D-mannose, sorbiose, and the like; disaccharides, such as lactose, trehalose, cellobiose, and the like; cyclodextrins, such as 2-hydroxypropyl-P-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; b) amino acids, such as glycine, arginine, aspartic acid, glutamic acid, cysteine, lysine and the like; c) organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamine hydrochloride, and the like: d)
  • the antibodies of the present invention can suitably be formulated with one or more of the following excipients: solvents, buffering agents, preservatives, humectants, chelating agents, antioxidants, stabilizers, emulsifying agents, suspending agents, gelforming agents, ointment bases, penetration enhancers, and skin protective agents.
  • solvents are e.g. water, alcohols, vegetable or marine oils (e.g. edible oils like almond oil, castor oil, cacao butter, coconut oil, com oil, cottonseed oil, linseed oil, olive oil, palm oil, peanut oil, poppy seed oil, rapeseed oil, sesame oil, soybean oil, sunflower oil, and tea seed oil), mineral oils, fatty oils, liquid paraffin, polyethylene glycols, propylene glycols, glycerol, liquid polyalkylsiloxanes, and mixtures thereof.
  • vegetable or marine oils e.g. edible oils like almond oil, castor oil, cacao butter, coconut oil, com oil, cottonseed oil, linseed oil, olive oil, palm oil, peanut oil, poppy seed oil, rapeseed oil, sesame oil, soybean oil, sunflower oil, and tea seed oil
  • mineral oils e.g. water, alcohols, vegetable or marine oils (e.g. edible oils like almond oil, castor oil, cacao butter, coconut oil
  • buffering agents are e.g. citric acid, acetic acid, tartaric acid, lactic acid, hydrogenphosphoric acid, diethyl amine etc.
  • preservatives for use in compositions are parabenes, such as methyl, ethyl, propyl p-hydroxybenzoate, butylparaben, isobutylparaben, isopropylparaben, potassium sorbate, sorbic acid, benzoic acid, methyl benzoate, phenoxyethanol, bronopol, bronidox, MDM hydantoin, iodopropynyl butylcarbamate, EDTA, benzalconium chloride, and benzylalcohol, or mixtures of preservatives.
  • humectants are glycerin, propylene glycol, sorbitol, lactic acid, urea, and mixtures thereof.
  • antioxidants are butylated hydroxy anisole (BHA), ascorbic acid and derivatives thereof, tocopherol and derivatives thereof, cysteine, and mixtures thereof.
  • emulsifying agents are naturally occurring gums, e.g. gum acacia or gum tragacanth; naturally occurring phosphatides, e.g. soybean lecithin, sorbitan monooleate derivatives: wool fats; wool alcohols; sorbitan esters; monoglycerides; fatty alcohols; fatty acid esters (e.g. triglycerides of fatty acids); and mixtures thereof.
  • naturally occurring gums e.g. gum acacia or gum tragacanth
  • naturally occurring phosphatides e.g. soybean lecithin
  • sorbitan monooleate derivatives wool fats; wool alcohols; sorbitan esters; monoglycerides; fatty alcohols; fatty acid esters (e.g. triglycerides of fatty acids); and mixtures thereof.
  • suspending agents are e.g., celluloses and cellulose derivatives such as, e.g., carboxymethyl cellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carraghenan, acacia gum, arabic gum, tragacanth, and mixtures thereof.
  • gel bases examples include: liquid paraffin, polyethylene, fatty oils, colloidal silica or aluminum, zinc soaps, glycerol, propylene glycol, tragacanth, carboxyvinyl polymers, magnesium-aluminum silicates, Carbopol®, hydrophilic polymers such as, e.g. starch or cellulose derivatives such as, e.g., carboxymethylcellulose, hydroxyethylcellulose and other cellulose derivatives, water-swellable hydrocolloids, carragenans, hyaluronates (e.g. hyaluronate gel optionally containing sodium chloride), and alginates including propylene glycol alginate.
  • liquid paraffin such as, e.g. starch or cellulose derivatives such as, e.g., carboxymethylcellulose, hydroxyethylcellulose and other cellulose derivatives, water-swellable hydrocolloids, carragenans, hyaluronates (e.g. hyal
  • ointment bases are e.g., beeswax, paraffin, cetanol, cetyl palmitate, vegetable oils, sorbitan esters of fatty acids (Span), polyethylene glycols, and condensation products between sorbitan esters of fatty acids and ethylene oxide, e g., polyoxyethylene sorbitan monooleate (Tween).
  • hydrophobic or water-emulsifying ointment bases are paraffins, vegetable oils, animal fats, synthetic glycerides, waxes, lanolin, and liquid polyalkylsiloxanes.
  • hydrophilic ointment bases are solid macrogols (polyethylene glycols).
  • Other examples of ointment bases are triethanolamine soaps, sulphated fatty alcohol and polysorbates.
  • excipients examples include polymers such as carmelose, sodium carmelose, hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, pectin, xanthan gum, locust bean gum, acacia gum, gelatin, carbomer, emulsifiers like vitamin E, glyceryl stearates, cetanyl glucoside, collagen, carrageenan, hyaluronates and alginates and chitosans.
  • the dose of antibody required in humans to be effective in the treatment or prevention of allergic inflammation differs with the type and severity of the allergic condition to be treated, the type of allergen, the age and condition of the patient, etc.
  • Typical doses of antibody to be administered are in the range of 1 pg to 1 g, preferably 1-1000 pg, more preferably 2-500, even more preferably 5-50, most preferably 10-20 pg per unit dosage form.
  • infusion of antibodies of the present invention can range from 10-500 mg/m 2 .
  • nucleic acids there are a variety of techniques available for introducing nucleic acids into viable cells.
  • the techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host.
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.
  • the currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection.
  • an administration device comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions, sch as a cyclic nucleotide analog or derivative of the disclosure, and/or additional therapeutic agents.
  • treating as referred to herein encompasses enhancing treatment, or improving treatment efficacy.
  • Treatment can include modulation of immune signaling, which can include inhibition of an inflammatory response, tumor regression as well as inhibition of tumor growth, metastasis or tumor cell proliferation, or inhibition or reduction of otherwise deleterious effects associated with the tumor.
  • Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular disease.
  • the invention comprehends a treatment method comprising any one of the methods or uses herein discussed.
  • therapeutically effective amount refers to a sufficient amount of a drug, agent, or compound to provide a desired therapeutic effect.
  • Therapy or treatment according to the invention can be performed alone or in conjunction with another therapy, and can be provided at home, the doctor’s office, a clinic, a hospital’s outpatient department, or a hospital. Treatment generally begins at a hospital so that the doctor can observe the therapy’s effects closely and make any adjustments that are needed. The duration of the therapy depends on the age and condition of the patient, the stage of the cancer, and how the patient responds to the treatment.
  • a person having a greater risk of developing an inflammatory response e.g., a person who is genetically predisposed or predisposed to allergies or a person having a disease characterized by episodes of inflammation
  • LC-MS/MS liquid chromatographytandem mass spectrometry
  • Applicants coupled it with a reversed-phase high pressure liquid chromatography (RP-HPLC) step prior to LC-MS/MS.
  • RP-HPLC reversed-phase high pressure liquid chromatography
  • This enabled us to scale up the amount of biological material (cultured cells) used as source material from which Applicants extracted polar metabolites.
  • Applicants were able to positively identify many second messengers downstream of dsDNA or dsRNA that are not identifiable in whole cell extracts with traditional approaches. Because it is analogous to other approaches with two separation steps, Applicants term this approach “2 dimensional LC-MS/MS.”
  • RNA molecules whose synthesis appears to be stimulated by double stranded RNA (dsRNA). Cyclic CMP-AMP (cCAMP) and cyclic AMP- CMP-CMP (cACCMP).
  • cCAMP Cyclic CMP-AMP
  • cACCMP cyclic AMP- CMP-CMP
  • Applicants first utilized 2D LC-MSMS to enrich and deconvolute observable metabolites.
  • Cells treated with dsRNA or dsDNA were grown and subsequently lysed in MeOH/CHCL, with polar phase metabolites fractionated by RP-HPLC (45 fractions).
  • HPLC fractions (2’ each) were resuspended in water, with 1 uL of each fraction assayed by LC-MS/MS to achieve more identifiable novel metabolites.
  • FIG. 1 As depicted in FIG. 2, the 2D metabolomics approach revealed a diverse array of dsRNA-induced second messengers, identified in FIG. 2 by retention time.
  • Applicants used linkage and base specific nucleases to enable the diagnosis of molecule structure of unknown molecules.
  • dsRNA double stranded RNA
  • the individual fractions that had been characterized by LC-MS/MS were treated with the enzymes rsAP, RNAseA, RNAseTl, RNAse T2, Nuclease SI and PDEII (FIG. 3B).
  • the sensitivity or resistance of the dsRNA-induced small molecules to each enzyme gave insight into the potential structures for each individual molecule.
  • Example analogs of cCAMP molecules e.g., 3’,3’-cCAMP
  • alternative phosphodiester linkages e.g., AMP 3’ to CMP 5’ and/or CMP 3’ to AMP 5’
  • FIG. 6 Example analogs of cCAMP molecules (e.g., 3’,3’-cCAMP) having alternative fluorinated substituents (e.g., mono- or di- fluorinated versions) are depicted in FIG. 7 which could have different (e.g., increased) cell permeability.
  • Example analogs of the cCAMP molecules (e g., 3’,3’-cCAMP) having alternative phosphodiester substituents are depicted in FIG. 8 (e.g., mono- or bis-phosphorothioate versions) and in FIG. 9 (e.g., mono- or bis-methylphosphonate versions) which could have different (e.g., increased) resistance to cellular nucleases.
  • Example analogs of cCAMP molecules (e.g., 3 ’,3’- cCAMP) having alternative nucleotide bases (e.g., mono- or poly-substituted nucleotide bases) are depicted in FIG. 10 which could have various purpose (e.g., labeling and the like).
  • Example analogs of cACCMP molecules e.g., 3’,3’,3;-cACCMP
  • alternative fluorinated substituents e.g., mono- or poly -fluorinated versions
  • FIG. 13 which could have different (e.g., increased) cell permeability
  • Example analogs of the cACCMP molecules (e.g., 3’,3’,3’- cACCMP) having alternative phosphodiester substituents are depicted in FIG. 14 (e.g., mono- or poly-phosphorothioate versions) and in FIG. 15 (e.g., mono- orpoly-methylphosphonate versions) which could have different (e.g., increased) resistance to cellular nucleases.
  • Example analogs of cACCMP molecules e.g., 3’,3’,3’-cACCMP
  • nucleotide bases e.g., mono- or poly-substituted nucleotide bases
  • FIG. 16 which could have various purpose (e.g., labeling and the like).
  • FIG. 17 Examples of combinations of cCAMP nucleotides and/or isonucleotides are shown in FIG. 17.
  • FIG. 18 shows examples of 3’,3’-cCAMP with an isonucleotidic AMP group
  • FIG. 19 shows examples of 3’,3’-cCAMP with an isonucleotidic CMP group.
  • Examples of combinations of cACCMP nucleotides and/or isonucleotides are shown in FIG. 20.
  • FIG. 21 shows examples of 3’,3’-cACCMP with an isonucleotidic AMP group.
  • Applicants can perform any modification or combination of modifications (e.g., various fluorinated, phosphorothioate, methyl phosphonate, and/or modified nucleotide base versions) on any cCAMP of any analog configuration (e.g., 2’, 3’; 3’, 3’; and the like)(nucleotides and/or isonucleotides) or any cACCMP of any analog configuration (e g., 3’, 3’; 2’, 3’, 3’; and the like)(nucleotides and/or isonucleotides).
  • modifications e.g., various fluorinated, phosphorothioate, methyl phosphonate, and/or modified nucleotide base versions

Abstract

The present invention provides novel cyclic nucleotide derivatives and analogs based on the discovery of novel second messengers generated in human cells in response to challenges to the innate immune system. Compositions find use in modulation of innate immune signaling.

Description

CYCLIC NUCLEOTIDES AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/413,769 filed October 6, 2022. The entire contents of the above-identified application are hereby fully incorporated herein by reference.
TECHNICAL FIELD
[0002] The subject matter disclosed herein is generally directed to cyclic nucleotides and methods of use.
BACKGROUND
[0003] Second messenger molecules, released by the cell in response to extracellular signaling molecules can trigger intracellular signal transduction cascades, and include cyclic adenosine monophosphate and cyclic guanosine monophosphate. Novel second messengers generated in human cells in response to challenges to the innate immune system.
[0004] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.
SUMMARY
[0005] In an aspect, the present disclosure provides compositions comprising a cyclic dinucleotide molecule, a cyclic trinucleotide molecule, an analog or derivative thereof, or any combination thereof. In one example embodiment, a composition comprises a cyclic dinucleotide molecule comprising a cytidine moiety and an adenosine moiety (cCAMP molecule), a cyclic trinucleotide molecule comprising an adenosine moiety and two cytidine moieties (cACCMP molecule), an analog or derivative thereof, or any combination thereof. In one example embodiment, a composition comprises an n’,n’ cyclic cytidine monophosphate-adenosine monophosphate analog (n’,n’ cCAMP analog), wherein each n’ is 2’ or 3’, an n’,n’,n’ cyclic adenosine monophosphate-cytidine monophosphate cytidine analog (n’,n’,n’ cACCMP analog), wherein each n’ is 2’ or 3’, an analog or derivative thereof, or any combination thereof. In one example embodiment, a composition is according to:
Figure imgf000004_0001
(Id); or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups and detectable labels, wherein each R4 is optionally further substituted.
[0006] In one example embodiment, a composition is according to:
Figure imgf000005_0001
Figure imgf000006_0001
Figure imgf000007_0001
Figure imgf000008_0001
Figure imgf000009_0001
(Ilh); or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 is optionally further substituted.
[0007] In one example embodiment, a composition is an isonucleotide analog of any one of the previous embodiments. In one example embodiment, an isonucleotide analog of any one of the previous embodiments comprises one or more sugar ring(s), each comprising the adenosine or the cytosine group at the 2’ carbon position versus the 1’ carbon position, and the R1 group at the 1 ’ carbon position versus the 2’ carbon position.
[0008] In one example embodiment, a composition is a cCAMP isonucleotide analog according to:
Figure imgf000010_0001
Figure imgf000011_0001
(Illd); or an analog or derivative thereof; wherein:
R6 or R7 is an adenine moiety or an analog or derivative thereof, and, when not an adenine moiety or an analog or derivative thereof, R6 or R7 is selected from hydrogen, hydroxyl, and fluorine groups;
R8 or R9 is a cytidine moiety or an analog or derivative thereof, and, when not a cytidine moiety or an analog or a derivative thereof, R8 or R9 is selected from hydrogen, hydroxyl, and fluorine groups; each RIO is independently selected from oxygen and sulfur groups; and each R11 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R11 is optionally further substituted.
[0009] In one example embodiment, the composition is a cACCMP isonucleotide analog
Figure imgf000012_0001
wherein:
R6 or R7 is an adenine group or an analog or derivative thereof, and, when not an adenine group or an analog or derivative thereof, R6 or R7 is selected from hydrogen, hydroxyl, and fluorine groups; at each occurrence, R8 or R9 is a cytidine group or an analog or derivative thereof, and, at each occurrence, when not a cytidine group or an analog or a derivative thereof, R8 or R9 is selected from hydrogen, hydroxyl, and fluorine groups; each RIO is independently selected from oxygen and sulfur groups; and each R11 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R11 is optionally further substituted.
[0010] In one example embodiment, the adenine moiety of an isonucleotide analog of any one of the previous embodiments is according to:
Figure imgf000013_0001
analog or derivative thereof; and/or the cytidine moiety of an isonucleotide analog of any one of the previous embodiments is according to:
Figure imgf000013_0002
analog or derivative thereof; wherein each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and a detectable labels, and wherein each R4 can be optionally further substituted.
[0011] In one example embodiment, a composition is according to any one of the previous embodiments, wherein Rl, R6 or R7, and/or R8 or R9 is/are a fluorine group; R2 or R10 is a sulfur group; R3 or Rl 1 is selected from a methyl group, a hydroxyl group, and alkoxyl (-OR5) groups, wherein R5 is a C1-C10 alkyl group optionally substituted with a C1-C10 alkyl ester group, optionally wherein R5 is a
Figure imgf000013_0003
group; and/or R4 is selected from a hydrogen group, a methyl group, and an alkyne group selected from cycloalkyne
Figure imgf000013_0004
groups, optionally wherein the cycloalkyne group is a cyclooctyne group, or a detectable label, optionally selected from biotin, fluorophore, and a radiolabel.
[0012] In one example embodiment, a composition is according to any one of the previous embodiments, wherein the composition is a prodrug composition.
[0013] In one example embodiment, an adenine moiety of a composition according to Formula
III, Illa, Illb, inc, Illd, or IV, is according to:
[0014] In one example embodiment, R11 of a composition according to Formula III, Illa, Illb, inc, Hid, or IV, is selected from a hydroxyl and alkoxyl (-OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group. In one example embodiment,
Figure imgf000014_0001
group, and/or the composition is a prodrug composition.
[0015] In an aspect, the present disclosure provides pharmaceutical compositions comprising one or more composition(s) of any one of the previous embodiments, or pharmaceutically acceptable salt(s) thereof. In an aspect, the present disclosure provides methods of modulating immune signaling in a cell. In one example embodiment, a method comprises administering one or more composition(s) of any one of the previous embodiments to the cell. In an aspect, the present disclosure provides methods of modulating an immune signaling in a subject in need thereof. In one example embodiment, a method of any one of the previous embodiments comprises administering an effective amount of one or more pharmaceutical composition(s) of any one of the previous embodiments to the subject. In one example embodiment, a method of any one of the previous embodiments administers composition(s) or pharmaceutical composition(s) of any one of the previous embodiments via a delivery vehicle comprising liposomes, lipid particles, or nanoparticles. In an aspect, the present disclosure provides methods of identifying one or more pathways modulated by one or more composition(s) of any one of the previous embodiments. In one example embodiment, a method comprises delivering the composition(s) to a cell, and evaluating differential expression of one or more genes of the one or more pathways by RNAseq to thereby identify the one or more pathways.
[0016] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention can be utilized, and the accompanying drawings of which: [0018] FIG. 1 - depiction of methodology approach for discovery of example molecules.
[0019] FIG. 2 - depiction of 2D metabolomics approach reveals a diverse array of dsRNA- induced second messengers. [0020] FIG. 3A-3B - (A) schematic showing method of discovery using linkage and basespecific nuclease to enable the diagnosis of molecule structure of unknown molecules; (B) digestion with enzymes enables assignment of structure to example novel metabolites.
[0021] FIG. 4 - depiction of analytical results and predicted structure identifying 3’,3’- cCAMP.
[0022] FIG. 5A-5B - (A) sensitivity and resistance to enzymes rule out a linear structure for putative cCAMP; (B) sensitivity and resistance to enzymes suggest cCAMP is 3’,3’-cCAMP.
[0023] FIG. 6 - example alternative phosphodiester linkage isomers of cCAMP, circles indicate chemical modification relative to natural 3’,3’-cCAMP.
[0024] FIG. 7 - example fluorinated analogs of cCAMP, circles indicate chemical modification relative to natural 3 ’,3 ’-cCAMP.
[0025] FIG. 8 - example phosphorothioate analogs of cCAMP, circles indicate chemical modification relative to natural 3 ’,3 ’-cCAMP.
[0026] FIG. 9 - example methylphosphonate analogs of cCAMP, circles indicate chemical modification relative to natural 3 ’,3 ’-cCAMP.
[0027] FIG. 10 - example base modified analogs of cCAMP, circles indicate chemical modification relative to natural 3 ’,3 ’-cCAMP.
[0028] FIG. 11 - depiction of analytical results and predicted structure identifying 3’,3’,3’- cACCMP
[0029] FIG. 12 - example alternative phosphodiester linkage isomers of cACCMP, circles indicate chemical modification relative to natural 3’,3’,3’-cCAMP.
[0030] FIG. 13 - example fluorinated analogs of cACCMP, circles indicate chemical modification relative to natural 3 ’,3 ’,3 ’-cACCMP.
[0031] FIG. 14 - example phosphorothioate analogs of cACCMP, circles indicate chemical modification relative to natural 3’, 3’3’, -cACCMP.
[0032] FIG. 15 - example methylphosphonate analogs of cACCMP, circles indicate chemical modification relative to natural 3 ’,3 ’,3’- cACCMP.
[0033] FIG. 16 - example base modified analogs of cACCMP, circles indicate chemical modification relative to natural 3 ’,3 ’,3’- cACCMP. [0034] FIG. 17 - example cCAMP nucleotides and/or isonucleotides, where: if R1 is an adenine group, then R2 is either H, OH, or F; if R2 is an adenine group, then R1 is H or OH; if R3 is a cytosine group, then R4 is either H, OH or F; if R4 is a cytosine group, then R3 is H or OH; and/or where R5 is either an H or a handle, an acyl group with carbon tail, etc.
[0035] FIG. 18 - example 3’,3’-cCAMP with an isonucleotidic AMP group.
[0036] FIG. 19 - example 3’,3’-cCAMP with an isonucleotidic CMP group.
[0037] FIG. 20 - example cACCMP nucleotides and/or isonucleotides, where: if R1 is an adenine group, then R2 is either H, OH, or F; if R2 is an adenine group, then R1 is H or OH; if R3 is a cytosine group, then R4 is either H, OH or F; if R4 is a cytosine group, then R3 is H or OH; and/or where R5 is either an H or a handle, an acyl group with carbon tail, etc.
[0038] FIG. 21 - example 3 ’,3 ’,3 -cACCMP nucleotide prodrug and an example isoadenine isonucleotidic variant of a 3’,3’,3’-c-ACCMP nucleotide prodrug, circle indicates chemical modification of isonucleotidic variant relative to natural 3’, 3’, 3’- cACCMP prodrug.
[0039] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.
DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS
General Definitions
[0040] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology can be found in Molecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2nd edition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew etal. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).
[0041] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.
[0042] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
[0043] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.
[0044] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, +7-5% or less, +/- 1% or less, and +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.
[0045] As used herein, a “biological sample” can contain whole cells and/or live cells and/or cell debris. The biological sample can contain (or be derived from) a “bodily fluid”. The present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids can be obtained from a mammal organism, for example by puncture, or other collecting or sampling procedures.
[0046] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
[0047] As used herein, unless otherwise stated, the term “group” (also referred to herein as “moiety” or the like) refers to a chemical entity that is monovalent (i.e., comprises one terminus that can be covalently bonded to other chemical species), divalent, or polyvalent (i.e., comprises two or more termini that can be covalently bonded to other chemical species). The term “group” also includes radicals (e.g., monovalent and multivalent, such as, for example, divalent radicals, trivalent radicals, and the like). Illustrative, non-limiting examples of groups include:
Figure imgf000018_0001
[0048] As used herein, unless otherwise indicated, the term “aliphatic group” refers to branched or unbranched hydrocarbon groups that, optionally, contain one or more degree(s) of unsaturation. Degrees of unsaturation include, but are not limited to, carbon-carbon double bonds and carbon-carbon triple bonds. Non-limiting examples, of aliphatic groups with one or more degree(s) of unsaturation include alkenyl groups, alkynyl groups, and aliphatic cyclic groups, and the like. An aliphatic group can be an alkyl group. In various examples, an aliphatic group is a Ci to C20 aliphatic group, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., Ci, C2, C3, C4, C5, C6, C7, C8, C9, C10, Cll, C12, C13, C14, C15, C16, C17, C18, C19, and C20, Cl to C10. C3 to C10, and C5 to C10). An aliphatic group can be unsubstituted or substituted with one or more substituent(s). Examples of substituent groups include, but are not limited to, various substituents such as, for example, halide groups (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups (e.g., a Cl to C30 alkyl groups (e.g., methyl, ethyl, propyl, and the like), monocycloalkyl groups (e.g., cyclohexyl, cyclopentyl, and the like), polycycloalkyl groups (e.g., bicyclic groups, and the like), alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group and the like), aryl groups (e.g., phenyl group and the like), polyaryl groups (e.g., pyrenyl group, and the like), halogenated aryl groups, hydroxyl groups, amine groups, nitro groups, cyano groups, isocyano groups, silyl groups, alkoxide groups, alcohol groups, ether groups, ketone groups, carboxylate groups, carboxylic acid groups, ester groups, amide groups, thioether groups, structural analogs thereof, and the like, and any combination thereof. Examples of alkyl groups include, but are not limited to, methyl groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert-butyl groups, cyclohexyl groups, and structural analogs thereof. A substituent group of an aliphatic group can be further substituted with one or more substituent group(s) described herein.
[0049] As used herein, unless otherwise indicated, the term “alkyl group” refers to branched or unbranched hydrocarbon groups that include only single bonds between carbon atoms (not including substituent(s), if any). In various examples, an alkyl group is a saturated group. In various examples, an alkyl group is a Ci to C20 alkyl group, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., Ci, C2, C3, C4, C5, C6, C7, C8, C9, C10, Cll, C12, C13, C14, C15, C16, C17, C18, C19, and C20, Cl to C10. C3 to C10, and C5 to C10). In various examples, an alkyl group is a cycloalkyl group, e.g., a monocycloalkyl group or a polycycloalkyl group (e.g. bicyclic and the like). In various examples, an alkyl group is unsubstituted or substituted with one or more substituent group(s). Examples of substituent groups include, but are not limited to, various substituents such as, for example, halide groups (-F, -Cl, - Br, and -I), aliphatic groups (e.g., alkyl groups (e.g., a Cl to C20 alkyl groups (e.g., methyl, ethyl, propyl, and the like), monocycloalkyl groups (e.g., cyclohexyl, cyclopentyl, and the like), polycycloalkyl groups (e.g., bicyclic groups, and the like), alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group and the like), aryl groups (e.g., phenyl group and the like), polyaryl groups (e.g., pyrenyl group, and the like), halogenated aryl groups, hydroxyl groups, amine groups, nitro groups, cyano groups, isocyano groups, silyl groups, alkoxide groups, alcohol groups, ether groups, ketone groups, carboxylate groups, carboxylic acid groups, ester groups, amide groups, thioether groups, structural analogs thereof, and the like, and any combination thereof. Examples of alkyl groups include, but are not limited to, methyl groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert-butyl groups, cyclohexyl groups, and structural analogs thereof. A substituent group of an alkyl group can be further substituted with one or more substituent group(s) described herein.
[0050] As used herein, unless otherwise indicated, the term “alkenyl group” refers to branched or unbranched hydrocarbon groups comprising one or more C-C double bond(s). Examples of alkenyl groups include, but are not limited to, an ethenyl (vinyl) group, 1 -propenyl groups, 2- propenyl (allyl) groups, 1-, 2-, and 3-butenyl groups, isopropenyl groups, and the like. In various examples, an alkenyl group is a Ci to C20 alkenyl group, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., Ci, C2, C3, C4, C5, C6, C7, C8, C$>, C10, Cu, C12, C13, C14, C15, C16, C17, C18, C19, and C20, Cl to C10. C3 to C10, and C5 to C10). An alkenyl group can be unsubstituted or substituted with one or more substituent(s). Examples of substituent groups include, but are not limited to, various substituents such as, for example, halide groups (- F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups (e.g., a Cl to C30 alkyl groups (e.g., methyl, ethyl, propyl, and the like), monocycloalkyl groups (e.g., cyclohexyl, cyclopentyl, and the like), polycycloalkyl groups (e.g., bicyclic groups, and the like), alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group and the like), aryl groups (e.g., phenyl group and the like), polyaryl groups (e.g., pyrenyl group, and the like), halogenated aryl groups, hydroxyl groups, amine groups, nitro groups, cyano groups, isocyano groups, silyl groups, alkoxide groups, alcohol groups, ether groups, ketone groups, carboxylate groups, carboxylic acid groups, ester groups, amide groups, thioether groups, structural analogs thereof, and the like, and any combination thereof. Examples of alkyl groups include, but are not limited to, methyl groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert-butyl groups, cyclohexyl groups, and structural analogs thereof. A substituent group of an alkenyl group can be further substituted with one or more substituent group(s) described herein.
[0051] As used herein, unless otherwise indicated, the term “alkynyl group” refers to branched or unbranched hydrocarbon groups comprising one or more C-C triple bond(s). Examples of alkynyl groups include, but are not limited to an ethyne group, 1- and 2-propyne groups, 1-, 2-, and 3-butyne groups, and the like. In various examples, an alkynyl group is a Ci to C20 alkynyl group, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., Cl, C2, C3, C4, C5, C6, C7, C8, C9, C10, Cll, C12, C13, C14, C15, C16, C17, C18, C19, and C20, Cl to C10, C3 to C10, and C5 to C10). An alkynyl group can be unsubstituted or substituted with one or more substituent(s). An alkynyl group can be unsubstituted or substituted with one or more substituent(s). Examples of substituent groups include, but are not limited to, various substituents such as, for example, halide groups (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups (e.g., a Cl to C30 alkyl groups (e.g., methyl, ethyl, propyl, and the like), monocycloalkyl groups (e.g., cyclohexyl, cyclopentyl, and the like), poly cycloalkyl groups (e.g., bicyclic groups, and the like), alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group and the like), aryl groups (e.g., phenyl group and the like), polyaryl groups (e.g., pyrenyl group, and the like), halogenated aryl groups, hydroxyl groups, amine groups, nitro groups, cyano groups, isocyano groups, silyl groups, alkoxide groups, alcohol groups, ether groups, ketone groups, carboxylate groups, carboxylic acid groups, ester groups, amide groups, thioether groups, structural analogs thereof, and the like, and any combination thereof. Examples of alkyl groups include, but are not limited to, methyl groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert-butyl groups, cyclohexyl groups, and structural analogs thereof. A substituent group of an alkynyl group can be further substituted with one or more substituent group(s) described herein.
[0052] As used herein, unless otherwise indicated, the term “aryl group” refers to C5 to C30 aromatic or partially aromatic carbocyclic groups, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., C5, C6, C7, C8, C9, C10, Cll, C12, C13, C14, C15, C16, C17, C18, C19 C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, and C30). Aryl groups can comprise polyaryl groups such as, for example, fused ring groups, biaryl groups, and the like, and any combination thereof. In various examples, the aryl group is unsubstituted or substituted with one or more substituent group(s). Examples of substituent groups include, but are not limited to, substituents such as, for example, halide groups (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group and the like), aryl groups, halogenated aryl groups, hydroxyl groups, amine groups, nitro groups, cyano groups, isocyano groups, silyl groups, alkoxide groups, alcohol groups, ether groups, ketone groups, carboxylate groups, carboxylic acid groups, ester groups, amide groups, thioether groups, structural analogs thereof, and the like, and any combination thereof. Aryl groups can contain hetero atoms, such as, for example, oxygen, nitrogen (e.g., pyridinyl groups and the like), sulfur, and the like, and any combination thereof. Examples of aryl groups include, but are not limited to, phenyl groups, biaryl groups (e.g., biphenyl groups and the like), fused ring groups (e.g., naphthyl groups and the like), hydroxybenzyl groups, tolyl groups, xylyl groups, furanyl groups, benzofuranyl groups, indolyl groups, imidazolyl groups, benzimidazolyl groups, pyridinyl groups, structural analogs thereof, and the like. A substituent group of an aryl group can be further substituted with one or more substituent group(s) described herein. [0053] As used herein, unless otherwise stated, the term “analog” refers to any compound or group that can be envisioned to arise from an original compound or group, respectively, if one atom or group of atoms, functional groups, or substructures is replaced with another atom or group of atoms, functional groups, substructures, or the like. Examples of analogs include, but are not limited to isomers, homologs, and the like. In various examples, an analog is not a functional analog (e.g., does not exhibit significantly different physical, chemical, biochemical, or pharmacological properties from the original compound or group). In various examples, the term “derivative” refers to any compound or group that is derived from an original compound or group, respectively, by a chemical reaction, where the compound or group is modified or partially substituted such that at least one structural feature of the original compound or group is retained. [0054] “Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The absolute stereochemistry is specified according to the Cahn-Ingold-Prelog R-S system When a compound is an enantiomer, the stereochemistry at each chiral carbon can be specified by either R or S. Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line. Certain of the compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry at each asymmetric atom, as (R)- or (S)-. The present chemical entities, pharmaceutical compositions and methods are meant to include all such possible isomers, including racemic mixtures, optically substantially pure forms and intermediate mixtures. In some chemical structures, stereocenters can be identified with "wavy" bonds indicating that the stereocenter can be in the R or S configuration, unless otherwise specified. However, stereocenters without a wavy bond (i.e., a "straight" bond) can also be in the (R) or (S) configuration, unless otherwise specified. Compositions comprising compounds can comprise stereocenters which each can independently be in the (R) configuration, the (S) configuration, or racemic mixtures.
[0055] All chiral, diastereomeric, racemic, and geometric isomeric forms of a structure are intended, unless specific stereochemistry or isomeric form is specifically indicated. All processes used to prepare compounds and intermediates made therein are encompassed by the present disclosure. All tautomers of shown or described compounds are also encompassed by the present disclosure.
[0056] As used herein, a bond substitution coming out of a ring, e.g.,
Figure imgf000023_0001
means that the substitution can be at any of the available position on the ring.
[0057] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one example embodiment”, “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one example embodiment of the present invention. Thus, appearances of the phrases “in one example embodiment,” “in one example embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but can. Furthermore, the particular features, structures or characteristics can be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.
[0058] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.
OVERVIEW
[0059] Embodiments disclosed herein provide cyclic nucleotides generated in cells in response to innate immune stimulation with double stranded RNA (dsRNA). The molecules disclosed herein can be useful as chemical tools or therapeutic agents, as cellular receptors for the molecules are likely modulated by the cyclic nucleotides. Cyclic dinucleotide molecules and cyclic trinucleotide molecules are provided. In particular, cyclic CMP -AMP molecules (cCAMP molecules) and cyclic AMP-CMP-CMP molecules (cACCMP molecules) are provided, including n’,n’ cCAMP analogs and n’,n’,n’ cACCMP analogs wherein each n is 2’ or 3’. Compositions of the cyclic nucleotide molecules, including analogs or derivatives thereof are also provided.
[0060] Nonnatural analogs and derivatives of the molecules are also provided. Example nonnatural analogs include isonucleotide analogs. Example nonnatural analogs include isomers with phosphodiester linkages. Such isomers with nonnatural phosphodiester linkages can modulate target specificity or biological activity. Cyclic analogs can comprise additional functional groups, for example, one or more fluorines, to enhance cell permeability. Cyclic nucleotide derivatives and analogs detailed herein are provided with enhanced nuclease resistance relative to the naturally occurring cyclic nucleotides, for example, via phosphorothioation or methylphosphonation. Additionally, cyclic nucleotides modified via labels, e.g., biotin, or other modification, e.g., methylation are also provided and can be useful for applications including chemical probes.
[0061] Molecules and compositions, including pharmaceutical compositions, can be utilized in methods, including methods of modulating immune signaling in a cell. In one example embodiment, the molecule or composition is administered via a delivery vehicle. Further, methods of identifying one or more pathways modulated by the cyclic nucleotide molecules and compositions are provided.
CYCLIC NUCLEOTIDE COMPOSITIONS
[0062] Cyclic nucleotide compositions comprising a cyclic dinucleotide molecule comprising a cytidine moiety and an adenosine moiety, a cyclic trinucleotide molecule comprising an adenosine moiety and two cytidine moieties, an analog or derivative thereof, or any combination thereof are detailed herein. cCAMP Molecules
[0063] In an aspect, the compositions detailed herein comprise a cyclic dinucleotide molecule, an analog or derivative thereof, or any combination thereof. In an example embodiment, the cyclic dinucleotide molecule comprises one adenosine monophosphate and one cytidine monophosphate (cCAMP molecule). In an example embodiment, the cCAMP molecule comprises a phosphodiester linkage analog (e.g., a phosphodiester linkage isomer). In an example embodiment, the cyclic dinucleotide molecule is an n’,n’ cCAMP analog, wherein each n’ is 2’ or 3’ (e.g., where n’,n’ indicates the carbon numbers of the AMP and CMP sugar rings, respectively, linked by a phosphodiester linkage to the 5’ sugar ring carbon of the CMP and AMP sugar rings, respectively). In an example embodiment, a composition comprising a cCAMP molecule, analog or derivative thereof, or any combination thereof, is a pharmaceutically active composition. In an example embodiment, a composition comprising a cCAMP molecule, analog or derivative thereof, or any combination thereof, is a prodrug composition.
[0064] In one example embodiment, the cCAMP molecule is a 3’3’ cCAMP analog according to the formula:
Figure imgf000025_0001
(la) - 3’3’cCAMP analogs; or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups and detectable labels, wherein each R4 is optionally further substituted.
[0065] In one example embodiment, the cCAMP molecule is a 2’, 3’ cCAMP analog according to the formula:
Figure imgf000026_0001
(Ib) - 2’, 3’ cCAMP analogs; or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups and detectable labels, wherein each R4 is optionally further substituted.
[0066] In one example embodiment, the cCAMP molecule is a 3 ’,2’ cCAMP analog according to the formula:
Figure imgf000026_0002
(Ic) - 3 ’,2’ cCAMP analogs; or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups and detectable labels, wherein each R4 is optionally further substituted.
[0067] In one example embodiment, the cCAMP molecule is a 2’, 2’ cCAMP analog according to the formula:
Figure imgf000027_0001
(Id) - 2’, 2’ cCAMP analogs; or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from, oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups and detectable labels, wherein each R4 is optionally further substituted.
[0068] Compositions according to any one of Formula la, lb, Ic, or Id can have various substituent groups. In one example embodiment, R1 is a fluorine group. In one example embodiment, R2 is a sulfur group. In one example embodiment, R3 is a methyl group. In one example embodiment, R3 is selected from a hydroxyl group and alkoxyl (-OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group. In one example o embodiment, R5 is a
Figure imgf000027_0002
group. In one example embodiment, the composition is a prodrug composition. In one example embodiment, R5 is a
Figure imgf000028_0001
group and the composition is a prodrug composition. In one example embodiment, R4 is a hydrogen group. In one example embodiment, R4 is a methyl group.
In one example embodiment, R4 is an alkyne group selected from cycloalkyne and
Figure imgf000028_0002
groups, optionally wherein the cycloalkyne group is a cyclooctyne group. In one example embodiment, R4 is a detectable label, optionally selected from biotin, fluorophore, and a radiolabel. cACCMP Molecules
[0069] In an aspect, the compositions detailed herein comprise a cyclic trinucleotide molecule, an analog or derivative thereof, or any combination thereof. In one example embodiment, the cyclic trinucleotide molecule comprises one adenosine monophosphate and two cytidine monophosphates (cACCMP molecule). In embodiments, the cyclic trinucleotide is an n’,n’,n’ cACCMP analog, wherein each n’ is 2’ or 3’ (e.g., where n’,n’,n’ indicates the carbon numbers of the AMP, first CMP, and second CMP sugar rings, respectively, linked by a phosphodiester linkage to the 5’ sugar ring carbon of the first CMP, the second CMP, and the AMP sugar rings, respectively). In one example embodiment, a composition comprising a cACCMP molecule, analog or derivative thereof, or any combination thereof, is a pharmaceutical composition. In one example embodiment, a composition comprising a cACCMP molecule, analog or derivative thereof, or any combination thereof, is a prodrug composition.
[0070] In one example embodiment, the cACCMP molecule is a 3 ’,3 ’,3 ’-cACCMP analog according to the formula:
Figure imgf000029_0001
(Ila) - 3’,3’,3’-cACCMP analogs; or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1-C10 alkyl group optionally substituted with a C1-C10 alkyl ester group, and wherein each R3 is optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 is optionally further substituted.
[0071] In one example embodiment, the cACCMP molecule is a 2’, 3’, 3’-cACCMP analog according to the formula:
Figure imgf000030_0001
(lib) - 2’, 3’, 3’-cACCMP analogs; or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from, oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 is optionally further substituted.
[0072] In one example embodiment, the cACCMP molecule is a 2’,2’,3’-cACCMP analog according to the formula:
Figure imgf000031_0001
(lie) - 2’,2’,3’-cACCMP analogs; or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 can be optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 can be optionally further substituted.
[0073] In one example embodiment, the cACCMP molecule is a 2’,2’,2’-cACCMP analog according to the formula:
Figure imgf000032_0001
(lid) - 2’,2’,2’-cACCMP analogs; or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 can be optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 can be optionally further substituted.
[0074] In one example embodiment, the cACCMP molecule is a 2’,3’,2’-cACCMP analog according to the formula:
Figure imgf000033_0001
(lie) - 2’,3’,2’-cACCMP analogs; or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 is optionally further substituted.
[0075] In one example embodiment, the cACCMP molecule is a 3’, 2’, 2’-cACCMP analog according to the formula:
Figure imgf000034_0001
(Ilf) - 3’, 2’, 2’-cACCMP analogs; or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 is optionally further substituted.
[0076] In one example embodiment, the cACCMP molecule is a 3’, 2’, 3’- cACCMP analog according to the formula:
Figure imgf000035_0001
(Ilg) - 3’, 2’, 3’- cACCMP analogs; or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 is optionally further substituted.
[0077] In one example embodiment, the cACCMP molecule is a 3 ’,3 ’,2’ -cACCMP analog according to the formula:
Figure imgf000036_0001
(Ilh) - 3’,3’,2’-cACCMP analogs; or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1-C10 alkyl group optionally substituted with a C1-C10 alkyl ester group, and wherein each R3 is optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 is optionally further substituted.
Compositions according to any one of Formula Ila, lib, lie, lid, lie, Ilf, Ilg, or Ilh can have various substituent groups. In one example embodiment, R1 is a fluorine group. In one example embodiment, R2 is a sulfur group. In one example embodiment, R3 is a methyl group. In one example embodiment, R3 is selected from a hydroxyl group and alkoxyl (-OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group. In one example o embodiment, R5 is a
Figure imgf000036_0002
group. In one example embodiment, the composition is a prodrug composition. In one example embodiment, R5 is a
Figure imgf000037_0001
group and the composition is a prodrug composition. In one example embodiment, R4 is a hydrogen group. In one example embodiment, R4 is a hydrogen group. In one example embodiment, R4 is a methyl group. In one example embodiment, R4 is an alkyne group selected from a cycloalkyne and
Figure imgf000037_0002
groups, optionally wherein the cycloalkyne group is a cyclooctyne group. In one example embodiment, R4 is a detectable label, optionally selected from biotin, fluorophore, and a radiolabel.
Analogs and Derivatives
[0078] In an example embodiment, analogs and derivatives of the cyclic nucleotides include modifications or substitutions at one or more positions of the purine or pyrimidine moieties, one or more positions of the sugar ring, including sugar substitutions, and modifications and/or replacements of the phosphodiester moiety or linkage. Isomeric configurations are also contemplated for use. Each of the modifications described herein can be used in any combination independently with each of the other modifications described herein.
Isonucleotide Analogs
[0079] In an aspect, the compositions detailed herein comprise a nucleotide analog of a cyclic dinucleotide molecule, a cyclic trinucleotide molecule, an analog or derivative thereof, or any combination thereof. In one example embodiment, a composition comprises an isonucleotide analog of a cCAMP, a cACCMP, an analog or derivative thereof, or any combination thereof. In one example embodiment, the composition comprises an isonucleotide analog of a cyclic nucleotide molecule according to any one of Formula la, lb, Ic, Id, Ila, lib, lie, lid, lie, Ilf, Ilg, or Ilh. In one example embodiment, the isonucleotide analog comprises one or more sugar ring(s) each comprising the adenosine group or the cytosine group at the 2’ carbon position and the R1 group at the 1’ carbon position. In one example embodiment, the R3 of the isonucleotide analog, an analog or derivative thereof, or any combination thereof, is selected from a hydroxyl group and alkoxyl (-OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group. In one example embodiment, R5 is a
Figure imgf000038_0001
group, and/or the composition is a prodrug composition.
[0080] In one example embodiment, the composition is a cCAMP isonucleotide analog according to:
Figure imgf000038_0002
(III) - 3’,3’-cCAMP isonucleotide analogs; or an analog or derivative thereof, wherein:
R6 or R7 is an adenine moiety or an analog or derivative thereof, and, when not an adenine moiety or an analog or derivative thereof, R6 or R7 is selected from hydrogen, hydroxyl, and fluorine groups;
R8 or R9 is a cytidine moiety or an analog or derivative thereof, and, when not a cytidine moiety or an analog or a derivative thereof, R8 or R9 is selected from hydrogen, hydroxyl, and fluorine groups; each RIO is independently selected from oxygen and sulfur groups; and each R11 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R11 is optionally further substituted.
[0081] In one example embodiment, a adenine moiety is according to:
Figure imgf000038_0003
wherein each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and a detectable labels, wherein each R4 can be optionally further substituted. In one example embodiment, R4 is a hydrogen group. In one example embodiment, R4 is a methyl group. In one example embodiment, R4 is an alkyne group selected from cycloalkyne and
Figure imgf000039_0001
groups, optionally wherein the cycloalkyne group is a cyclooctyne group. In one example embodiment, R4 is a detectable label, optionally selected from biotin, fluorophore, and a radiolabel. In one example embodiment, R11 is selected from a hydroxyl group and alkoxyl (-OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group. In one example o embodiment, R5 is a
Figure imgf000039_0002
group. In one example embodiment, the composition is a prodrug composition. In one example embodiment, R5 is a o
Figure imgf000039_0003
group and the composition is a prodrug composition.
[0082] In one example embodiment, the composition is a cACCMP isonucleotide analog of
Formula (III), further according to:
Figure imgf000039_0004
(Illa) - 3’,3’-c-(2’R2’dCMP-isonucAMP) analogs;
Figure imgf000040_0001
(IIIc) - 3’,3’-c-(2’R2’dAMP-isonucCMP) analogs;
Figure imgf000041_0001
(IV) - 3’,3’,3’-cACCMP isonucleotide analogs; or an analog or derivative thereof; wherein: R6 or R7 is an adenine group or an analog or derivative thereof, and, when not an adenine group or an analog or derivative thereof, R6 or R7 is selected from hydrogen, hydroxyl, and fluorine groups; at each occurrence, R8 or R9 is a cytidine group or an analog or derivative thereof, and, at each occurrence, when not a cytidine group or an analog or a derivative thereof, R8 or R9 is selected from hydrogen, hydroxyl, and fluorine groups; each RIO is independently selected from oxygen and sulfur groups; and each R11 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1-C10 alkyl group optionally substituted with a C1-C10 alkyl ester group, and wherein each R11 can be optionally further substituted.
[0084] In one example embodiment, a adenine moiety is according to:
Figure imgf000042_0003
wherein each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and a detectable labels, and wherein each R4 can be optionally further substituted. In one example embodiment, R4 is a hydrogen group. In one example embodiment, R4 is a methyl group. In one example embodiment, R4 is an alkyne group selected from cycloalkyne and
Figure imgf000042_0001
groups, optionally wherein the cycloalkyne group is a cyclooctyne group. In one example embodiment, R4 is a detectable label, optionally selected from biotin, fluorophore, and a radiolabel. In one example embodiment, R3 is selected from a hydroxyl and alkoxyl (-OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group. In one example embodiment, R5 is a
Figure imgf000042_0002
group. In one example embodiment, the composition is a prodrug composition. In one example embodiment, R5 is a
Figure imgf000043_0001
group and the composition is a prodrug composition.
[0085] In one example embodiment, the 3’,3’,3’-cACCMP isonucleotide analog comprises an isonucleotidic AMP, one or more isonucleotidic CMP(s), or any combination thereof.
[0086] In one example embodiment, the 3’,3’,3’-cACCMP isonucleotide analog is according to:
Figure imgf000043_0002
Phosphodiester Linkage Analogs
[0087] In one example embodiment, a cyclic nucleotide molecule analog or derivative comprises a cyclic n’,n’ CMP -AMP analog (e.g., isomer) or a cyclic n’,n’,n’ cyclic AMP-CMP- CMP (cACCMP) analog (e.g., isomer). In one example embodiment, the cCAMP molecule is a n’,n’ cCAMP analog, where n’,n’ indicates the carbon numbers of the AMP and CMP sugar rings, respectively, linked by a phosphodiester linkage to the 5’ sugar ring carbon of the CMP and AMP sugar rings, respectively. In one example embodiment, the cACCMP molecule is a n’,n’,n’ cACCMP analog, where n’,n’,n’ indicates the carbon numbers of AMP, first CMP, and second CMP sugar rings, respectively, linked by a phosphodiester linkage to the 5’ sugar ring carbon of the first CMP, second CMP, and AMP sugar rings, respectively. Thus, in one example embodiment, the compositions comprise a 2’, 3’ cCAMP, a 2’, 2’ cCAMP, a 3’2’ cCAMP, a 2’, 3 ’,3 ’-cACCMP, a 2’, 2’, 3 ’-cACCMP, a 2’,2’,2’-cACCMP, a 2’,3’,2’-cACCMP, a 3’,2’,2’- cACCMP a 3’,2’,3’-cACCMP, a 3’,3’,2’-cACCMP, an analog (e.g., an isonucleotide analog, a phosphodiester linkage analog, a fluorinated analog, a phosphodiester analog, a modified nucleotide base analog, or the like) or derivative thereof, or any combination thereof Fluorinated Analogs
[0088] In one example embodiment, a cyclic nucleotide analog or derivative comprises one or more fluorine modifications. In one example embodiment, the cyclic nucleotide analog or derivative is according to any one of Formula la, lb, Ic, Id, Ila, lib, lie, lid, He, Ilf, Ilg, Uh, III, IIIa-IIID, or IV, wherein R1 or one of R6 and R7 and/or one of R8 and R9 = fluorine (F). The cyclic nucleotide analog or derivative can be a mono-fluorinated or di-fluorinated cCAMP molecule, a mono-fluorinated, di-fluorinated, or tri-fluorinated cACCMP molecule, analog (e.g., an isonucleotide analog, a phosphodiester linkage analog, a phosphodiester analog, a modified nucleotide base analog, or the like) or derivative thereof, or any combination thereof. In one example embodiment, the fluorinated cyclic nucleotide molecule, analog or derivative thereof, or any combination thereof, has increased cell permeability relative to a cyclic nucleotide that lacks fluorination. Additional modifications at the Ri can comprise amino, methoxy, and azido groups.
Phosphodiester Analogs
[0089] In one example embodiment, a cyclic nucleotide analog or derivative comprises one or more phosphorothioate modifications. In one example embodiment, the cyclic nucleotide analog or derivative is according to any one of Formula la, lb, Ic, Id, Ila, lib, lie, lid, lie, Ilf, Ilg, Ilh, III, IIIa-IIID, or IV, wherein R2 or RIO = sulfur (S). The cyclic nucleotide analog or derivative can be a mono-phosphorothioate or di- phosphorothioate cCAMP molecule, or a mono-phosphorothioate di-phosphorothioate, or tri-phosphorothioate cACCMP molecule, analog (e.g., an isonucleotide analog, a phosphodiester linkage analog, a fluorinated analog, a methylphosphonate modified analog, a modified nucleotide base analog, or the like) or derivative thereof, or any combination thereof. In one example embodiment, the phosphorothioate cyclic nucleotide molecule, analog (e.g., an isonucleotide analog) or derivative thereof, or any combination thereof, has increased resistance to cellular nucleases relative to a cyclic nucleotide molecule that lacks the phosphorothioate modification. [0090] In one example embodiment, a cyclic nucleotide molecule comprises one or more methylphosphonate modifications. In one example embodiment, the cyclic nucleotide molecule is according to any one of Formula la, lb, Ic, Id, Ila, lib, lie, lid, lie, Ilf, Ilg, Ilh, III, IIIa-IIID, or IV, wherein R3 or R11 = a C1-C5 alkyl group, preferably a methyl group. The cyclic nucleotide can be a mono-methylphosphonate or di-methylphosphonate cCAMP molecule, a mono- methylphosphonate, di-methylphosphonate, or tri-methylphosphonate cACCMP, an analog (e.g., an isonucleotide analog, a phosphodiester linkage analog, a fluorinated analog, a phosphorothioate modified analog, a modified nucleotide base analog, or the like) or derivative thereof, or any combination thereof. In one example embodiment, the methylphosphonate cyclic nucleotide molecule, an analog (e.g., an isonucleotide analog) or derivative thereof, or any combination thereof, has increased resistance to cellular nucleases relative to a cyclic nucleotide molecule that lacks the methylphosphonate modifications.
Modified Nucleotide Base Analogs
[0091] In one example embodiment, a cyclic nucleotide molecule comprises one or more nucleotide base modifications. In one example embodiment, the cyclic nucleotide molecule is according to any one of Formula la, lb, Ic, Id, Ila, lib, lie, lid, lie, Ilf, Ilg, Ilh, III, IIIa-IIID, or IV, III, IIIa-IIID, or IV, wherein R4 = independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, detectable labels, and handles, wherein each R4 can be optionally further substituted. The cyclic nucleotide can be a mono-, or di-, or tri -nucleotide base-substituted cCAMP molecule, a mono-, di-, tri-, tetra- or penta-nucleotide base-substituted cACCMP molecule, an analog (e.g., an isonucleotide analog, a phosphodiester linkage analog, a fluorinated analog, a phosphodiester analog, or the like) or derivative thereof, or any combination thereof.
[0092] In one example embodiment, the cyclic nucleotide molecule is substituted with a chemical handle.
Click Chemistry Modifications
[0093] A cyclic nucleotide can comprise a handle via a click chemistry substitution at R4 of any one of Formula la, lb, Ic, Id, Ila, lib, lie, lid, lie, Ilf, Ilg, Ilh, III, IIIa-IIID, or IV, III, IIIa- IIID, or IV. Exemplary click chemistry molecules can comprise trans-cyclooctene, cyclooctyne, or terminal alkyne, [e.g. reagent N-(lR,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl- l,8-diamino-3,6-di oxaoctane (“BCN amine”). See, e.g., Fantoni, et al., A Hitchhiker’s Guide to Click-Chemistry with Nucleic Acids, Chem. Rev. 2021, 121, 12, 7122-7154, doi: 10.1021/acs.chemrev.0c0092B, incorporated herein by reference.
[0094] In one example embodiment, one or more of R4 is an alkyne selected from a cycloalkyne and
Figure imgf000046_0001
. in one example embodiment, the cycloalkyne is a cyclooctyne.
Detectable Labels
[0095] A detectable label can comprise radiolabels, biotin labels, fluorophores or other optical tags, including amine-reactive dyes. In an example embodiment, one can use a detectably labeled cyclic dinucleotide or trinucleotide according to the present disclosure for identification of binding proteins. In an example embodiment, one can use photoaffinity probes as described in Hou et al., Protocol for identification and validation of 2’3’-cGAMP-binding proteins by photoaffinity probes, STAR Protocols, Volume 3, Issue 1, 18 March 2022, Pages 101076; doi: 10.1016/j.xpro.2021.101076 (describing cGAMP probes comprising a diazirine as the photocrosslinkable group and a terminal alkyne as the clickable group that when irradiated at 365 nm, can form a covalent connection with binding or interacting proteins, which can be visualized in protein gel by conjugation with fluorescence tag (Rh-N3) or be identified/validated after conjugation to affinity tag (Biotin-N3) via click chemistry), referring to Hou et al., Cell Chem. Biol. 29, 1, 133-144. e20 (2022), both of which are incorporated herein by reference in its entirety.
Additional Modifications
[0096] The cyclic nucleotide molecules can comprise one or more non-naturally occurring nucleotide or nucleotide analog such as a nucleotide with phosphorothioate linkage, boranophosphate linkage, a locked nucleic acid (LNA) nucleotides comprising a methylene bridge between the 2’ and 4’ carbons of the ribose ring, an amino-LNA or thio-LNA, peptide nucleic acids (PNA), or bridged nucleic acids (BNA). Other examples of modified nucleotides include 2'- O-methyl analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, or 2'- fluoro analogs. Further examples of modification to one or more nucleotides in the cyclic nucleotides disclosed herein include further linkage of chemical moieties at the 2' position, including but not limited to peptides, nuclear localization sequence (NLS), peptide nucleic acid (PNA), polyethylene glycol (PEG), triethylene glycol, or tetraethyleneglycol (TEG). Synthetic nucleic acid analogies comprising a different sugar backbone, e.g. xeno nucleic acid (XNA) are also included as analogs that can be incorporated in the compositions detailed herein., and can include 1,5-anhydrohexitol nucleic acid (HNA), Cyclohexene nucleic acid (CeNA), Threose nucleic acid (TNA), and glycol nucleoic acid (GNA)., and 2'-Fluoro-arabinonucleic Acid (FANA). XNAs can be chosen for particular applications, including, e.g., probing biomolecular interactions.
METHODS
[0097] Methods of use of the compositions are provided herein. Methods include use of the cyclic nucleotides in modulating immune signaling in the cell, which can comprise administering any of the compositions as disclosed herein to a cell. Methods of modulating immune signaling in a subject in need thereof can comprise administration of an effective amount a pharmaceutical composition as detailed herein. Further methods of identifying novel cyclic nucleotide molecules, pathways modulated by the molecules, and cell receptors of the molecules are also provided.
Modulation
[0098] In one example embodiment, the cyclic nucleotides, their analogs and derivatives can be utilized to module immune signaling in a cell. The term “modulate” broadly denotes a qualitative and/or quantitative alteration, change or variation in that which is being modulated. Where modulation can be assessed quantitatively - for example, where modulation comprises or consists of a change in a quantifiable variable such as a quantifiable property of a cell or where a quantifiable variable provides a suitable surrogate for the modulation - modulation specifically encompasses both increase (e g., activation) or decrease (e.g., inhibition) in the measured variable. The term encompasses any extent of such modulation, e.g., any extent of such increase or decrease, and can more particularly refer to statistically significant increase or decrease in the measured variable. By means of example, modulation can encompass an increase in the value of the measured variable by at least about 10%, e.g., by at least about 20%, preferably by at least about 30%, e.g., by at least about 40%, more preferably by at least about 50%, e.g., by at least about 75%, even more preferably by at least about 100%, e.g., by at least about 150%, 200%, 250%, 300%, 400% or by at least about 500%, compared to a reference situation without said modulation; or modulation can encompass a decrease or reduction in the value of the measured variable by at least about 10%, e.g., by at least about 20%, by at least about 30%, e.g., by at least about 40%, by at least about 50%, e.g., by at least about 60%, by at least about 70%, e.g., by at least about 80%, by at least about 90%, e.g., by at least about 95%, such as by at least about 96%, 97%, 98%, 99% or even by 100%, compared to a reference situation without said modulation. Preferably, modulation can be specific or selective, hence, one or more desired phenotypic aspects of an immune cell or immune cell population can be modulated without substantially altering other (unintended, undesired) phenotypic aspect(s).
[0099] As used herein, "modulating" or "to modulate" generally means either reducing or inhibiting the expression or activity of, or alternatively increasing the expression or activity of a target or antigen. In particular, "modulating" or "to modulate" can mean either reducing or inhibiting the activity of, or alternatively increasing a (relevant or intended) biological activity of, a target or antigen as measured using a suitable in vitro, cellular or in vivo assay (which will usually depend on the target involved), by at least 5%, at least 10%, at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more, compared to activity of the target in the same assay under the same conditions but without the presence of an agent. An "increase" or "decrease" refers to a statistically significant increase or decrease respectively. For the avoidance of doubt, an increase or decrease will be at least 10% relative to a reference, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, a t least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or more, up to and including at least 100% or more, in the case of an increase, for example, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, or more. "Modulating" can also involve effecting a change (which can either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of a target or antigen. "Modulating" can also mean effecting a change with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the target or antigen (or in which its substrate(s), ligand(s) or pathway(s) are involved, such as its signaling pathway or metabolic pathway and their associated biological or physiological effects) is involved. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist can be determined in any suitable manner and/or using any suitable assay known or described herein (e.g., in vitro or cellular assay), depending on the target or antigen involved.
[0100] Modulating can, for example, also involve allosteric modulation of the target and/or reducing or inhibiting the binding of the target to one of its substrates or ligands and/or competing with a natural ligand, substrate for binding to the target. Modulating can also involve activating the target or the mechanism or pathway in which it is involved. Modulating can for example also involve effecting a change in respect of the folding or confirmation of the target, or in respect of the ability of the target to fold, to change its conformation (for example, upon binding of a ligand), to associate with other (sub)units, or to disassociate. Modulating can for example also involve effecting a change in the ability of the target to signal, phosphorylate, dephosphorylate, and the like.
[0101] The term “agent” broadly encompasses any condition, substance or agent capable of modulating one or more phenotypic aspects of a cell or cell population as disclosed herein. Such conditions, substances or agents can be of physical, chemical, biochemical and/or biological nature. In an example embodiment, the agent is a cyclic nucleotide, derivative, analog, or any combination thereof, as described herein. The term “candidate agent” refers to any condition, substance or agent that is being examined for the ability to modulate one or more phenotypic aspects of a cell or cell population as disclosed herein in a method comprising applying the candidate agent to the cell or cell population (e.g., exposing the cell or cell population to the candidate agent or contacting the cell or cell population with the candidate agent) and observing whether the desired modulation takes place.
[0102] Agents can include any potential class of biologically active conditions, substances or agents, such as for instance antibodies, proteins, peptides, nucleic acids, oligonucleotides, small molecules, or combinations thereof, as described herein.
[0103] The methods of phenotypic analysis can be utilized for evaluating environmental stress and/or state, for screening of chemical libraries, and to screen or identify structural, syntenic, genomic, and/or organism and species variations. For example, a culture of cells, can be exposed to an environmental stress, such as but not limited to heat shock, osmolarity, hypoxia, cold, oxidative stress, radiation, starvation, a chemical (for example a therapeutic agent or potential therapeutic agent) and the like. After the stress is applied, a representative sample can be subjected to analysis, for example at various time points, and compared to a control, such as a sample from an organism or cell, for example a cell from an organism, or a standard value. By exposing cells, or fractions thereof, tissues, or even whole animals, to different members of the chemical libraries, and performing the methods described herein, different members of a chemical library can be screened fortheir effect on immune phenotypes thereof simultaneously in a relatively short amount of time, for example using a high throughput method.
[0104] A further aspect of the invention relates to a method for identifying the modulating of one or more phenotypic aspects of a cell or cell population as disclosed herein, comprising: a) applying a candidate agent to the cell or cell population; b) detecting modulation of one or more phenotypic aspects of the cell or cell population by the candidate agent, thereby identifying the agent. The phenotypic aspects of the cell or cell population that is modulated can be a gene signature or biological program specific to a cell type or cell phenotype or phenotype specific to a population of cells (e.g., an inflammatory phenotype or suppressive immune phenotype). In certain embodiments, steps can include administering candidate modulating agents to cells, detecting identified cell (sub)populations for changes in signatures, or identifying relative changes in cell (sub) populations which can comprise detecting relative abundance of particular gene signatures. [0105] Aspects of the present disclosure relate to the correlation of an agent with the spatial proximity and/or epigenetic profile of the nucleic acids in a sample of cells. In some embodiments, the disclosed methods can be used to screen the compositions for modulation of chromatin architecture epigenetic profiles, and/or relationships thereof.
[0106] In some embodiments, screening of test agents involves testing a combinatorial library containing a large number of potential modulator compounds. A combinatorial chemical library can be a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide library, is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (for example the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
[0107] In certain embodiments, the present invention provides for gene signature screening. The concept of signature screening was introduced by Stegmaier et al. (Gene express! on -based high-throughput screening (GE-HTS) and application to leukemia differentiation. Nature Genet. 36, 257-263 (2004)), who realized that if a gene-expression signature was the proxy for a phenotype of interest, it could be used to find small molecules that effect that phenotype without knowledge of a validated drug target. The signatures or biological programs of the present invention can be used to screen for drugs that reduce the signature or biological program in cells as described herein. The signature or biological program can be used for GE-HTS. In certain embodiments, pharmacological screens can be used to identify drugs that are selectively toxic to cells having a signature.
[0108] The Connectivity Map (cmap) is a collection of genome-wide transcriptional expression data from cultured human cells treated with bioactive small molecules and simple pattern-matching algorithms that together enable the discovery of functional connections between drugs, genes and diseases through the transitory feature of common gene-expression changes (see, Lamb et al., The Connectivity Map: Using Gene-Expression Signatures to Connect Small Molecules, Genes, and Disease. Science 29 Sep 2006: Vol. 313, Issue 5795, pp. 1929-1935, DOI: 10.1126/science.1132939; and Lamb, J., The Connectivity Map: a new tool for biomedical research. Nature Reviews Cancer January 2007: Vol. 7, pp. 54-60). In certain embodiments, Cmap can be used to screen for small molecules capable of modulating a signature or biological program of the present invention in silica.
Differential expression
[0109] Detection of differential expression of one or more genes for identification of one or more pathways modulated by the compositions is provided. It is to be understood that also when referring to proteins (e.g. differentially expressed proteins), such can fall within the definition of “gene” signature. Levels of expression or activity or prevalence can be compared between different cells in order to characterize or identify for instance signatures specific for cell (sub)populations. Increased or decreased expression or activity of signature genes can be compared between different cells in order to characterize or identify for instance specific cell (sub)populations. The detection of a signature in single cells can be used to identify and quantitate for instance specific cell (sub)populations. A signature can include a gene or genes, protein or proteins, or epigenetic element(s) whose expression or occurrence is specific to a cell (sub)population, such that expression or occurrence is exclusive to the cell (sub)population. A gene signature as used herein, can thus refer to any set of up- and down-regulated genes that are representative of a cell type or subtype. A gene signature as used herein, can also refer to any set of up- and down-regulated genes between different cells or cell (sub)populations derived from a gene-expression profile. For example, a gene signature can comprise a list of genes differentially expressed in a distinction of interest.
[0110] The signature as defined herein (being it a gene signature, protein signature or other genetic or epigenetic signature) can be used to indicate the presence of a cell type, a subtype of the cell type, the state of the microenvironment of a population of cells, a particular cell type population or subpopulation, and/or the overall status of the entire cell (sub)population. Furthermore, the signature can be indicative of cells within a population of cells in vivo.
[OHl] The signature according to certain embodiments of the present invention can comprise or consist of one or more genes, proteins and/or epigenetic elements, such as for instance 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In certain embodiments, the signature can comprise or consist of two or more genes, proteins and/or epigenetic elements, such as for instance 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In certain embodiments, the signature can comprise or consist of three or more genes, proteins and/or epigenetic elements, such as for instance 3, 4, 5, 6, 7, 8, 9, 10 or more. In certain embodiments, the signature can comprise or consist of four or more genes, proteins and/or epigenetic elements, such as for instance 4, 5, 6, 7, 8, 9, 10 or more. In certain embodiments, the signature can comprise or consist of five or more genes, proteins and/or epigenetic elements, such as for instance 5, 6, 7, 8, 9, 10 or more. In certain embodiments, the signature can comprise or consist of six or more genes, proteins and/or epigenetic elements, such as for instance 6, 7, 8, 9, 10 or more. In certain embodiments, the signature can comprise or consist of seven or more genes, proteins and/or epigenetic elements, such as for instance 7, 8, 9, 10 or more. In certain embodiments, the signature can comprise or consist of eight or more genes, proteins and/or epigenetic elements, such as for instance 8, 9, 10 or more. In certain embodiments, the signature can comprise or consist of nine or more genes, proteins and/or epigenetic elements, such as for instance 9, 10 or more. In certain embodiments, the signature can comprise or consist of ten or more genes, proteins and/or epigenetic elements, such as for instance 10, 11, 12, 13, 14, 15, or more. It is to be understood that a signature according to the invention can for instance also include genes or proteins as well as epigenetic elements combined.
[0112] In certain embodiments, a signature is characterized as being specific for a particular target cell or target cell (sub)population if it is upregulated or only present, detected or detectable in that particular target cell or target cell (sub)population, or alternatively is downregulated or only absent, or undetectable in that particular target cell or target cell (sub)population. In this context, a signature consists of one or more differentially expressed genes/proteins or differential epigenetic elements when comparing different cells or cell (sub)populations, including comparing different target cell or target cell (sub)populations, as well as comparing target cell or target cell (sub)populations with non-target cell or non-target cell (sub)populations. It is to be understood that “differentially expressed” genes/proteins include genes/proteins which are up- or down-regulated as well as genes/proteins which are turned on or off. When referring to up-or down-regulation, in certain embodiments, such up- or down-regulation is preferably at least two-fold, such as two-fold, three-fold, four-fold, five-fold, or more, such as for instance at least ten-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or more. Alternatively, or in addition, differential expression can be determined based on common statistical tests, as is known in the art.
[0113] As discussed herein, differentially expressed genes/proteins, or differential epigenetic elements can be differentially expressed on a single cell level, or can be differentially expressed on a cell population level. Preferably, the differentially expressed genes/ proteins or epigenetic elements as discussed herein, such as constituting the gene signatures as discussed herein, when as to the cell population or subpopulation level, refer to genes that are differentially expressed in all or substantially all cells of the population or subpopulation (such as at least 80%, preferably at least 90%, such as at least 95% of the individual cells). This allows one to define a particular subpopulation of target cells. As referred to herein, a “subpopulation” of cells preferably refers to a particular subset of cells of a particular cell type which can be distinguished or are uniquely identifiable and set apart from other cells of this cell type. The cell subpopulation can be phenotypically characterized and is preferably characterized by the signature as discussed herein. A cell (sub)population as referred to herein can constitute of a (sub)population of cells of a particular cell type characterized by a specific cell state.
[0114] When referring to induction, or alternatively suppression of a particular signature, preferable is meant induction or alternatively suppression (or upregulation or downregulation) of at least one gene/protein and/or epigenetic element of the signature, such as for instance at least two, at least three, at least four, at least five, at least six, or all genes/proteins and/or epigenetic elements of the signature. Detection
[0115] Detection of differential expression can comprise single cell RNA sequencing. In certain embodiments, the invention involves single cell RNA sequencing (see, e.g., Kalisky, T., Blainey, P. & Quake, S. R. Genomic Analysis at the Single-Cell Level. Annual review of genetics 45, 431-445, (2011); Kalisky, T. & Quake, S. R. Single-cell genomics. Nature Methods 8, 311- 314 (2011); Islam, S. et al. Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Research, (2011); Tang, F. et al. RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nature Protocols 5, 516-535, (2010); Tang, F. et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nature Methods 6, 377-382, (2009); Ramskold, D. et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nature Biotechnology 30, 777-782, (2012); and Hashimshony, T., Wagner, F., Sher, N. & Yanai, I. CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification. Cell Reports, Cell Reports, Volume 2, Issue 3, p666-673, 2012).
[0116] In certain embodiments, the invention involves plate based single cell RNA sequencing (see, e.g., Picelli, S. et al., 2014, “Full-length RNA-seq from single cells using Smart-seq2” Nature protocols 9, 171-181, doi:10.1038/nprot.2014.006).
[0117] In certain embodiments, the invention involves high-throughput single-cell RNA-seq. In this regard reference is made to Macosko et al., 2015, “Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets” Cell 161, 1202-1214; International patent application number PCT/US2015/049178, published as WQ2016/040476 on March 17, 2016; Klein et al., 2015, “Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells” Cell 161, 1187-1201; International patent application number PCT/US2016/027734, published as WO2016168584A1 on October 20, 2016; Zheng, et al., 2016, “Haplotyping germline and cancer genomes with high-throughput linked-read sequencing” Nature Biotechnology 34, 303-311; Zheng, et al., 2017, “Massively parallel digital transcriptional profiling of single cells” Nat. Commun. 8, 14049 doi: 10.1038/ncommsl4049; International patent publication number WO2014210353A2; Zilionis, et al., 2017, “Single-cell barcoding and sequencing using droplet microfluidics” Nat Protoc. Jan;12(l):44-73; Cao et al., 2017, “Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing” bioRxiv preprint first posted online Feb. 2, 2017, doi: dx.doi.org/10.1101/104844; Rosenberg et al., 2017, “Scaling single cell transcriptomics through split pool barcoding” bioRxiv preprint first posted online Feb. 2, 2017, doi: dx. doi. org/10.1101/105163; Rosenberg et al., “Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding” Science 15 Mar 2018; Vitak, et al., “Sequencing thousands of single-cell genomes with combinatorial indexing” Nature Methods, 14(3):302— 308, 2017; Cao, et al., Comprehensive single-cell transcriptional profiling of a multicellular organism. Science, 357(6352):661-667, 2017; Gierahn et al., “Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput” Nature Methods 14, 395-398 (2017); and Hughes, et al., “Highly Efficient, Massively-Parallel Single-Cell RNA-Seq Reveals Cellular States and Molecular Features of Human Skin Pathology” bioRxiv 689273; doi: doi.org/10.1101/689273, all the contents and disclosure of each of which are herein incorporated by reference in their entirety.
[0118] In certain embodiments, the invention involves single nucleus RNA sequencing. In this regard reference is made to Swiech et al., 2014, “In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9” Nature Biotechnology Vol. 33, pp. 102-106; Habib et al., 2016, “Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons” Science, Vol. 353, Issue 6302, pp. 925-928; Habib et al., 2017, “Massively parallel single-nucleus RNA-seq with DroNc-seq” Nat Methods. 2017 Oct;14(10):955-958; International patent application number PCT/US2016/059239, published as WO2017164936 on September 28, 2017; International patent application number PCT7US2018/060860, published as WO/2019/094984 on Can 16, 2019; International patent application number PCT/US2019/055894, published as WO/2020/077236 on April 16, 2020; and Drokhlyansky, et al., “The enteric nervous system of the human and mouse colon at a single-cell resolution,” bioRxiv 746743; doi: doi.org/10.1101/746743, which are herein incorporated by reference in their entirety.
[0119] In certain embodiments, the invention involves the Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq) as described, (see, e.g., Buenrostro, et al., Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature methods 2013; 10 (12): 1213-1218; Buenrostro et al., Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-490 (2015); Cusanovich, D. A., Daza, R., Adey, A., Pliner, H., Christiansen, L., Gunderson, K. L., Steemers, F. J., Trapnell, C. & Shendure, J. Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing. Science. 2015 Can 22;348(6237):910-4. doi: 10.1126/science.aabl601. Epub 2015 Can 7; US20160208323 Al; US20160060691A1; and WO2017156336A1).
[0120] Interrogating protein interactions using thermal proteome profiling (TPP) for identification of receptors of cyclic nucleotides are contemplated. See, Mateus et al., Mol Syst Biol 2020 Mar; 16(3): e9232 (detailing principles of TPP and methodology), incorporated herein by reference in its entirety. Steps of TPP can include preparing cellular material and inducing perturbation by administering the cyclic nucleotides to the cell, treating samples with heat, collection of soluble protein fraction, and conducting mass spectrometry -based proteomic analysis. [0121] Exemplary methods of detection and identification of novel cyclic nucleotides can comprise stimulating innate immune signaling of a population of cells, extracting polar metabolites from the cells, fractionating extract via reversed-phase high pressure liquid chromatography, and analyzing the fractions by liquid chromatography-tandem mass spectrometry (LC-MS/MS) thereby detecting novel second messenger small molecules, e.g., cyclic nucleotides. Stimulating the cells typically comprises exposing the cells to double stranded RNA or double stranded DNA. Elucidating the chemical structures of the small molecules identified comprises digestion with one or more enzymes to assign structure to novel metabolites, which can comprise one or more of rsAP, RNAseA, RNaseTl, RNAse T2, Nuclease SI and/or PDEII, as described in more detail in the examples.
Administration
[0122] It will be appreciated that administration of therapeutic entities in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures can be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2): 1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell el al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238- 311 (1998) and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists.
[0123] The medicaments of the invention are prepared in a manner known to those skilled in the art, for example, by means of conventional dissolving, lyophilizing, mixing, granulating or confectioning processes. Methods well known in the art for making formulations are found, for example, in Remington: The Science and Practice of Pharmacy, 20th ed., ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York.
[0124] Administration of medicaments of the invention can be by any suitable means that results in a compound concentration that is effective for treating or inhibiting (e.g., by delaying) the development of a disease. The compound is admixed with a suitable carrier substance, e.g., a pharmaceutically acceptable excipient that preserves the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable excipient is physiological saline. The suitable carrier substance is generally present in an amount of 1-95% by weight of the total weight of the medicament. The medicament can be provided in a dosage form that is suitable for administration. Thus, the medicament can be in form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, delivery devices, injectables, implants, sprays, or aerosols. Pharmaceutical Compositions
[0125] The compositions of the present disclosure include pharmaceutical composition comprising one or more molecules or compositions as detailed herein, or pharmaceutically acceptable salts thereof. [0126] The agents disclosed herein (e.g., cyclic nucleotides) can be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such compositions comprise a therapeutically effective amount of the agent and a pharmaceutically acceptable carrier. Such a composition can also further comprise (in addition to an agent and a carrier) diluents, fdlers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Compositions comprising the agent can be administered in the form of salts provided the salts are pharmaceutically acceptable. Salts can be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry.
[0127] The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. The term “pharmaceutically acceptable salt” further includes all acceptable salts such as acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide, bromide, methylnitrate, calcium edetate, methylsulfate, camsylate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate, N- methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, edisylate, pamoate (embonate), estolate, palmitate, esylate, pantothenate, fumarate, phosphate/di phosphate, gluceptate, polygalacturonate, gluconate, salicylate, glutamate, stearate, glycollylarsanilate, sulfate, hexylresorcinate, subacetate, hydrabamine, succinate, hydrobromide, tannate, hydrochloride, tartrate, hydroxynaphthoate, teoclate, iodide, tosylate, isothionate, triethiodide, lactate, panoate, valerate, and the like which can be used as a dosage form for modifying the solubility or hydrolysis characteristics or can be used in sustained release or pro-drug formulations. It will be understood that, as used herein, references to specific agents (e.g., neuromedin U receptor agonists or antagonists), also include the pharmaceutically acceptable salts thereof.
[0128] The composition of the invention can also advantageously be formulated in order to release 2’ -ATP mimetics or derivatives, and/or agonist in the subject in a timely controlled fashion. In a particular embodiment, the composition of the invention is formulated for controlled release.
[0129] The agents of the present invention can be modified, such that they acquire advantageous properties for therapeutic use (e.g., stability and specificity), but maintain their biological activity.
[0130] In particular embodiments, the agents (e.g., cyclic nucleotides) include a protecting group covalently joined to the N-terminal amino group. In exemplary embodiments, a protecting group covalently joined to the N-terminal amino group of the agonists reduces the reactivity of the amino terminus under in vivo conditions. Amino protecting groups include — Cl-10 alkyl, — Cl- 10 substituted alkyl, — C2-10 alkenyl, — C2-10 substituted alkenyl, aryl, — Cl-6 alkyl aryl, — C(O)— (CH2)l-6— COOH, — C(O)— Cl-6 alkyl, — C(O)-aryl, — C(O)— O— Cl-6 alkyl, or — C(O) — O-aryl. In particular embodiments, the amino terminus protecting group is selected from the group consisting of acetyl, propyl, succinyl, benzyl, benzyloxycarbonyl, and t- butyloxy carbonyl. In other embodiments, deamination of the N-terminal amino acid is another modification that can be used for reducing the reactivity of the amino terminus under in vivo conditions.
[0131] Chemically modified compositions of the agents (wherein the agent is linked to a polymer are also included within the scope of the present invention. The polymer selected is usually modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be controlled. Included within the scope of polymers is a mixture of polymers. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable. The polymer or mixture thereof can include but is not limited to polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (for example, glycerol), and polyvinyl alcohol.
[0132] In certain embodiments, the present invention provides for one or more therapeutic agents. In certain embodiments, the one or more agents comprises a small molecule inhibitor, small molecule degrader (e.g., PROTAC), genetic modifying agent, antibody, antibody fragment, antibody-like protein scaffold, aptamer, protein, or any combination thereof.
[0133] The terms “therapeutic agent”, “therapeutic capable agent” or “treatment agent” are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
[0134] As used herein, “treatment” or “treating,” or “palliating” or “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, the compositions can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom can not have yet been manifested. As used herein "treating" includes ameliorating, curing, preventing it from becoming worse, slowing the rate of progression, or preventing the disorder from re-occurring (i.e., to prevent a relapse). In certain embodiments, the present invention provides for one or more therapeutic agents against combinations of targets identified. Targeting the identified combinations can provide for enhanced or otherwise previously unknown activity in the treatment of disease.
[0135] One type of small molecule applicable to the present invention is a degrader molecule. Proteolysis Targeting Chimera (PROTAC) technology is a rapidly emerging alternative therapeutic strategy with the potential to address many of the challenges currently faced in modern drug development programs. PROTAC technology employs small molecules that recruit target proteins for ubiquitination and removal by the proteasome (see, e.g., Zhou et al., Discovery of a Small-Molecule Degrader of Bromodomain and Extra- Terminal (BET) Proteins with Picomolar Cellular Potencies and Capable of Achieving Tumor Regression. J. Med. Chem. 2018, 61, 462-481; Bondeson and Crews, Targeted Protein Degradation by Small Molecules, Annu Rev Pharmacol Toxicol. 2017 Jan 6; 57: 107-123; and Lai et al., Modular PROTAC Design for the Degradation of Oncogenic BCR-ABL Angew Chem Int Ed Engl. 2016 Jan 11; 55(2): 807-810).
[0136] In certain embodiments, combinations of targets are modulated (e.g., one or more targets related to cyclic nucleotide generation or modulation). In certain embodiments, an agent against one of the targets in a combination can already be known or used clinically. In certain embodiments, targeting the combination can require less of the agent as compared to the current standard of care and provide for less toxicity and improved treatment.
[0137] Methods of administrating the pharmacological compositions, including agonists, antagonists, antibodies or fragments thereof, to an individual include, but are not limited to, intradermal, intrathecal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, by inhalation, and oral routes. The compositions can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (for example, oral mucosa, rectal and intestinal mucosa, and the like), ocular, and the like and can be administered together with other biologically-active agents. Administration can be systemic or local. In addition, it can be advantageous to administer the composition into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Pulmonary administration can also be employed by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. It can also be desirable to administer the agent locally to the area in need of treatment; this can be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant.
[0138] Various delivery systems are known and can be used to administer the pharmacological compositions including, but not limited to, encapsulation in liposomes, microparticles, microcapsules; minicells; polymers; capsules; tablets; and the like. In one example embodiment, the agent can be delivered in a vesicle, in particular a liposome. In a liposome, the agent is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. No. 4,837,028 and U.S. Pat. No. 4,737,323. In yet another embodiment, the pharmacological compositions can be delivered in a controlled release system including, but not limited to: a delivery pump (See, for example, Saudek, et al., New Engl. J. Med. 321 : 574 (1989) and a semi-permeable polymeric material (See, for example, Howard, et al., J. Neurosurg. 71 : 105 (1989)). Additionally, the controlled release system can be placed in proximity of the therapeutic target (e g., a tumor), thus requiring only a fraction of the systemic dose. See, for example, Goodson, In: Medical Applications of Controlled Release, 1984. (CRC Press, Boca Raton, Fla.).
Nanoparticle
[0139] Delivery of therapeutic agents can be accomplished via nanoparticle. Anderson et al. (US 20170079916) provides a modified dendrimer nanoparticle for the delivery of therapeutic, prophylactic and/or diagnostic agents to a subject, comprising: one or more zero to seven generation alkylated dendrimers; one or more amphiphilic polymers; and one or more therapeutic, prophylactic and/or diagnostic agents encapsulated therein. One alkylated dendrimer can be selected from the group consisting of poly(ethyleneimine), poly(polyproylenimine), diaminobutane amine polypropylenimine tetramine and poly(amido amine).
[0140] Anderson et al. (US20050123596) provides examples of microparticles that are designed to release their payload when exposed to acidic conditions, wherein the microparticles comprise at least one agent to be delivered, a pH triggering agent, and a polymer, wherein the polymer is selected from the group of polymethacrylates and polyacrylates. See also, Anderson et al (US 20020150626) providing lipid-protein-sugar particles for delivery of nucleic acids, wherein the polynucleotide is encapsulated in a lipid-protein-sugar matrix by contacting the polynucleotide with a lipid, a protein, and a sugar; and spray drying mixture of the polynucleotide, the lipid, the protein, and the sugar to make microparticles.
Liposomes and Lipids
[0141] Semi-solid and soft nanoparticles have been manufactured, and are within the scope of the present invention. A prototype nanoparticle of semi-solid nature is the liposome. Various types of liposome nanoparticles are currently used clinically as delivery systems for anticancer drugs and vaccines. Nanoparticles with one half hydrophilic and the other half hydrophobic are termed Janus particles and are particularly effective for stabilizing emulsions. They can self-assemble at water/oil interfaces and act as solid surfactants.
[0142] Berg et al. (US20160174546) a nanolipid delivery system, in particular a nano-particle concentrate, comprising: a composition comprising a lipid, oil or solvent, the composition having a viscosity of less than 100 cP at 25°C and a Kauri Butanol solvency of greater than 25 Kb; and at least one amphipathic compound selected from the group consisting of an alkoxylated lipid, an alkoxylated fatty acid, an alkoxylated alcohol, a heteroatomic hydrophilic lipid, a heteroatomic hydrophilic fatty acid, a heteroatomic hydrophilic alcohol, a diluent, and combinations thereof, wherein the compound is derived from a starting compound having a viscosity of less than 1000 cP at 50° C, wherein the concentrate is configured to provide a stable nano emulsion having a D50 and a mean average particle size distribution of less than 100 nm when diluted.
[0143] Liu et al. (US 20140301951) provides a protocell nanostructure comprising: a porous particle core comprising a plurality of pores; and at least one lipid bilayer surrounding the porous particle core to form a protocell, wherein the protocell is capable of loading one or more cargo components to the plurality of pores of the porous particle core and releasing the one or more cargo components from the porous particle core across the surrounding lipid bilayer.
[0144] Bader et al. (US 20150250725), provides a method for producing a lipid particle comprising the following: i) providing a first solution comprising denatured apolipoprotein, ii) adding the first solution to a second solution comprising at least two lipids and a detergent but no apolipoprotein, and iii) removing the detergent from the solution obtained in ii) and thereby producing a lipid particle.
[0145] In another embodiment, the delivery system can be an administration device. As used herein, an administration device can be any pharmaceutically acceptable device adapted to deliver a composition of the invention (e.g., to a subject's nose). A nasal administration device can be a metered administration device (metered volume, metered dose, or metered-weight) or a continuous (or substantially continuous) aerosol -producing device. Suitable nasal administration devices also include devices that can be adapted or modified for nasal administration. In some embodiments, the nasally administered dose can be absorbed into the bloodstream of a subject. [0146] A metered nasal administration device delivers a fixed (metered) volume or amount (dose) of a nasal composition upon each actuation. Exemplary metered dose devices for nasal administration include, by way of example and without limitation, an atomizer, sprayer, dropper, squeeze tube, squeeze-type spray bottle, pipette, ampule, nasal cannula, metered dose device, nasal spray inhaler, breath actuated bi-directional delivery device, pump spray, pre-compression metered dose spray pump, monospray pump, bispray pump, and pressurized metered dose device. The administration device can be a single-dose disposable device, single-dose reusable device, multidose disposable device or multi-dose reusable device. The compositions of the invention can be used with any known metered administration device.
[0147] A continuous aerosol-producing device delivers a mist or aerosol comprising droplet of a nasal composition dispersed in a continuous gas phase (such as air). A nebulizer, pulsating aerosol nebulizer, and a nasal continuous positive air pressure device are exemplary of such a device. Suitable nebulizers include, by way of example and without limitation, an air driven jet nebulizer, ultrasonic nebulizer, capillary nebulizer, electromagnetic nebulizer, pulsating membrane nebulizer, pulsating plate (disc) nebulizer, pulsating/vibrating mesh nebulizer, vibrating plate nebulizer, a nebulizer comprising a vibration generator and an aqueous chamber, a nebulizer comprising a nozzle array, and nebulizers that extrude a liquid formulation through a self- contained nozzle array.
[0148] In certain embodiments, the device can be any commercially available administration devices that are used or can be adapted for nasal administration of a composition of the invention (see, e.g., US patent publication US20090312724A1).
[0149] The amount of the agents (e.g., cyclic nucleotides, analogs and derivatives) which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques by those of skill within the art. In addition, in vitro assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the overall seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Ultimately, the attending physician will decide the amount of the agent with which to treat each individual patient. In certain embodiments, the attending physician will administer low doses of the agent and observe the patient's response. Larger doses of the agent can be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. In general, the daily dose range lie within the range of from about 0.001 mg to about 100 mg per kg body weight of a mammal, preferably 0.01 mg to about 50 mg per kg, and most preferably 0.1 to 10 mg per kg, in single or divided doses. On the other hand, it can be necessary to use dosages outside these limits in some cases. In certain embodiments, suitable dosage ranges for intravenous administration of the agent are generally about 5-500 micrograms (pg) of active compound per kilogram (Kg) body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. In certain embodiments, a composition containing an agent of the present invention is subcutaneously injected in adult patients with dose ranges of approximately 5 to 5000 pg/human and preferably approximately 5 to 500 pg/human as a single dose. It is desirable to administer this dosage 1 to 3 times daily. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient. Ultimately the attending physician will decide on the appropriate duration of therapy using compositions of the present invention. Dosage will also vary according to the age, weight and response of the individual patient.
[0150] Methods for administering antibodies for therapeutic use is well known to one skilled in the art. In certain embodiments, small particle aerosols of antibodies or fragments thereof can be administered (see e.g., Piazza et al., J. Infect. Dis., Vol. 166, pp. 1422-1424, 1992; and Brown, Aerosol Science and Technology, Vol. 24, pp. 45-56, 1996). In certain embodiments, antibodies are used as agonists to depress inflammatory diseases. In certain embodiments, antibodies can be administered in liposomes, i.e., immunoliposomes (see, e.g., Maruyama et al., Biochim. Biophys. Acta, Vol. 1234, pp. 74-80, 1995). In certain embodiments, immunoconjugates, immunoliposomes or immunomicrospheres containing an agent of the present invention is administered by inhalation. [0151] In certain embodiments, antibodies can be topically administered to mucosa, such as the oropharynx, nasal cavity, respiratory tract, gastrointestinal tract, eye such as the conjunctival mucosa, vagina, urogenital mucosa, or for dermal application. In certain embodiments, antibodies are administered to the nasal, bronchial or pulmonary mucosa. In order to obtain optimal delivery of the antibodies to the pulmonary cavity in particular, it can be advantageous to add a surfactant such as a phosphoglyceride, e.g. phosphatidylcholine, and/or a hydrophilic or hydrophobic complex of a positively or negatively charged excipient and a charged antibody of the opposite charge.
[0152] Other excipients suitable for pharmaceutical compositions intended for delivery of antibodies to the respiratory tract mucosa can be a) carbohydrates, e.g., monosaccharides such as fructose, galactose, glucose. D-mannose, sorbiose, and the like; disaccharides, such as lactose, trehalose, cellobiose, and the like; cyclodextrins, such as 2-hydroxypropyl-P-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; b) amino acids, such as glycine, arginine, aspartic acid, glutamic acid, cysteine, lysine and the like; c) organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamine hydrochloride, and the like: d) peptides and proteins, such as aspartame, human serum albumin, gelatin, and the like; e) alditols, such mannitol, xylitol, and the like, and f) polycationic polymers, such as chitosan or a chitosan salt or derivative.
[0153] For dermal application, the antibodies of the present invention can suitably be formulated with one or more of the following excipients: solvents, buffering agents, preservatives, humectants, chelating agents, antioxidants, stabilizers, emulsifying agents, suspending agents, gelforming agents, ointment bases, penetration enhancers, and skin protective agents.
[0154] Examples of solvents are e.g. water, alcohols, vegetable or marine oils (e.g. edible oils like almond oil, castor oil, cacao butter, coconut oil, com oil, cottonseed oil, linseed oil, olive oil, palm oil, peanut oil, poppy seed oil, rapeseed oil, sesame oil, soybean oil, sunflower oil, and tea seed oil), mineral oils, fatty oils, liquid paraffin, polyethylene glycols, propylene glycols, glycerol, liquid polyalkylsiloxanes, and mixtures thereof.
[0155] Examples of buffering agents are e.g. citric acid, acetic acid, tartaric acid, lactic acid, hydrogenphosphoric acid, diethyl amine etc. Suitable examples of preservatives for use in compositions are parabenes, such as methyl, ethyl, propyl p-hydroxybenzoate, butylparaben, isobutylparaben, isopropylparaben, potassium sorbate, sorbic acid, benzoic acid, methyl benzoate, phenoxyethanol, bronopol, bronidox, MDM hydantoin, iodopropynyl butylcarbamate, EDTA, benzalconium chloride, and benzylalcohol, or mixtures of preservatives.
[0156] Examples of humectants are glycerin, propylene glycol, sorbitol, lactic acid, urea, and mixtures thereof. [0157] Examples of antioxidants are butylated hydroxy anisole (BHA), ascorbic acid and derivatives thereof, tocopherol and derivatives thereof, cysteine, and mixtures thereof.
[0158] Examples of emulsifying agents are naturally occurring gums, e.g. gum acacia or gum tragacanth; naturally occurring phosphatides, e.g. soybean lecithin, sorbitan monooleate derivatives: wool fats; wool alcohols; sorbitan esters; monoglycerides; fatty alcohols; fatty acid esters (e.g. triglycerides of fatty acids); and mixtures thereof.
[0159] Examples of suspending agents are e.g., celluloses and cellulose derivatives such as, e.g., carboxymethyl cellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carraghenan, acacia gum, arabic gum, tragacanth, and mixtures thereof.
[0160] Examples of gel bases, viscosity-increasing agents or components which are able to take up exudate from a wound are: liquid paraffin, polyethylene, fatty oils, colloidal silica or aluminum, zinc soaps, glycerol, propylene glycol, tragacanth, carboxyvinyl polymers, magnesium-aluminum silicates, Carbopol®, hydrophilic polymers such as, e.g. starch or cellulose derivatives such as, e.g., carboxymethylcellulose, hydroxyethylcellulose and other cellulose derivatives, water-swellable hydrocolloids, carragenans, hyaluronates (e.g. hyaluronate gel optionally containing sodium chloride), and alginates including propylene glycol alginate.
[0161] Examples of ointment bases are e.g., beeswax, paraffin, cetanol, cetyl palmitate, vegetable oils, sorbitan esters of fatty acids (Span), polyethylene glycols, and condensation products between sorbitan esters of fatty acids and ethylene oxide, e g., polyoxyethylene sorbitan monooleate (Tween).
[0162] Examples of hydrophobic or water-emulsifying ointment bases are paraffins, vegetable oils, animal fats, synthetic glycerides, waxes, lanolin, and liquid polyalkylsiloxanes. Examples of hydrophilic ointment bases are solid macrogols (polyethylene glycols). Other examples of ointment bases are triethanolamine soaps, sulphated fatty alcohol and polysorbates.
[0163] Examples of other excipients are polymers such as carmelose, sodium carmelose, hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, pectin, xanthan gum, locust bean gum, acacia gum, gelatin, carbomer, emulsifiers like vitamin E, glyceryl stearates, cetanyl glucoside, collagen, carrageenan, hyaluronates and alginates and chitosans. [0164] The dose of antibody required in humans to be effective in the treatment or prevention of allergic inflammation differs with the type and severity of the allergic condition to be treated, the type of allergen, the age and condition of the patient, etc. Typical doses of antibody to be administered are in the range of 1 pg to 1 g, preferably 1-1000 pg, more preferably 2-500, even more preferably 5-50, most preferably 10-20 pg per unit dosage form. In certain embodiments, infusion of antibodies of the present invention can range from 10-500 mg/m2.
[0165] There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection.
[0166] In another aspect, provided is an administration device, pharmaceutical pack or kit, comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions, sch as a cyclic nucleotide analog or derivative of the disclosure, and/or additional therapeutic agents.
Treatment
[0167] It will be understood by the skilled person that treating as referred to herein encompasses enhancing treatment, or improving treatment efficacy. Treatment can include modulation of immune signaling, which can include inhibition of an inflammatory response, tumor regression as well as inhibition of tumor growth, metastasis or tumor cell proliferation, or inhibition or reduction of otherwise deleterious effects associated with the tumor.
[0168] Use of products, such as cyclic nucleotides for the purpose of immune regulation in diseases where immune modulation is central to pathogenesis are contemplated.
[0169] Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular disease. The invention comprehends a treatment method comprising any one of the methods or uses herein discussed.
[0170] The phrase "therapeutically effective amount" as used herein refers to a sufficient amount of a drug, agent, or compound to provide a desired therapeutic effect. [0171] Therapy or treatment according to the invention can be performed alone or in conjunction with another therapy, and can be provided at home, the doctor’s office, a clinic, a hospital’s outpatient department, or a hospital. Treatment generally begins at a hospital so that the doctor can observe the therapy’s effects closely and make any adjustments that are needed. The duration of the therapy depends on the age and condition of the patient, the stage of the cancer, and how the patient responds to the treatment. Additionally, a person having a greater risk of developing an inflammatory response (e.g., a person who is genetically predisposed or predisposed to allergies or a person having a disease characterized by episodes of inflammation) can receive prophylactic treatment to inhibit or delay symptoms of the disease.
[0172] Further embodiments are illustrated in the following Examples which are given for illustrative purposes only and are not intended to limit the scope of the invention.
EXAMPLES
Example 1 -
[0173] In searching for novel second messengers generated in human cells in response to challenges to the innate immune system, Applicants realized that current liquid chromatographytandem mass spectrometry (LC-MS/MS) approaches were significantly underpowered to detect novel second messenger small molecules (data not shown). For example, stimulation of innate immune signaling with double stranded DNA (dsDNA) leads to production of cyclic GMP-AMP (2’,3’-cGAMP), which robustly activates interferon signaling. However, Applicants found that even at peak activation of the cGAMP pathway that cGAMP was not detectable in whole cell polar extracts by traditional LC-MC/MC.
[0174] To enhance the sensitivity of LC-MS/MS, Applicants coupled it with a reversed-phase high pressure liquid chromatography (RP-HPLC) step prior to LC-MS/MS. This enabled us to scale up the amount of biological material (cultured cells) used as source material from which Applicants extracted polar metabolites. By fractionating these extracts first with RP-HPLC and then analyzing fractions by LC-MS/MS, Applicants were able to positively identify many second messengers downstream of dsDNA or dsRNA that are not identifiable in whole cell extracts with traditional approaches. Because it is analogous to other approaches with two separation steps, Applicants term this approach “2 dimensional LC-MS/MS.”
[0175] To annotate the possible chemical structures of the many novel small molecules that this approach identified, Applicants took advantage of well -characterized enzymes that are standard tools in molecular biology. The sensitivity or resistance of a particular unidentified molecule allowed us to rule in or out possible structures for many of these molecules.
[0176] Applicants describe here two cyclic nucleotide molecules whose synthesis appears to be stimulated by double stranded RNA (dsRNA). Cyclic CMP-AMP (cCAMP) and cyclic AMP- CMP-CMP (cACCMP).
[0177] Applicants first utilized 2D LC-MSMS to enrich and deconvolute observable metabolites. Cells treated with dsRNA or dsDNA were grown and subsequently lysed in MeOH/CHCL, with polar phase metabolites fractionated by RP-HPLC (45 fractions). HPLC fractions (2’ each) were resuspended in water, with 1 uL of each fraction assayed by LC-MS/MS to achieve more identifiable novel metabolites. (FIG. 1). As depicted in FIG. 2, the 2D metabolomics approach revealed a diverse array of dsRNA-induced second messengers, identified in FIG. 2 by retention time. Next, Applicants used linkage and base specific nucleases to enable the diagnosis of molecule structure of unknown molecules. Example nuclease cleavage shown in FIG. 3A. After, Applicants identified novel molecules induced in cells by double stranded RNA (dsRNA), the individual fractions that had been characterized by LC-MS/MS were treated with the enzymes rsAP, RNAseA, RNAseTl, RNAse T2, Nuclease SI and PDEII (FIG. 3B). The sensitivity or resistance of the dsRNA-induced small molecules to each enzyme gave insight into the potential structures for each individual molecule. FIG. 4 indicates the approach to identification of the predicted structure of an example second messenger, 3’,3’-cCAMP, identified at m/z 633.092. Sensitivity and resistance to enzymes via enzymatic digestion ruled out a linear structure for the unknown molecule (FIG. 5 A), with further evaluation suggesting that the cCAMP molecule is 3’ 3 ’-cCAMP (FIG. 5B).
[0178] Example analogs of cCAMP molecules (e.g., 3’,3’-cCAMP) having alternative phosphodiester linkages (e g., AMP 3’ to CMP 5’ and/or CMP 3’ to AMP 5’) are depicted in FIG. 6 which could have different signaling properties or in vivo stability. Example analogs of cCAMP molecules (e.g., 3’,3’-cCAMP) having alternative fluorinated substituents (e.g., mono- or di- fluorinated versions) are depicted in FIG. 7 which could have different (e.g., increased) cell permeability. Example analogs of the cCAMP molecules (e g., 3’,3’-cCAMP) having alternative phosphodiester substituents are depicted in FIG. 8 (e.g., mono- or bis-phosphorothioate versions) and in FIG. 9 (e.g., mono- or bis-methylphosphonate versions) which could have different (e.g., increased) resistance to cellular nucleases. Example analogs of cCAMP molecules (e.g., 3 ’,3’- cCAMP) having alternative nucleotide bases (e.g., mono- or poly-substituted nucleotide bases) are depicted in FIG. 10 which could have various purpose (e.g., labeling and the like).
[0179] The same methodology was also used to identify and predict the structure of another example second messenger, 3’,3’,3’-cACCMP, identified at m/z 468.5621 (FIG. 11). Example analogs of cACCMP molecules (e.g., 3’,3’,3’-cACCMP) having alternative phosphodiester linkages (e.g., AMP 2’ to CMP 5’, CMP 2’ to CMP 5’, and/or AMP 2’ to CMP 5’) are depicted in FIG. 12 which could have different signaling properties or in vivo stability. Example analogs of cACCMP molecules (e.g., 3’,3’,3;-cACCMP) having alternative fluorinated substituents (e.g., mono- or poly -fluorinated versions) are depicted in FIG. 13 which could have different (e.g., increased) cell permeability. Example analogs of the cACCMP molecules (e.g., 3’,3’,3’- cACCMP) having alternative phosphodiester substituents are depicted in FIG. 14 (e.g., mono- or poly-phosphorothioate versions) and in FIG. 15 (e.g., mono- orpoly-methylphosphonate versions) which could have different (e.g., increased) resistance to cellular nucleases. Example analogs of cACCMP molecules (e.g., 3’,3’,3’-cACCMP) having alternative nucleotide bases (e.g., mono- or poly-substituted nucleotide bases) are depicted in FIG. 16 which could have various purpose (e.g., labeling and the like).
[0180] Examples of combinations of cCAMP nucleotides and/or isonucleotides are shown in FIG. 17. FIG. 18 shows examples of 3’,3’-cCAMP with an isonucleotidic AMP group, and FIG. 19 shows examples of 3’,3’-cCAMP with an isonucleotidic CMP group). Examples of combinations of cACCMP nucleotides and/or isonucleotides are shown in FIG. 20. FIG. 21 shows examples of 3’,3’-cACCMP with an isonucleotidic AMP group.
[0181] Applicants can perform any modification or combination of modifications (e.g., various fluorinated, phosphorothioate, methyl phosphonate, and/or modified nucleotide base versions) on any cCAMP of any analog configuration (e.g., 2’, 3’; 3’, 3’; and the like)(nucleotides and/or isonucleotides) or any cACCMP of any analog configuration (e g., 3’, 3’, 3’; 2’, 3’, 3’; and the like)(nucleotides and/or isonucleotides).
* * *
[0182] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and can be applied to the essential features herein before set forth.

Claims

CLAIMS What is claimed is:
1. A composition comprising a cyclic dinucleotide comprising a cytidine moiety and an adenosine moiety or a cyclic trinucleotide comprising an adenosine moiety and two cytidine moieties, or an analog or derivative thereof, or any combination thereof.
2. The composition of claim 1, comprising an n’, n’ cyclic cytidine monophosphate-adenosine monophosphate (n’,n’ cCAMP), an n’,n’,n’ cyclic adenosine monophosphate-cytidine monophosphate cytidine, (n’, n’, n’cACCMP), wherein each n’ is 2’ or 3’, an analog or derivative thereof, or any combination thereof.
3. The composition of claim 1 or claim 2, according to:
Figure imgf000073_0001
Figure imgf000074_0001
(Id); or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from, oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups and detectable labels, wherein each R4 is optionally further substituted. The composition of claim 1 or claim 2, according to:
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
(Ilh); or an analog or derivative thereof; wherein: each R1 is independently selected from hydrogen, oxygen, methoxy, amine, azide, and fluorine groups; each R2 is independently selected from oxygen and sulfur groups; each R3 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each R3 is optionally further substituted; and each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and detectable labels, wherein each R4 is optionally further substituted. The composition of any one of claims 1-4, wherein the composition is an isonucleotide analog of the cyclic dinucleotide comprising the cytidine moiety and the adenosine moiety or the cyclic trinucleotide comprising the adenosine moiety and the two cytidine moieties, or an analog or derivative thereof, or any combination thereof. The composition of claim 5, wherein the isonucleotide analog comprises one or more sugar ring(s), each comprising the adenosine or the cytosine group at the 2’ carbon position versus the 1 ’ carbon position and the R1 group at the 1 ’ carbon position versus the 2’ carbon position. The composition of claim 5 or claim 6, wherein the composition is according to:
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
(Hid); or an analog or derivative thereof; wherein: R6 or R7 is an adenine moiety or an analog or derivative thereof, and, when not an adenine moiety or an analog or derivative thereof, R6 or R7 is selected from hydrogen, hydroxyl, and fluorine groups;
R8 or R9 is a cytidine moiety or an analog or derivative thereof, and, when not a cytidine moiety or an analog or a derivative thereof, R8 or R9 is selected from hydrogen, hydroxyl, and fluorine groups; each RIO is independently selected from, oxygen and sulfur groups; and each R11 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1-C10 alkyl group optionally substituted with a C1-C10 alkyl ester group, and wherein each R11 is optionally further substituted. The composition of claim 5 or claim 6, wherein the composition is according to:
Figure imgf000082_0001
(IV); or an analog or derivative thereof; wherein:
R6 or R7 is an adenine group or an analog or derivative thereof, and, when not an adenine group or an analog or derivative thereof, R6 or R7 is selected from hydrogen, hydroxyl, and fluorine groups; at each occurrence, R8 or R9 is a cytidine group or an analog or derivative thereof, and, at each occurrence, when not a cytidine group or an analog or a derivative thereof, R8 or R9 is selected from hydrogen, hydroxyl, and fluorine groups; each RIO is independently selected from oxygen and sulfur groups; and each Rl 1 is independently selected from hydrogen, C1-C5 alkyl, hydroxyl, and alkoxyl (- OR5) groups, wherein R5 is a C1 -C10 alkyl group optionally substituted with a C1 -C10 alkyl ester group, and wherein each Rl 1 is optionally further substituted. The composition of any one of claims 5-8, wherein: the adenine moiety is according to:
Figure imgf000083_0001
analog or derivative thereof; and/or the cytidine moiety is according to:
Figure imgf000083_0002
analog or derivative thereof; wherein each R4 is independently selected from hydrogen, C1-C5 alkyl, alkyne, and azide groups, and a detectable labels, and wherein each R4 can be optionally further substituted. The composition of any of claims 1-9, wherein Rl, R6 or R7, and/or R8 or R9 is/are a fluorine group. The composition of any of claims 1-10, wherein R2 or RIO is a sulfur group. The composition of any of claims 1-11, wherein R3 or R11 is selected from methyl group, hydroxyl group, and alkoxyl (-OR5) groups, and wherein R5 is a
Figure imgf000084_0001
group. The composition of any of claims 1-12, wherein R4 is selected from hydrogen group, methyl group, and alkyne groups selected from cycloalkyne and
Figure imgf000084_0002
groups, optionally wherein the cycloalkyne group is a cyclooctyne group. The composition of any of claims 1-13, wherein R4 is a detectable label, optionally selected from biotin, fluorophore, and a radiolabel. The composition of any of claims 1-14, wherein the composition is a prodrug composition. A pharmaceutical composition comprising one or more composition(s) of any one of the previous claims, or pharmaceutically acceptable salt(s) thereof. A method of modulating immune signaling in a cell, the method comprising administering one or more composition(s) of any one of the previous claims to the cell. A method of modulating an immune signaling in a subject in need thereof, the method comprising administering an effective amount of one or more pharmaceutical composition(s) of claim 16 to the subject. The method of claim 17 or claim 18, wherein the composition(s) or pharmaceutical composition(s) is/are administered via a delivery vehicle comprising liposomes, lipid particles, or nanoparticles. A method of identifying one or more pathways modulated by one or more composition(s) of any one of claims 1-15, the method comprising delivering the composition(s) to a cell, and evaluating differential expression of one or more genes of the one or more pathways by RNAseq to thereby identify the one or more pathways.
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