WO2025240977A1 - Compositions de composés sulfonyl-purine et leur utilisation pour modifier des sites protéiques fonctionnels - Google Patents

Compositions de composés sulfonyl-purine et leur utilisation pour modifier des sites protéiques fonctionnels

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WO2025240977A1
WO2025240977A1 PCT/US2025/030073 US2025030073W WO2025240977A1 WO 2025240977 A1 WO2025240977 A1 WO 2025240977A1 US 2025030073 W US2025030073 W US 2025030073W WO 2025240977 A1 WO2025240977 A1 WO 2025240977A1
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substituted
group
alkyl
compound
aralkyl
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Ku-Lung HSU
Zhihong Li
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/22—Benzimidazoles; Hydrogenated benzimidazoles with hetero atoms directly attached to ring nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/04—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/28—Oxygen atom
    • C07D473/30—Oxygen atom attached in position 6, e.g. hypoxanthine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/32—Nitrogen atom
    • C07D473/34—Nitrogen atom attached in position 6, e.g. adenine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01035—3-Hydroxyacyl-CoA dehydrogenase (1.1.1.35)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/02—Thioester hydrolases (3.1.2)
    • C12Y301/02022—Palmitoyl-protein hydrolase (3.1.2.22)

Definitions

  • sulfonyl-purine and sulfonyl-purine analog- 10 containing compounds as well as to their use in covalently modifying peptides and proteins, in proteomics, and for modulating the biological activity of peptides and proteins.
  • these compounds also referred to herein as sulfonyl-purine (SuPUR) ligands or probes, can form covalent conjugates with nucleophilic groups in peptides and proteins (e.g., with phenols and amines of tyrosine and lysine side chains), thereby forming covalently modified peptides and proteins.
  • Exemplary 15 covalently modified proteins described include amyloid-beta (A ⁇ )-binding alcohol dehydrogenase (ABAD) and alpha/beta ( ⁇ / ⁇ )-hydrolase domain 10 (ABHD10).
  • a ⁇ amyloid-beta
  • ABAD alpha/beta
  • ABHD10 alpha/beta
  • a ⁇ is the pathological protein in AD, partly due to the interaction between A ⁇ and ABAD.
  • ABAD is a mitochondrial protein that is an important regulator of energy balance and, upon A ⁇ binding, activates signaling cascades leading to neuronal death.
  • ABAD ABAD-induced ABAD
  • ROS reactive 25 oxygen species
  • ABHD10 is a member of the serine hydrolase superfamily and plays an important role in the metabolism of the immunosuppressant mycophenolate mofetil (MMF) by facilitating the removal of acyl glucuronide metabolite (AcMPAG) in the liver.
  • MMF immunosuppressant mycophenolate mofetil
  • ABHD10 detoxifies the body and reduces the 30 risk of MMF-induced side effects, such as leucopenia and gastrointestinal toxicity.
  • ABHD10 also exhibits a similar detoxifying effect on probenecid acyl glucuronide (PRAG), the primary metabolite of the uricosuric agent probenecid, which can cause severe allergic or anaphylactic reactions.
  • PRAG probenecid acyl glucuronide
  • ABHD10 has S-depalmitoylase activity, which acts on peroxiredoxin-5 (PRDX5), an important antioxidant protein. Therefore, ABHD10 can be classified as a member of the acyl protein 35 thioesterase (APT) family of regulatory proteins.
  • APT thioesterase
  • Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.
  • R 1 is phenyl or substituted phenyl
  • Z and Y are N
  • R 2 and R 6 are selected from the group consisting of fluoro, chloro, bromo, methyl, perfluoromethyl, methoxy, and perfluoromethoxy.
  • R 4 is cyano. 15
  • R is selected from the group consisting of: - 5 - Attorney Docket No.: 3436/3 PCT , - 6 - Attorney Docket No.: 3436/3 PCT - 7 - Attorney Docket No.: 3436/3 PCT 5 - 8 - Attorney Docket No.: 3436/3 PCT
  • the presently disclosed subject matter provides a pharmaceutical composition comprising a compound having a structure of Formula (I) and a pharmaceutically acceptable carrier.
  • the presently disclosed subject matter provides a method of covalently 5 modifying a peptide or protein, the method comprising contacting a sample comprising the peptide or protein with a compound of Formula (I) or a pharmaceutical composition thereof.
  • the contacting provides a covalent modified peptide or protein, wherein said covalently modified peptide or protein comprises one or more covalently modified tyrosine or covalently modified lysine residues, wherein the covalently modified tyrosine or covalently modified lysine residues 10 comprise a structure: wherein R 1 is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, 15 optionally wherein R 1 is selected from phenyl and substituted phenyl.
  • the presently disclosed subject matter provides a method of inhibiting amyloid-beta (A ⁇ )-binding alcohol dehydrogenase (ABAD), the method comprising contacting a sample comprising ABAD with a compound of Formula (I), thereby covalently modifying one or more 25 amino acid residues in the ABAD, wherein the compound of Formula (I) has a structure: - 9 - Attorney Docket No.: 3436/3 PCT Formula (I), wherein: each represents a single or double bond; subject to the proviso that three represent a double bond and no two adjacent are both double bonds; Z is selected from CH, N, and NH; Y is selected from CH, N, and NH; X 1 and X 2 are independently selected from the group 5 consisting of H, O, F, Cl, Br,-NH 2 , -NHX 5 , and -N(X 5 ) 2 , wherein each X 5 is selected from the group consisting of alkyl, aralkyl
  • R is selected from the group consisting of: , , - 10 - Attorney Docket No.: 3436/3 PCT In some embodiments, R is: .
  • the covalently modified ABAD comprises a covalently modified non- catalytic tyrosine residue, optionally wherein said covalently modified non-catalytic tyrosine residue is 5 tyrosine 168 (Tyr168).
  • the covalently modified ABAD exhibits reduced catalytic activity compared to a corresponding unmodified ABAD.
  • the covalently modified ABAD exhibits decreased binding for beta-amyloid (A ⁇ ) compared to a corresponding unmodified ABAD.
  • a ⁇ beta-amyloid
  • the covalently modified ABAD exhibits a reduction in reactive oxygen species (ROS) induced by beta-amyloid (A ⁇ ) compared to a corresponding 10 unmodified ABAD.
  • the presently disclosed subject matter provides a method of inhibiting alpha/beta-hydrolase domain 10 (ABHD10), the method comprising contacting a sample comprising ABHD10 with a compound of Formula (I), thereby covalently modifying one or more amino acid residues in the ABHD10, wherein the compound has a structure of Formula (I): 15 Formula (I), wherein: each represents a single or double bond; subject to the proviso that three represent a double bond and no two adjacent are both double bonds; Z is selected from CH, N, and NH; Y is selected from CH, N, and NH; X 1 and X 2 are independently selected from the group consisting of H, O, F, Cl, Br,-NH 2 , -NHX 5 , and -N(X
  • R is selected from the group consisting of: , 5 - 12 - Attorney Docket No.: 3436/3 PCT
  • R is:
  • the covalently modified ABHD10 comprises a covalently modified non- catalytic tyrosine residue, optionally wherein said covalently modified non-catalytic tyrosine residue is 5 tyrosine 215 (Tyr215).
  • the covalently modified ABHD10 exhibits reduced catalytic activity compared to a corresponding unmodified ABHD10.
  • the presently disclosed subject matter provides a method of identifying a reactive amino acid residue of a protein, optionally wherein the reactive amino acid residue is a tyrosine residue or a lysine residue, the method comprising: (a) providing a protein sample comprising 10 isolated proteins, living cells, or a cell lysate; (b) contacting the protein sample with a probe compound for a period of time sufficient for the probe compound to covalently react with at least one reactive amino acid residue in the protein sample, thereby forming at least one modified reactive amino acid residue; and (c) analyzing proteins in the protein sample to identify at least one modified reactive amino acid residue, thereby identifying at least one reactive amino acid residue of a protein; wherein the probe 15 compound has a structure of Formula (I): Formula (I), wherein: each represents a single or double bond; subject to the proviso that three represent a double bond and no two adjacent are both double bonds; Z is selected from CH, N, and NH; Y is selected from CH, N,
  • Y and Z are each selected from N and NH. 5 Accordingly, it is an object of the presently disclosed subject matter to provide compounds of Formula (I), e.g., SuPUR compounds, related pharmaceutical compositions, methods of identifying reactive amino acid residues, and methods of covalently modifying peptides and proteins, e.g., ABAD and ABHD10. This and other objects are achieved in whole or in part by the presently disclosed subject matter. 10 An object of the presently disclosed subject matter having been stated above, other objects and advantages of the presently disclosed subject matter will become apparent to those of ordinary skill in the art after a study of the following description of the presently disclosed subject matter and non- limiting Figures and Examples.
  • Figure 1A is a schematic drawing comparing generic chemical structures of, from left to right, sulfonyl-fluoride compounds used in sulfonyl-fluoride exchange (SuFEx) chemistry, sulfonyl-triazole 25 compounds used in sulfonyl-triazole exchange (SuTEx) chemistry, and exemplary sulfonyl-purine compounds for use in sulfonyl-purine (SuPUR) chemistry.
  • the leaving groups of these compounds upon reaction with a nucleophilic moiety are F, triazole, and purine, respectively.
  • R 1 in each of the compounds represents a sulfonyl substituent that remains attached with the sulfonyl group to a nucleophilic moiety (e.g., a nucleophilic moiety of a peptide or protein) covalently modified by the 30 SuFEx, SuTEx, or SuPUR compound.
  • a nucleophilic moiety e.g., a nucleophilic moiety of a peptide or protein
  • R 2 represents a substituent attached to the triazole leaving group.
  • Figure 1B is a schematic drawing showing the chemical structure of an exemplary sulfonyl- purine (SuPUR) probe compound referred to herein as “AHL-PuP-2”.
  • Figure 1C is a schematic drawing showing the chemical structure of an exemplary sulfonyl- 35 purine (SuPUR) ligand compound referred to herein as “ZH-2-049-2”.
  • Figure 1D is a schematic drawing showing an exemplary workflow for sulfonyl-purine (SuPUR)-based proteomic studies.
  • Figure 1E is an image of a gel from a gel-based activity-based protein profiling (APBB) assay performed in the cell soluble fraction (left) and the membrane soluble fraction of cells using sulfonyl- 5 purine (SuPUR) chemistry.
  • APBB gel-based activity-based protein profiling
  • the total number of proteins labeled was 1800 and the total number of quality peptides was 9905. 40.39% of the labeled peptides were labeled at a lysine (Lys or K) residue, while 55.41% of the labeled peptides were labeled at a tyrosine (Tyr or Y) residue.
  • Figure 2A is an image of portions of a gel assay of the cell soluble fraction of amyloid-beta (A ⁇ )-binding alcohol dehydrogenase (ABAD)-transfected HEK-293 cells incubated first with dimethyl sulfoxide (DMSO) or 2.5 micromolar ( ⁇ M), 25 ⁇ M, 100 ⁇ M, or 200 ⁇ M of an exemplary sulfonyl- purine (SuPUR) ligand (referred to herein as “ZH-2-049-2”) and then with a SuPUR probe (referred to herein as “AHL-PuP-2”) for one hour.
  • DMSO dimethyl sulfoxide
  • ⁇ M 2.5 micromolar
  • ZH-2-049-2 an exemplary sulfonyl- purine ligand
  • AHL-PuP-2 SuPUR probe
  • FIG. 2B is an image of portions of a gel assay showing that the sulfonyl-purine (SuPUR) probe compound reacts with wild-type (WT) amyloid-beta (A ⁇ )-binding alcohol dehydrogenase (ABAD), but not with a mutant ABAD where the tyrosine at residue 168 is replaced by a glycine 25 (Y168G).
  • SuPUR sulfonyl-purine
  • Figure 3B is a schematic drawing showing select residues in the catalytic pocket of amyloid- beta (A ⁇ )-binding alcohol dehydrogenase (ABAD), including tyrosine-168 (Tyr168), lysine-172 (Lys172), phenylalanine-201 (Phe201) with an exemplary sulfonyl-purine (SuPUR) ligand (ZH-2-049- 35 2) in the pocket and possible non-covalent interactions between the catalytic pocket residues and the ligand.
  • a ⁇ amyloid- beta
  • ABAD amyloid- beta
  • Tyr168 tyrosine-168
  • Lysine-172 Lysine-172
  • Phe201 phenylalanine-201
  • SuPUR exemplary sulfonyl-purine
  • Figure 3D is a graph showing the ratio of the quantified fluorescence signal corresponding to the amyloid-beta (A ⁇ )-binding alcohol dehydrogenase (ABAD) band compared to the quantified fluorescence signal corresponding to a control protein band ( ⁇ -FLAG) in the gel shown in Figure 3C.
  • Figure 3E is a graph showing the concentration-dependent inhibition of amyloid-beta (A ⁇ )- binding alcohol dehydrogenase (ABAD) provided by exemplary sulfonyl-purine (SuPUR) compounds 10 ZH-1-049-2 (data in circles), ZH-2-025 (data in squares), and ZH-2-029 (data in triangles).
  • Inhibition is expressed as a percentage compared ABAD not treated with a SuPUR compound and the concentration of the SuPUR compound is expressed as the log of the micromolar ( ⁇ M) concentration of the compound.
  • ZH-2-025 shows potent activity for ABAD with a 50% inhibitory concentration (IC 50 ) of 430 nanomolar (nM).
  • IC 50 50% inhibitory concentration
  • ZH-1-049-2 shows an IC 50 of 776 nM and ZH-2-029 an IC 50 of 571 nM.
  • Figure 4A is a schematic drawing showing select residues in the catalytic pocket of amyloid- beta (A ⁇ )-binding alcohol dehydrogenase (ABAD), including tyrosine-168 (Tyr168), lysine-172 (Lys172), alanine-154(Ala154), glycine-199 (Gly199), glycine-93 (Gly93), and phenylalanine-201 (Phe201) with an exemplary dichlorophenyl-substituted sulfonyl-purine (SuPUR) ligand (ZH-2-025) in the pocket and possible non-covalent interactions between the catalytic pocket residues and the ligand, 20 including possible halogen bonding interactions between the chloride atoms of the ligand and Gly93.
  • ABAD amyloid- beta
  • Tyr168 tyrosine-168
  • Lys172 Lysine-172
  • alanine-154(Ala154) g
  • Figure 4B is a schematic drawing showing the chemical structures of various sulfonyl-purine (SuPUR) ligands comprising ortho-substituted phenyl sulfonyl substituents.
  • Figure 4C is an image of portions of a gel-based screening assay showing the amyloid-beta (A ⁇ )-binding alcohol dehydrogenase (ABAD) reactivity of the sulfonyl-purine (SuPUR) ligands shown 25 in Figure 4B.
  • ZH-2-025, ZH-2-085, ZH-2-093, and ZH-2-101 showed the highest activity.
  • Figure 4D is a graph showing the results of a bioluminescent screening assay (sold under the tradename NAD-GLOTM) for the activity of amyloid-beta (A ⁇ )-binding alcohol dehydrogenase (ABAD) treated with 500 nanomolar (nM) concentrations of exemplary sulfonyl-purine (SuPUR) compounds.
  • a ⁇ amyloid-beta
  • nM nanomolar
  • 30 Figure 4E is a series of schematic drawings showing the chemical structures of three exemplary sulfonyl-purine (SuPUR) ligands containing dihalophenyl moieties and their calculated electrostatic potential (ESP) surfaces.
  • ESP surfaces were calculated at the M062X/def2TZVPP level of theory by Gaussian 16 C.02 software.
  • FIG. 5A is a graph showing the results of a bioluminescent assay for nicotinamide adenine dinucleotide (NAD) (sold under the tradename NAD-GLOTM) showing that NAD+ is a substrate of amyloid-beta (A ⁇ )-binding alcohol dehydrogenase (ABAD).
  • NAD nicotinamide adenine dinucleotide
  • Figure 5B is a graph showing the ability of exemplary sulfonyl-purine (SuPUR) ligands to 5 inhibit amyloid-beta (A ⁇ )-binding alcohol dehydrogenase (ABAD) binding to nicotinamide adenine dinucleotide (NAD+) as determined via a bioluminescent assay (sold under the tradename NAD- GLOTM).
  • the 50% inhibitory concentration (IC 50 ) of ZH-2-025, ZH-2-029, ZH-2-101, and ZH-2-115 was 53.7 nanomolar (nM), 164.8 nM, 72.8 nM, and 706.3 nM, respectively.
  • Figure 5C is a composite image of a gel showing the results of an assay determining the ability 10 of a sulfonyl-purine (SuPUR) ligand (ZH-2-025) to disrupt the interaction of amyloid-beta (A ⁇ )-binding alcohol dehydrogenase (ABAD) and amyloid-beta 1-42 (A ⁇ 1-42).
  • Figure 5D is a graph showing the quantification of relevant protein signal compared to lane Input for the different gel lanes of the output side of the gel shown in Figure 5C.
  • Figure 5E is a graph of results of a cell proliferation assay (WST-1 assay) showing the cell 15 toxicity of sulfonyl-purine (SuPUR) ligand ZH-2-025 in HEK293T and SH-SY5Y cells.
  • WST-1 assay cell proliferation assay
  • Figure 5F is a composite image of a gel showing the results of a pull down assay showing that exemplary sulfonyl-purine (SuPUR) ligand ZH-2-029 can disrupt the interaction between amyloid-beta (A ⁇ )-binding alcohol dehydrogenase (ABAD) and amyloid-beta 1-42 (A ⁇ 1-42).
  • Figure 5G is a graph of results of a cell proliferation assay (WST-1 assay) showing that exemplary sulfonyl-purine (SuPUR) ligand ZH-2-029 exhibits moderate cell toxicity.
  • Figure 5H is a composite gel image of cellular thermal shift assay (SETSA) showing that exemplary sulfonyl-purine (SuPUR) ligand ZH-2-029 can stabilize amyloid-beta (A ⁇ )-binding alcohol dehydrogenase (ABAD), particularly for the dimer.
  • Figure 5I is a series of micrograph images of reactive oxygen species (ROS) production in cells treated under different conditions (i.e., sulfonyl-purine (SuPUR) compound ZH-2-029 and amyloid- beta 1-42 (A ⁇ 1-42), SuPUR compound ZH-2-036 and A ⁇ 1-42; A ⁇ 1-42, or dimethyl sulfoxide (DMSO, control)).
  • ROS reactive oxygen species
  • the non-fluorescent probe dichlorodihydrofluorescein diacetate (H 2 DCF-DA) was used to measure cytosolic ROS, e.g., hydrogen peroxide. See top row. To determine levels of the 30 superoxide anion radicals DHE was used, which is oxidized to the fluorescent ethidium cation by O 2 radical. See bottom row. For detection of mitochondria-associated ROS, dihydrorhodamine (DHR), which localized to the mitochondria. See middle row.
  • Figure 6 is an image of a gel assay of the cell soluble and cell membrane fractions of HEK-293 cells treated with a sulfonyl-purine (SuPUR) ligand (ZH-1-049-2) for one hour at 0.02, 0.2, 2.0, 10, 50, 35 or 100 micromolar ( ⁇ M) concentrations and then treated with 1 ⁇ M fluorescently-tagged - 17 - Attorney Docket No.: 3436/3 PCT fluorophosphate (FP-Rh) for one hour.
  • the SuPUR ligand showed selective labelling activity for ABHD10 in the membrane fraction.
  • Figure 7A is a schematic diagram showing the chemical structures of some exemplary sulfonyl- purine (SuPUR) ligands assayed for ability to covalently modify and inhibit alpha/beta ( ⁇ / ⁇ )-hydrolase 5 domain 10 (ABHD10).
  • Figure 7B is an image of gel analysis of the screening of sulfonyl-purine (SuPUR) ligand activity in the cell membrane fraction of HEK-293 cells. The cell membrane fraction was treated with the SuPUR ligand indicated at the top of the image at a 0.2 micromolar ( ⁇ M) or 2 ⁇ M concentration for one hour and then with 1 ⁇ M fluorescently-tagged fluorophosphate (FP-Rh) for one hour.
  • ⁇ M micromolar
  • FP-Rh fluorescently-tagged fluorophosphate
  • Figure 7C is an image of gel analysis of the screening of sulfonyl-purine (SuPUR) ligand activity in the cell soluble fraction of HEK-293 cells.
  • the cell soluble fraction was treated with the SuPUR ligand indicated at the top of the image at a 0.2 micromolar ( ⁇ M) or 2 ⁇ M concentration for one hour and then with 1 ⁇ M fluorescently-tagged fluorophosphate (FP-Rh) for one hour.
  • Figure 8A is an image of a gel showing the concentration-dependent activity of exemplary 15 sulfonyl-purine (SuPUR) ligands ZH-2-097 and ZH-2-103 in a cell soluble fraction of HEK-293 cells.
  • FIG. 8B is a graph showing the relationship between sulfonyl-purine (SuPUR) ligand 20 concentration (log of the micromolar ( ⁇ M) ligand concentration) and the percentage (%) of alpha/beta ( ⁇ / ⁇ )-hydrolase domain 10 (ABHD10) inhibition determined via gel-based screening in a cell soluble fraction of HEK-293 cells. Data is provided for ZH-2-097 (circles) and ZH-20103 (squares).
  • FIG. 8C is a schematic drawing showing the chemical structures of exemplary sulfonyl-purine (SuPUR) ligands: ZH-2-025, ZH-2-097, and ZH-2-103.
  • Figure 8D is an image of a gel showing the concentration-dependent activity of exemplary sulfonyl-purine (SuPUR) ligands ZH-2-025, ZH-2-097, and ZH-2-103 in the membrane soluble fraction of HEK-293 cells.
  • FIG. 9A is a schematic drawing showing the chemical structures of exemplary sulfonyl- purine (SuPUR) ligands: ZH-2-097 and ZH-2-036.
  • Figure 9B is a schematic drawing showing select residues in the catalytic pocket of alpha/beta ( ⁇ / ⁇ )-hydrolase domain 10 (ABHD10), including tyrosine-215 (Tyr215), serine-152 (Ser152), serine- 35 86 (Ser86), histidine-279 (His279), arginine-280 (Arg280), and arginine-282 (Arg-282) with an - 18 - Attorney Docket No.: 3436/3 PCT exemplary an sulfonyl-purine (SuPUR) ligand, ZH-2-097 in the pocket and with possible interactions between the catalytic pocket residues and the ligand shown in dashed lines.
  • ABHD10 alpha/beta ( ⁇ / ⁇ )-hydrolase domain 10
  • Figure 9C is an image of a gel showing the concentration-dependent activity of exemplary sulfonyl-purine (SuPUR) ligands ZH-2-097 and ZH-2-103 in the membrane and soluble fractions of 5 HEK-293 cells.
  • Cells were treated with the SuPUR compound for 2 hours and then with 1 micromolar ( ⁇ M) fluorescently tagged fluorophosphate (FP-Rh) for one hour.
  • Figure 10A is a heat map of hydrolase proteins modified by an exemplary sulfonyl-purine (SuPUR) compound ZH-2-097 (0.2 micromolar ( ⁇ M), 2 ⁇ M, 5 ⁇ M or 10 ⁇ M).
  • FIG. 10B is a graph showing the abundance ratio of peptide-spectrum matches (PSMs) detected from particular amino acid residue ranges in alpha/beta ( ⁇ / ⁇ )-hydrolase domain 10 (ABHD10) following covalent modification of ABHD10 with exemplary sulfonyl-purine (SuPUR) compound ZH- 2-097 at different concentrations (0.2 micromolar ( ⁇ M), 2 ⁇ M, 5 ⁇ M or 10 ⁇ M).
  • ABHD10 modified with ZH-2-036 which has an imidazopyridine leaving group is 15 shown.
  • Figure 10C is a graph showing the one-way ANOVA analysis of the data shown in Figure 10B.
  • Figure 10D is a schematic drawing showing a Venn diagram of overlapping hydrolase proteins detected based on activity-based protein profiling studies performed in cell membrane and cell soluble studies performed with exemplary sulfonyl-purine (SuPUR) ligand ZH-2-097.
  • Figure 10E is a graph showing the abundance ratio of peptide-spectrum matches (PSMs) detected from studies used to identify the binding site of exemplary sulfonyl-purine (SuPUR) compound ZH-2-097 in alpha/beta ( ⁇ / ⁇ )-hydrolase domain 10 (ABHD10).
  • PSMs peptide-spectrum matches
  • ZH-2-097 showed selective labeling of ABHD10 at tyrosine-87 (Y87) and tyrosine-215 (Y215) in a concentration dependent manner.
  • Figure 11 is an MS2 spectrum annotation of AHL-PuP-2 labeled site (Y380) found in a Keratin, 25 type I cytoskeletal 18 (KRT18) polypeptide (see e.g., Accession No. P05783 of the GENBANK® biosequence database).
  • Figures 12A-12C are the results of analysis showing SuPUR chemistry mediates regio-selective protein-ligand interactions across the human proteome.
  • Figure 12A shows the number of modified tyrosine (Y) and lysine (K) sites identified in the HEK293T proteome using AHL-PuP-2 (100 ⁇ M, 1 h, 30 rt; left panel) or ZH-2-087 (100 ⁇ M, 1 h, rt; right panel).
  • Figure 12B is a bar graph (top) showing a comparison of AHL-PuP-2 modified sites and ZH-2-087 modified sites. Below the bar graph is a summary of the modified amino acids with each SuPUR ligand.
  • Figure 13 is two plots of 13 C NMR data for ZH-1-049-2 and ZH-2-055 showing significant 35 chemical shifts in the C5 and C4 signals between the N9- and N7-substituted products, attributed to differences in electronic density.
  • - 19 Attorney Docket No.: 3436/3 PCT
  • Figures 14A and 14B are volcano plots of SuPUR TMT-ABPP analysis of ZH-1-049-2 and ZH-2-036 (25 ⁇ M, 1 hour, in vitro) using AHL-PuP-2 as a probe and showing the distribution of probe- modified peptides in comparison between the compound-treated group and DMSO group in HEK293T soluble proteome ( Figure 14A) and membrane proteome ( Figure 14B).
  • Figures 14C and 14D are volcano plots of SuPUR TMT-ABPP analysis of ZH-2-055 and ZH- 5-019 (25 ⁇ M, 1 hour, in vitro) using ZH-2-087 as a probe showing the distribution of probe-modified peptides in comparison between the compound-treated group and DMSO group in HEK293T soluble proteome ( Figure 14C) and membrane proteome ( Figure 14D).
  • Figure 15 is a proposed mechanism of ABHD10 inhibition by ZH-2-097 that involves covalent targeting a non-catalytic tyrosine, Tyr215.
  • Figure 16 is a heatmap showing LC-MS/MS analysis of HEK293T cells treated with ZH-2-097 (0.2 ⁇ M-10 ⁇ M) and ZH-2-036 (control) versus DMSO revealing selective inhibition of ABHD10 in 15 serine hydrolase family.
  • Figures 17A and 17B are volcano plots showing the distribution of probe modified peptides in comparison between compound treated group and DMSO group in soluble ( Figure 17A) and membrane (Figure 17B) proteomes from HEK293T cells.
  • Figure 18 is a gel showing the results of gel-based ABPP of SuPUR analogs that were designed 20 by a nitrogen-walk strategy and the binding activities thereof analogs.
  • the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including in the claims.
  • the phrase “a protein” refers to one or more proteins, including a plurality of the same protein.
  • the phrase “at least one”, when employed herein to refer to an entity refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 10 30, 35, 40, 45, 50, 75, 100, or more of that entity, including but not limited to whole number values between 1 and 100 and greater than 100.
  • all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”.
  • the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations 25 of A, B, C, and D.
  • the term “comprising”, which is synonymous with “including” “containing”, or “characterized by”, is inclusive or open-ended and does not exclude additional, unrecited elements and/or method steps. “Comprising” is a term of art that means that the named elements and/or steps are present, but that other elements and/or steps can be added and still fall within the scope of the relevant subject 30 matter.
  • a pharmaceutical composition can “consist essentially of” a pharmaceutically active agent or a plurality of 35 pharmaceutically active agents, which means that the recited pharmaceutically active agent(s) is/are the only pharmaceutically active agent(s) present in the pharmaceutical composition.
  • compositions that in some embodiments comprises a given active agent also in some embodiments can consist essentially of that same active agent, and indeed can in some embodiments consist of that same active agent.
  • additional therapeutically active compound and “additional therapeutic agent”, as used in the context of the presently disclosed subject matter, refers to the use or administration of a 15 compound for an additional therapeutic use for a particular injury, disease, or disorder being treated.
  • Such a compound could include one being used to treat an unrelated disease or disorder, or a disease or disorder which may not be responsive to the primary treatment for the injury, disease, or disorder being treated.
  • the terms “administration of” and/or “administering” a compound should be 20 understood to refer to providing a compound of the presently disclosed subject matter to a subject in need of treatment.
  • aqueous solution can include other ingredients commonly used, such as sodium bicarbonate described herein, and further includes any acid or base solution used to adjust the pH of the aqueous solution while solubilizing a peptide.
  • binding refers to the adherence of molecules to one another, such as, but not limited to, enzymes to substrates, ligands to receptors, antibodies to antigens, DNA binding domains of proteins to DNA, and DNA or RNA strands to complementary strands.
  • Binding partner refers to a molecule capable of binding to another molecule.
  • biocompatible refers to a material that does not elicit a substantial 30 detrimental response in the host.
  • biologically active fragment and “bioactive fragment” of a peptide encompass natural and synthetic portions of a longer peptide or protein that are capable of specific binding to their natural ligand and/or of performing a desired function of a protein, for example, a fragment of a protein of larger peptide which still contains the epitope of interest and is immunogenic. 35
  • biological sample refers to samples obtained from a subject, including but not limited to skin, hair, tissue, blood, plasma, cells, sweat, and urine.
  • a “control” cell, tissue, sample, or subject is a cell, tissue, sample, or subject of the same type as a test cell, tissue, sample, or subject.
  • the control may, for example, be examined at precisely or nearly the same time the test cell, tissue, sample, or subject is examined.
  • the control may also, for example, be examined at a time distant from the time at which the test cell, tissue, sample, or subject is 5 examined, and the results of the examination of the control may be recorded so that the recorded results may be compared with results obtained by examination of a test cell, tissue, sample, or subject.
  • the control may also be obtained from another source or similar source other than the test group or a test subject, where the test sample is obtained from a subject suspected of having a condition, disease, or disorder for which the test is being performed.
  • a “test” cell is a cell being examined.
  • a “pathogenic” cell is a cell that, when present in a tissue, causes or contributes to a condition, disease, or disorder in the animal in which the tissue is located (or from which the tissue was obtained).
  • a tissue “normally comprises” a cell if one or more of the cell are present in the tissue in an animal not afflicted with a condition, disease, or disorder.
  • condition refers to physiological states in which diseased cells or cells of interest can be targeted with the compositions of the presently disclosed subject matter.
  • diagnosis refers to detecting a risk or propensity to a condition, disease, or disorder. In any method of diagnosis exist false positives and false negatives. Any one 20 method of diagnosis does not provide 100% accuracy.
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
  • an “effective amount” or “therapeutically effective amount” refers to an amount of a compound or composition sufficient to produce a selected effect, such as but not limited to alleviating symptoms of a condition, disease, or disorder.
  • an “effective amount” or “therapeutically effective amount” refers to an amount of a compound or composition sufficient to produce a selected effect, such as but not limited to alleviating symptoms of a condition, disease, or disorder.
  • the amount of each compound, when administered in combination with one or more other compounds, may be different from when that compound is administered alone.
  • an effective amount of a combination of compounds refers collectively to the combination as a whole, although the actual amounts of each compound may vary.
  • the term “more effective” means that the selected effect occurs to a greater extent by one treatment 35 relative to the second treatment to which it is being compared.
  • - 23 - Attorney Docket No.: 3436/3 PCT As used herein, an “essentially pure” preparation of a particular protein or peptide is a preparation wherein in some embodiments at least about 95% and in some embodiments at least about 99%, by weight, of the protein or peptide in the preparation is the particular protein or peptide.
  • fragment refers 5 to a portion of an amino acid sequence, comprising at least one amino acid, or a portion of a nucleic acid sequence comprising at least one nucleotide.
  • fragment refers 5 to a portion of an amino acid sequence, comprising at least one amino acid, or a portion of a nucleic acid sequence comprising at least one nucleotide.
  • fragment refers 5 to a portion of an amino acid sequence, comprising at least one amino acid, or a portion of a nucleic acid sequence comprising at least one nucleotide.
  • sulfonyl-purine refers to a synthetic compound that includes a sulfonyl group directly attached to a nitrogen atom of a purine 10 moiety or a purine analog moiety (e.g., another bicyclic, imidazole-containing fused ring moiety, such as a hypoxanthine, xanthine, imidazopyridine, or benzimidazole moiety).
  • a purine analog moiety e.g., another bicyclic, imidazole-containing fused ring moiety, such as a hypoxanthine, xanthine, imidazopyridine, or benzimidazole moiety.
  • the purine or purine analog moiety can be substituted or unsubstituted.
  • a SuPUR compound can undergo SuPUR chemistry, e.g., in which the sulfonyl group of the SuPUR compound acts as an electrophile in a covalent reaction between the SuPUR compound and a reactive nucleophilic group of another 15 compound, such as a reactive amino acid residue in a protein or peptide.
  • SuPUR compounds disclosed herein can include SuPUR “ligands” and SuPUR “probes.”
  • the terms “SuPUR probe” or “probe” can refer to a SuPUR compound that is broadly reactive and can be used to detect sites amenable to covalent reactions with SuPUR compounds.
  • SuPUR probes can include a tag for detection.
  • the tag for detection can be a moiety that can be directly 20 used for detection (e.g., a fluorophore, biotin or another affinity label, a radioisotope, etc.) or a moiety (e.g., an alkyne group) that can be chemically modified to incorporate a detectable group (e.g., biotin) after the SuPUR probe has undergone a covalent reaction with a SuPUR reactive site.
  • the terms “SuPUR ligand” or “ligand”, as used herein can refer to a SuPUR compound that does not include a tag for detection and/or that has been tailored to undergo covalent reactions more selectively 25 with a particular reactive site and/or protein and/or peptide of interest.
  • the term “ligand” as used herein can be used more generally to refer to any entity (e.g., a molecule) that specifically or selectively binds to or “is specifically or selectively 35 reactive with a second entity (e.g., a biomolecule, such as a peptide, protein, nucleic acid, lipid, etc.) when the ligand functions in a binding reaction which is determinative of the presence of the second - 24 - Attorney Docket No.: 3436/3 PCT entity in a heterogenous sample (i.e., a sample comprising a plurality of different entities, such as a plurality of different biomolecules).
  • a heterogenous sample i.e., a sample comprising a plurality of different entities, such as a plurality of different biomolecules.
  • injecting include administration of a compound of the presently disclosed subject matter by any number of routes and modes including, but not limited to, topical, oral, buccal, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, 10 sublingual, vaginal, ophthalmic, pulmonary, vaginal, and rectal approaches.
  • linkage refers to a connection between two groups. The connection can be either covalent or non-covalent, including but not limited to ionic bonds, hydrogen bonding, and hydrophobic/hydrophilic interactions.
  • linker refers to a molecule that joins two other molecules either 15 covalently or noncovalently, such as but not limited to through ionic or hydrogen bonds or van der Waals interactions.
  • the terms “measuring the level of expression” and “determining the level of expression” as used herein refer to any measure or assay which can be used to correlate the results of the assay with the level of expression of a gene or protein of interest. Such assays include measuring the level of 20 mRNA, protein levels, etc. and can be performed by assays such as northern and western blot analyses, binding assays, immunoblots, etc.
  • the level of expression can include rates of expression and can be measured in terms of the actual amount of an mRNA or protein present.
  • Parenteral administration thus includes, 35 but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through - 25 - Attorney Docket No.: 3436/3 PCT a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.
  • composition refers to a composition comprising at least one active 5 ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, without limitation, a human).
  • a mammal for example, without limitation, a human
  • “Pharmaceutically acceptable” means physiologically tolerable, for either human or veterinary 10 application.
  • “pharmaceutical compositions” include formulations for human and veterinary use.
  • the term “pharmaceutically acceptable carrier” means a chemical composition with which an appropriate compound or derivative can be combined and which, following the combination, can be used to administer the appropriate compound to a subject.
  • physiologically acceptable ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered. “Plurality” means at least two.
  • mass spectrometry refers to a technique for the identification 20 and/or quantitation of molecules in a sample.
  • MS includes ionizing the molecules in a sample, forming charged molecules; separating the charged molecules according to their mass-to-charge ratio; and detecting the charged molecules. MS allows for both the qualitative and quantitative detection of molecules in a sample.
  • the molecules can be ionized and detected by any suitable means known to one of skill in the art.
  • Some examples of mass spectrometry are “tandem mass spectrometry” or “MS/MS,” 25 which are the techniques wherein multiple rounds of mass spectrometry occur, either simultaneously using more than one mass analyzer or sequentially using a single mass analyzer.
  • the term “mass spectrometry” can refer to the application of mass spectrometry to protein analysis.
  • electrospray ionization ESI
  • matrix-assisted laser desorption/ionization MALDI
  • intact protein molecules can be ionized by the above 30 techniques, and then introduced to a mass analyzer.
  • protein molecules can be broken down into smaller peptides, for example, by enzymatic digestion by a protease, such as trypsin. Subsequently, the peptides are introduced into the mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry.
  • mass spectrometer is used to refer an apparatus for performing mass 35 spectrometry that includes a component for ionizing molecules and detecting charged molecules.
  • Various types of mass spectrometers can be employed in the methods of the presently disclosed subject - 26 - Attorney Docket No.: 3436/3 PCT matter.
  • whole protein mass spectroscopy analysis can be conducted using time-of-flight (TOF) or Fourier transform ion cyclotron resonance (FT-ICR) instruments.
  • TOF time-of-flight
  • FT-ICR Fourier transform ion cyclotron resonance
  • MALDI time-of-flight instruments can be employed, as they permit the acquisition of peptide mass fingerprints (PMFs) at high pace.
  • high throughput protein identification is a gel- 10 based process that includes the pre-fractionation and purification of proteins by one-dimensional protein gel electrophoresis. The gel can then be fractionated into several molecular weight fractions to reduce sample complexity, and proteins can be in-gel digested with trypsin.
  • the technique 20 uses gel electrophoresis to separate the proteins, which are then transferred from the gel to a membrane (typically nitrocellulose or PVDF) and stained, in membrane, with antibodies specific to the target protein.
  • a membrane typically nitrocellulose or PVDF
  • the expression “stable isotope labeling by amino acids in cell culture” (SILAC) is used herein to refer to an approach for incorporation of a label into proteins for mass spectrometry (MS)-based 25 quantitative proteomics.
  • SILAC comprises metabolic incorporation of a given “light” or “heavy” form of the amino acid into the proteins.
  • SILAC comprises the incorporation of amino acids with substituted stable isotopic nuclei (e.g. deuterium, 13 C, 15 N).
  • SILAC experiment two cell populations are grown in culture media that are identical, except that one of them contains a “light” and the other a “heavy” form of a particular amino acid (for example, 12 C and 13 C labeled L- 30 lysine, respectively).
  • a particular amino acid for example, 12 C and 13 C labeled L- 30 lysine, respectively.
  • the labeled analog of an amino acid is supplied to cells in culture instead of the natural amino acid, it is incorporated into all newly synthesized proteins. After a number of cell divisions, each instance of the amino acid is replaced by its isotope-labeled analog. Since there is little chemical difference between the labeled amino acid and the natural amino acid isotopes, the cells behave substantially similar to the control cell population grown in the presence of a normal amino acid.
  • prevention means to stop something from happening, or taking advance measures against something possible or probable from happening.
  • prevention generally refers to action taken to decrease the chance of getting a disease or condition. It is noted that “prevention” need not be absolute, and thus can occur as a matter of degree.
  • a “preventive” or “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs, or exhibits only early signs, of a condition, disease, or disorder.
  • a prophylactic or 5 preventative treatment is administered for the purpose of decreasing the risk of developing pathology associated with developing the condition, disease, or disorder.
  • Polypeptide refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof.
  • synthetic peptides or polypeptides refers to non-naturally occurring peptides or polypeptides. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid phase peptide synthesis methods are known to those of skill in the art.
  • the term “protein” typically refers to large polypeptides (e.g., greater than 50 amino acid residues).
  • proteome refers to the entire set of proteins expressed by a genome, cell, tissue, or organism at a particular time and/or under particular conditions.
  • purified and like terms relate to an enrichment of a molecule or 25 compound relative to other components normally associated with the molecule or compound in a native environment. The term “purified” does not necessarily indicate that complete purity of the particular molecule has been achieved during the process.
  • a “highly purified” compound as used herein refers to a compound that is in some embodiments greater than 90% pure, that is in some embodiments greater than 95% pure, and that is in some 30 embodiments greater than 98% pure.
  • the term “mammal” refers to any member of the class Mammalia, including, without limitation, humans, and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • the term 35 does not denote a particular age or sex.
  • subject refers to a member of species for which treatment and/or prevention of a disease or disorder using the compositions and methods of the presently disclosed subject matter might be desirable.
  • the term “subject” is intended to encompass in some embodiments any member of the Kingdom Animalia including, but not limited to the phylum Chordata 5 (e.g., members of Classes Osteichythyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Aves (birds), and Mammalia (mammals), and all Orders and Families encompassed therein.
  • the compositions and methods of the presently disclosed subject matter are particularly useful for warm-blooded vertebrates.
  • the presently disclosed subject matter concerns mammals and birds.
  • compositions and methods derived from 10 and/or for use in mammals such as humans and other primates, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economic importance (animals raised on farms for consumption by humans) and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), rodents (such as mice, 15 rats, and rabbits), marsupials, and horses.
  • carnivores other than humans such as cats and dogs
  • swine pigs, hogs, and wild boars
  • ruminants such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels
  • rodents such as
  • domesticated fowl e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans.
  • livestock including but not limited to domesticated swine 20 (pigs and hogs), ruminants, horses, poultry, and the like.
  • sample refers in some embodiments to a biological sample from a subject, including, but not limited to, normal tissue samples, diseased tissue samples, biopsies, blood, saliva, feces, semen, tears, and urine.
  • a sample can also be any other source of material obtained from a subject which contains proteins, cells, tissues, or fluid of interest.
  • sample can also be obtained from cell or 25 tissue culture.
  • standard refers to something used for comparison. For example, it can be a known standard agent or compound which is administered and used for comparing results when administering a test compound, or it can be a standard parameter or function which is measured to obtain a control value when measuring an effect of an agent or compound on a parameter or function.
  • Standard 30 can also refer to an “internal standard”, such as an agent or compound which is added at known amounts to a sample and is useful in determining such things as purification or recovery rates when a sample is processed or subjected to purification or extraction procedures before a marker of interest is measured.
  • Internal standards are often a purified marker of interest which has been labeled, such as with a radioactive isotope, allowing it to be distinguished from an endogenous marker.
  • a “subject” of analysis, diagnosis, or treatment is an animal. Such animals include mammals, in some embodiments, humans.
  • a “subject in need thereof” is a patient, animal, mammal, or human, who will benefit from the method of this presently disclosed subject matter.
  • the term “substantially pure” describes a compound, e.g., a protein or polypeptide, which has been separated from components which naturally accompany it.
  • a compound is substantially 5 pure when in some embodiments at least 10%, in some embodiments at least 20%, in some embodiments at least 50%, in some embodiments at least 60%, in some embodiments at least 75%, in some embodiments at least 90%, and in some embodiments at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column 10 chromatography, gel electrophoresis, or HPLC analysis.
  • a compound, e.g., a protein is also substantially purified when it is essentially free of naturally associated components or when it is separated from the native contaminants which accompany it in its natural state.
  • symptom refers to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease.
  • a “sign” is objective evidence of disease.
  • a bloody nose is a sign. It is evident to the patient, doctor, nurse, and other observers.
  • a “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.
  • a “therapeutically effective amount” of a compound is that amount of compound which is 20 sufficient to provide a beneficial effect to the subject to which the compound is administered.
  • the phrase “therapeutic agent” refers to an agent that is used to, for example, treat, inhibit, prevent, mitigate the effects of, reduce the severity of, reduce the likelihood of developing, slow the progression of, and/or cure, a disease or disorder.
  • treatment and “treating” as used herein refer to both therapeutic treatment and 25 prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition, prevent the pathologic condition, pursue or obtain beneficial results, and/or lower the chances of the individual developing a condition, disease, or disorder, even if the treatment is ultimately unsuccessful.
  • Those in need of treatment include those already with the condition as well as those prone to have or predisposed to having a condition, disease, or disorder, or those in 30 whom the condition is to be prevented.
  • All genes, gene names, and gene products disclosed herein are intended to correspond to homologs and/or orthologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this 35 disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates.
  • ABAD refers to the abhydrolase domain containing 10, depalmitoylase gene and its transcription and translation products.
  • ABAD is also known as hydroxysteroid 17-beta dehydrogenase 10 (HSD17B10).
  • HSD17B10 hydroxysteroid 17-beta dehydrogenase 10
  • exemplary human ABAD nucleic acid and amino acid sequences are presented in Accession Nos. NM_004493.3 and NP_004484.1 of the 5 GENBANK® biosequence database.
  • ABHD10 refers to the 3-hydroxyacyl-CoA dehydrogenase type-2 gene and its transcription and translation products.
  • Exemplary human ABHD10 nucleic acid and amino acid sequences are presented in Accession Nos. NM_018394.4 and NP_060864.1 of the GENBANK® biosequence database.
  • GSTP1 refers to the glutathione S-transferase pi 1 gene and its transcription and translation products.
  • Exemplary human GSTP1 nucleic acid and amino acid sequences are presented in Accession Nos. NM_000852.4 and NP_000843.1 of the GENBANK® biosequence database.
  • GNPNAT1 refers to the glucosamine-phosphate N-acetyltransferase 15 1 gene and its transcription and translation products.
  • Exemplary human GNPNAT1 nucleic acid and amino acid sequences are presented in Accession Nos. NM_198066.4 and NP_932332.1 of the GENBANK® biosequence database.
  • PARP2 refers to the poly(ADP-ribose) polymerase 2 gene and its transcription and translation products.
  • Exemplary human PARP2 nucleic acid and amino acid sequences 20 are presented in Accession Nos. NM_005484.4 and NP_005475.2 of the GENBANK® biosequence database.
  • ACAT2 refers to the acetyl-CoA acetyltransferase 2 gene and its transcription and translation products.
  • GNAS refers to the GNAS complex locus and its transcription and translation products.
  • Exemplary human GNAS nucleic acid and amino acid sequences are presented in Accession Nos. NM_000516.7 and NP_000507.1 of the GENBANK® biosequence database.
  • alkyl refers to C 1-20 inclusive, linear (i.e., “straight-chain”), branched, 30 or cyclic, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups.
  • Branched refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. In some 35 embodiments, the alkyl group is “lower alkyl.” “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C 1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.
  • the - 31 - Attorney Docket No.: 3436/3 PCT alkyl is “higher alkyl.” “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, “alkyl” refers, in particular, to C 1-8 straight-chain alkyls. In other embodiments, “alkyl” refers, in particular, to C 1-8 branched-chain alkyls. 5 Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more alkyl group substituents, which can be the same or different.
  • alkyl group substituent includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl.
  • nitrogen substituent is hydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), or aryl.
  • substituted alkyl includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, 15 hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.
  • aryl is used herein to refer to an aromatic moiety that can be a single aromatic ring, or multiple aromatic rings that are fused together, linked covalently, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety.
  • the common linking group also can be a carbonyl, as in benzophenone, or oxygen, as in diphenylether, or nitrogen, as in diphenylamine.
  • aryl specifically encompasses heterocyclic aromatic compounds.
  • the aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone, among others.
  • aryl means a cyclic aromatic comprising about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.
  • aryl group can be optionally substituted (a “substituted aryl”) with one or more aryl group substituents, which can be the same or different, wherein “aryl group substituent” includes alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, carbonyl, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NR’R’’, 30 wherein R’ and R’’ can each be independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralky
  • substituted aryl includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, 35 hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.
  • aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like.
  • heteroaryl refers to aryl groups wherein at least one atom of the backbone of the 5 aromatic ring or rings is an atom other than carbon.
  • heteroaryl groups have one or more non- carbon atoms selected from the group including, but not limited to, nitrogen, oxygen, and sulfur.
  • acyl specifically includes arylacyl groups, such as an acetylfuran and a phenacyl group.
  • acyl groups include acetyl and benzoyl.
  • Cyclic and cycloalkyl refer to a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.
  • the cycloalkyl group can be optionally partially unsaturated.
  • the cycloalkyl group also can be optionally substituted with an alkyl 15 group substituent as defined herein, oxo, and/or alkylene.
  • cyclic alkyl chain There can be optionally inserted along the cyclic alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, thus providing a heterocyclic group.
  • Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl, and cycloheptyl.
  • Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, 20 camphane, and noradamantyl.
  • heterocycle refers to cycloalkyl groups (i.e., non-aromatic, cyclic groups as described hereinabove) wherein one or more of the backbone carbon atoms of a cyclic ring is replaced by a heteroatom (e.g., nitrogen, sulfur, or oxygen).
  • heterocycles include, but are not limited to, tetrahydrofuran, tetrahydropyran, 25 morpholine, dioxane, piperidine, piperazine, and pyrrolidine.
  • heterocycles include, for example, the cyclic forms of sugars, such as ribose, glucose, galactose, and the like.
  • Alkylene refers to a straight or branched bivalent aliphatic hydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.
  • the alkylene group can be straight, branched or cyclic.
  • the alkylene group also can be optionally 30 unsaturated and/or substituted with one or more “alkyl group substituents.” There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as “alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described.
  • alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons.
  • Alkoxyl or “alkoxy” refers to an alkyl-O- group wherein alkyl is as previously described.
  • alkoxyl as used herein can refer to, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, 5 butoxyl, t-butoxyl, and pentoxyl.
  • oxyalkyl can be used interchangably with “alkoxyl”.
  • aryloxy and aryloxyl refer to an aryl-O-group, wherein aryl is as previously described.
  • aryloxy as used herein can refer to, for example, phenoxy, p-chlorophenoxy, p- fluorophenoxy, p-methylphenoxy, p-methoxyphenoxy, and the like.
  • Aralkyl refers to an aryl-alkyl- group wherein aryl and alkyl are as previously described and 10 include substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.
  • the aromatic portion of the aralkyl group can be substituted by one or more aryl group substituents and/or the alkyl portion of the aralkyl group can be substituted by one or more alkyl group substituents and the aralkyl group can be a “substituted aralkyl” group.
  • the term “amino” refers to the -NR’R” group, wherein R’ and R” are each independently 15 selected from the group including H and substituted and unsubstituted alkyl, cycloalkyl, heterocycle, aralkyl, aryl, and heteroaryl. In some embodiments, the amino group is -NH 2 .
  • alkylamino and “aminoalkyl” refer to a -NHR group where R is alkyl or substituted alkyl.
  • arylamino refers to a -NHR group where R is aryl or substituted aryl.
  • the term “carboxyl” can also be used to refer to a carboxylate or carboxylic acid group.
  • halo refers to fluoro, chloro, bromo, and iodo 35 groups.
  • - 34 - Attorney Docket No.: 3436/3 PCT
  • perhaloalkyl refers to an alkyl group wherein all of the hydrogen atoms are replaced by halo.
  • perhaloalkyl can refer to a “perfluroalkyl” group wherein all of the hydrogen atoms of the alkyl group are replaced by fluoro.
  • Perhaloalkyl groups include, but are not limited to, - CF 3 . 5
  • hydroxyl and “hydroxy” refer to the -OH group.
  • oxo refers to a compound described previously herein wherein a carbon atom is replaced by an oxygen atom.
  • thio refers to the -S- or -SH group.
  • alkylthio and “thioalkyl” refer to a -SR group where R is alkyl or substituted alkyl.
  • arylthiol refers to a -SR group where R is aryl or substituted aryl.
  • cyano refers to the -CN group.
  • nitro refers to the -NO 2 group.
  • a line crossed by a wavy line, e.g., in the structure: 15 indicates the site where the indicated substituent or structure can bond to another group.
  • Activity-based probes can comprise a reactive group for targeting a specific enzyme class and a reporter tag for detection e.g., by in-gel fluorescence scanning or by avidin-enrichment coupled with liquid chromatography mass spectrometry (LCMS), respectively. Tuning the reactivity of sulfur electrophiles can be important for developing biorthogonal chemical probe discovery.
  • LCMS liquid chromatography mass spectrometry
  • sulfonyl-purine and sulfonyl-purine analog e.g., sulfonyl-xanthine or sulfonyl-hypoxanthine
  • Sulfonyl-purine (SuPUR) or “SuPUR” chemistry.
  • the structure of a generic SuPUR compound is shown on the right side of Figure 1A.
  • the structure of an exemplary SuPUR probe compound is shown in Figure 1B, while a workflow of the use of the SuPUR probe in proteomic studies is shown in Figure 1D.
  • the SuPUR - 35 - Attorney Docket No.: 3436/3 PCT probe compound includes a terminal alkyne moiety that can act as a reporter tag, for instance, upon Click reaction with a desthiobiotin azide.
  • the structure of an exemplary SuPUR ligand compound is shown in Figure 1C.
  • the SuPUR approach demonstrates that purine and purine analogs can be 5 effective leaving groups for activating nucleophilic substitution reactions (e.g., of tyrosine and lysine sites of peptides and proteins; see Figures 1E and 1F) and that SuPUR compounds can be used as covalent ligands that form covalent conjugates with nucleophilic groups in peptides and proteins.
  • SuPUR compounds identified noncatalytic tyrosine and lysine residues that can be liganded by exemplary SuPUR ligands with site specificity and potency to disrupt protein function. 10 More particularly, as described hereinbelow, for example, a series of covalent SuPUR ligands were prepared and selective covalent labeling of amyloid-beta (A ⁇ )-binding alcohol dehydrogenase (ABAD) and alpha/beta ( ⁇ / ⁇ )-hydrolase domain 10 (ABHD10) was studied. Covalent modification of ABAD and ABHD10 with these compounds can inhibit the function of these enzymes, which are known to be closely related to mitochondrial metabolism and S-depalmitoylase.
  • a ⁇ amyloid-beta
  • ABHD10 alpha/beta domain 10
  • the presently disclosed subject matter provides a family of covalent ligands targeting an ABAD tyrosine (Tyr) residue to achieve the inhibition of ABAD, thereby advancing basic research in Alzheimer’s disease (AD) and/or for facilitating development of therapeutic agents (e.g., small molecule agents) for treating AD.
  • the SuPUR ligand 20 covalently modifies (e.g., selectively and covalently modifies) tyrosine-168 (Y168) of ABAD.
  • the presently disclosed subject matter provides a family of covalent ligands for targeting an ABHD10 tyrosine (Try) residue to provide an ABHD10 inhibitor with improved selectivity compared to inhibitors that target serine residues.
  • the SuPUR ligand covalent modifies (e.g., selectively and covalently modifies) tyrosine-87 (Y87) and/or tyrosine-215 (Y215) of 25 ABHD10. II.A. Compositions
  • the presently disclosed subject matter provides a compound (referred to herein as a SuPUR compound) comprising a sulfonyl-purine or sulfonyl-purine analog.
  • the sulfonyl- purine analog can comprise, for example, a fused imidazole-based moiety, such as, a hypoxanthine, a 30 xanthine, an imidazopyridine, or a benzimidazole moiety.
  • the purine or purine analog can be substituted or unsubstituted.
  • X 1 and X 2 are independently selected from the group consisting of H, O, -NH 2 , -NHX 5 , and -N(X 5 ) 2 .
  • X 1 and X 2 are independently selected from the group consisting of H, O, -NH 2 , -NHX 5 , and -N(X 5 ) 2 .
  • Z and Y are each selected from N and NH.
  • - 37 - Attorney Docket No.: 3436/3 PCT In some embodiments, X 1 is O, Z is NH, X 2 is H, and Y is N.
  • the compound of Formula (I) has a structure comprising Formula (Ia): Formula (Ia), where each represents a single or double bond; subject to the proviso that one represents 5 a double bond and one represents a single bond; and wherein X 3 and X 4 are as defined for Formula (I).
  • R 1 is phenyl or substituted phenyl.
  • the compound of Formula (Ia) is: 10 (referred to herein as “ZH-1-142”).
  • both X 1 and X 2 are O and both of Y and Z are NH.
  • the compound of Formula (I) has a structure of Formula (Ib): where each represents a single or double bond; subject to the proviso that one represents 15 a double bond and one represents a single bond; wherein X 3 and X 4 are as defined for Formula (I).
  • X 1 and X 2 are independently selected from the group consisting of H, O, -NH 2 , -NHX 5 , and -N(X 5 ) 2 .
  • Z and Y are each N.
  • one of Z and Y is CH. In some embodiments, both Z and Y are CH. In 5 some embodiments, X 1 and X 2 are each H. In some embodiments, the compound has a structure selected from: ZH-2-035 ZH-2-036. In some embodiments, Z and Y are each N.
  • X 1 and/or X 2 is/are H.
  • R 1 is C1-C6 alkyl or C1-C6 substituted alkyl.
  • R 1 is perhaloalkyl-substituted (e.g., -CF 3 -substituted) C1-C6 alkyl.
  • R 1 is alkoxy- substituted alkyl.
  • R 1 is a branched C1-C6 alkyl (e.g., isopropyl, sec-butyl (i.e., 20 butan-2-yl) or isobutyl (i.e., 2-methylpropyl).
  • R 1 is C3-C6 cycloalkyl, i.e., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.
  • the compound has a structure selected from: - 40 - Attorney Docket No.: 3436/3 PCT ZH-1-059 ZH-1-060 ZH-1-063 ZH-1-089
  • R 1 is phenyl or substituted phenyl.
  • Z and Y are each N.
  • the compound of Formula (III) has a structure of Formula (III’): - 41 - Attorney Docket No.: 3436/3 PCT Formula (III’), wherein X 1, X 2 , R 2 , R 3 , R 4 , R 5 , and R 6 are defined as described above for Formula (III), or a pharmaceutically acceptable salt thereof.
  • R 8 is selected from terminal alkyne-substituted alkyl (e.g., -CH 2 -C ⁇ CH), cycloalkyl, cycloalkyl- 10 substituted
  • R 2 and R 6 are selected from the group consisting of fluoro, chloro, bromo, methyl, perfluoromethyl, methoxy, and perfluoromethoxy.
  • R 4 is cyano.
  • R in the compound of Formula (I), R is selected from the group consisting 15 of: , - 42 - Attorney Docket No.: 3436/3 PCT - 43 - Attorney Docket No.: 3436/3 PCT 5
  • Z and Y are each N
  • X 1 and X 2 are each H.
  • the presently disclosed compounds are provided in the form of pharmaceutically acceptable salts or solvates. II.B.
  • compositions and Administration 10 The presently disclosed subject matter also relates, in some embodiments, to pharmaceutical compositions comprising, consisting essentially of, or consisting of one or more SuPUR compounds of - 44 - Attorney Docket No.: 3436/3 PCT the presently disclosed subject matter and a pharmaceutically acceptable carrier, diluent, and/or excipient.
  • Pharmaceutical compositions comprising the present compounds are administered to a subject in need thereof by any number of routes including, but not limited to, topical, oral, intravenous, 5 intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • the presently disclosed compositions are administered by injecting the composition subcutaneously, intraperitoneally, into adipose tissue, and/or intramuscularly into the subject.
  • a method for treating a subject in need of such treatment 10 comprises administering a pharmaceutical composition comprising at least one compound of the presently disclosed subject matter to a subject in need thereof.
  • Compounds identified by the methods of the presently disclosed subject matter can be administered with known compounds or other medications as well.
  • the pharmaceutical compositions useful for practicing the presently disclosed subject matter 15 may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day.
  • the term “physiologically acceptable” ester or salt means an ester or salt form 25 of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.
  • the compositions of the presently disclosed subject matter may comprise at least one active peptide, one or more acceptable carriers, and optionally other peptides or therapeutic agents.
  • the compositions of the presently disclosed subject matter may 30 comprise a pharmaceutically acceptable salt.
  • suitable carriers include, but are not limited to, water, normal saline, dextrose, mannitol, lactose or other sugars, lecithin, albumin, sodium glutamate, cysteine hydrochloride, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, ethoxylated isosteraryl alcohols, polyoxyethylene sorbitol 5 and sorbitan esters, microcrystalline cellulose, aluminum methahydroxide, bentonite, kaolin, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • compositions of the presently disclosed subject matter can further comprise an adjuvant.
  • the at least one adjuvant is selected from the group 10 consisting of montanide ISA-51 (Seppic, Inc.), QS-21 (Aquila Pharmaceuticals, Inc.), tetanus helper peptides, GM-CSF, cyclophosamide, bacillus Calmette-Guerin (BCG), corynbacterium parvum, levamisole, azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freunds adjuvant (complete and incomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, diphtheria toxin (DT).
  • montanide ISA-51 Seppic, Inc.
  • QS-21 Alquila Pharmaceuticals, Inc.
  • compositions may also contain minor amounts of nontoxic auxiliary pharmaceutical substances or excipients and/or additives, such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like).
  • auxiliary pharmaceutical substances or excipients and/or additives such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like).
  • Suitable additives include, but are not limited to, physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions (e.g., 0.01 to 10 mole percent) of chelants (such 20 as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA or CaNaDTPA-bisamide), or, optionally, additions (e.g., 1 to 50 mole percent) of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • chelants such 20 as, for example, DTPA or DTPA-bisamide
  • calcium chelate complexes as for example calcium DTPA or CaNaDTPA-bisamide
  • additions e.g., 1 to 50 mole percent
  • calcium or sodium salts for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate.
  • absorption enhancing or delaying agents such
  • compositions can be prepared in conventional forms, either as liquid solutions 25 or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Pharmaceutical compositions according to the presently disclosed subject matter can be prepared in a manner fully within the skill of the art.
  • the compositions of the presently disclosed subject matter, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising these compounds can be so that the compounds 30 can have a physiological effect.
  • Administration can occur enterally or parenterally; for example, orally, rectally, intracisternally, intravaginally, intraperitoneally, locally (e.g., with powders, ointments, or drops), or as a buccal or nasal spray or aerosol. Parenteral administration is preferred.
  • Particularly preferred parenteral administration methods include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion, and catheter 35 instillation into the vasculature), peri- and intra-target tissue injection, subcutaneous injection or - 46 - Attorney Docket No.: 3436/3 PCT deposition including subcutaneous infusion, intramuscular injection, and direct application to the target area, for example by a catheter or other placement device.
  • the injection or direct application can be in a single dose or in multiple doses.
  • the administration of the compound is by infusion
  • 5 the infusion can be a single sustained dose over a prolonged period of time or multiple infusions.
  • compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a 10 desired single- or multi-dose unit.
  • Such pharmaceutical compositions are generally suitable for administration to animals of all sorts.
  • Subjects to which administration of the pharmaceutical compositions of the presently disclosed subject matter is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such 15 as cattle, pigs, horses, sheep, cats, and dogs, birds including commercially relevant birds such as chickens, ducks, geese, and turkeys.
  • a pharmaceutical composition of the presently disclosed subject matter may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount 20 of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • the relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the presently disclosed subject matter will 25 vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • a pharmaceutical composition of the presently disclosed subject matter may further comprise one or more additional pharmaceutically active agents.
  • Particularly 30 contemplated additional agents include anti-emetics and scavengers such as cyanide and cyanate scavengers.
  • scavengers such as cyanide and cyanate scavengers.
  • Controlled- or sustained-release formulations of a pharmaceutical composition of the presently disclosed subject matter may be made using conventional technology.
  • additional ingredients include, but are not limited to, one or more of the 35 following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring - 47 - Attorney Docket No.: 3436/3 PCT agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or 5 hydrophobic materials.
  • dosages of the compound of the presently disclosed subject matter which may be administered to an animal range in amount from 1 ⁇ g to about 100 g 10 per kilogram of body weight of the animal. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration.
  • the dosage of the compound will vary from about 1 mg to about 10 g per kilogram of body weight of the animal. In another aspect, the dosage will vary from about 10 mg to about 1 g per kilogram of body weight of 15 the animal.
  • the compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less.
  • the frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, 20 such as, but not limited to, the type of cancer being diagnosed, the type and severity of the condition or disease being treated, the type and age of the animal, etc.
  • Suitable preparations include injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, suspension in, liquid prior to injection, may also be prepared.
  • the preparation may also be emulsified, or the polypeptides encapsulated in liposomes.
  • the active 25 ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine preparation may also include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants.
  • the presently disclosed subject matter also includes a kit comprising the composition of the presently disclosed subject matter and an instructional material which describes administering the composition to a subject.
  • this kit comprises a (in some embodiments sterile) solvent suitable for dissolving or suspending the composition of the presently disclosed subject matter prior to administering the compound to the subject.
  • an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of a composition of - 48 - Attorney Docket No.: 3436/3 PCT the presently disclosed subject matter in the kit for effecting alleviation of the various diseases or disorders recited herein.
  • the instructional material may describe one or more methods of using the compositions for diagnostic or identification purposes or of alleviation the diseases or disorders in a cell or a tissue of a mammal.
  • the instructional material of the kit of the presently 5 disclosed subject matter may, for example, be affixed to a container which contains a composition of the presently disclosed subject matter or be shipped together with a container which contains the composition.
  • the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.
  • the presently disclosed subject matter also related to methods for using the compositions of the 10 presently disclosed subject matter for various purposes.
  • the presently disclosed subject matter also relates to methods for treating and/or preventing diseases, disorders, and/or conditions associated with inflammation.
  • a “treatment effective amount” or a “therapeutic amount” is an amount of a therapeutic composition sufficient to produce a measurable response (e.g., a biologically or clinically relevant response in a subject being treated, such as but not limited to a reduction in scarring and/or fibrosis, particularly as compared to the same subject had the subject not received the composition).
  • compositions of the presently disclosed subject matter 20 can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject.
  • the selected dosage level will depend upon the activity of the composition, the route of administration, combination with other drugs or treatments, the severity of the disease, disorder, and/or condition being treated, and the condition and prior medical history of the subject being treated. However, it is within the skill of the art to start doses of the 25 compositions of the presently disclosed subject matter at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • the potency of a composition can vary, and therefore a “treatment effective amount” can vary.
  • Suitable methods for administration of the compositions of the presently disclosed subject matter include, but are not limited to intravenous administration, oral delivery, and delivery directly to a target tissue or organ (e.g., a topical application and/or a site of injury such as but not limited to a 5 muscle injury).
  • exemplary routes of administration include parenteral, enteral, intravenous, intraarterial, intracardiac, intrapericardial, intraosseal, intracutaneous, subcutaneous, intradermal, subdermal, transdermal, intrathecal, intramuscular, intraperitoneal, intrasternal, parenchymatous, oral, sublingual, buccal, inhalational, and intranasal.
  • a particular route of administration can be made based at least in part on the nature of the formulation and the ultimate target site where the 10 compositions of the presently disclosed subject matter are desired to act.
  • the method of administration encompasses features for regionalized delivery or accumulation of the compositions at the site in need of treatment.
  • the compositions are delivered directly into the site to be treated.
  • a composition of the presently disclosed subject matter is administered to the subject via a route selected 15 from the group consisting of intraperitoneal, intramuscular, intravenous, and intranasal, or any combination thereof.
  • compositions comprising the molecules described above, together with one or more pharmaceutically acceptable excipients or vehicles, and optionally other therapeutic and/or prophylactic ingredients.
  • excipients include liquids such as 20 water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, cyclodextrins, modified cyclodextrins (i.e., sufobutyl ether cyclodextrins), etc.
  • Suitable excipients for non-liquid formulations are also known to those of skill in the art.
  • compositions of the present invention include, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such 25 as acetates, propionates, malonates, benzoates, and the like.
  • auxiliary substances such as wetting or emulsifying agents, biological buffering substances, surfactants, and the like, may be present in such vehicles.
  • a biological buffer can be virtually any solution which is pharmacologically acceptable and which provides the formulation with the desired pH, i.e., a pH in the physiologically acceptable range.
  • buffer solutions include 30 saline, phosphate buffered saline, Tris buffered saline, Hank’s buffered saline, and the like.
  • the pharmaceutical compositions may be in the form of a liquid, suspension, cream, ointment, lotion, or the like, preferably in unit dosage form suitable for single administration of a precise dosage.
  • the compositions can in some embodiments include one or more pharmaceutically acceptable carriers and, in addition, may include other 35 pharmaceutical agents, adjuvants, diluents, buffers, etc. - 50 - Attorney Docket No.: 3436/3 PCT III.
  • SuPUR compounds can be used as covalent ligands to expand the landscape of proteins amenable to targeting by small molecules.
  • SuPUR compounds provide covalent ligands that combine features of recognition and reactivity, thereby providing for the selective targeting of sites on proteins of interest, including those that are difficult to address by reversible binding interactions alone and/or with other types of covalent ligands.
  • Scheme 1 SuPUR Reactions with Proteins with Reactive Tyrosine or Lysine Residues
  • Scheme 1 above shows the reaction of an exemplary SuPUR compound (e.g., an exemplary SuPUR ligand) with a protein having a reactive tyrosine (Y) or lysine (K).
  • the SuPUR compound comprises a sulfur electrophile, i.e., a sulfonyl group directed attached to a nitrogen atom of 15 a purine moiety.
  • the purine moiety acts as a leaving group (LG) in the reaction of the SuPUR compound with the nucleophilic phenol or amine moiety of the side chain of the tyrosine or lysine residue, resulting in a modified protein where a modified tyrosine or lysine residue is covalently attached to the sulfur atom from the SuPUR compound.
  • LG leaving group
  • AGs of SuPUR ligands can include a variety of optionally substituted alkyl, cycloalkyl (including heterocyclic), aryl (including heteroaryl), and aralkyl groups, while SuPUR “probes” can contain an AG group that comprises an alkyne group (e.g., a terminal alkyne group), a fluorophore moiety, another detectable moiety (e.g., an affinity label), or a combination thereof.
  • an alkyne group e.g., a terminal alkyne group
  • fluorophore moiety e.g., an affinity label
  • the alkyne group of an AG of a protein modified with a SuPUR probe can be contacted with an alkyne-reactive group (e.g., an azide) to attach a detectable group (e.g., a biotin) to the modified protein.
  • an alkyne-reactive group e.g., an azide
  • a detectable group e.g., a biotin
  • a SuPUR ligand of the presently disclosed subject matter can compete 5 with a SuPUR probe compound described herein for binding with a reactive amino acid residue, e.g., a reactive tyrosine and/or lysine residue.
  • the ligand molecule comprises an AG moiety that facilitates interaction of the compound with a particular amino acid residue of interest, e.g., a reactive tyrosine and/or lysine residue.
  • the ligand comprises an AG moiety that facilitates hydrophobic interaction, hydrogen bonding, or a combination thereof.
  • the ligand can comprise an AG moiety that can increase or decrease steric hinderance at the electrophilic sulfonyl group, thereby modifying the reactivity of the ligand.
  • SuPUR ligands are typically non-naturally occurring and/or form non-naturally occurring adducts after reaction with a biological target, e.g., a protein or peptide.
  • exemplary SuPUR ligands include, for example, the compounds referred to herein as ZH-1-049-0, ZH-1-049-2, ZH-1-059, ZH-1-060, ZH-1-063, ZH-1-065, 15 ZH-1-069, ZH-1-075, ZH-1-089, ZH-2-017, ZH-2-018, ZH-2-019, ZH-2-020, ZH-2-022, ZH-2-023, ZH-2-024, ZH-2-025, ZH-2-026, ZH-2-027, ZH-2-029, ZH-2-035, ZH-2-036, ZH-2-039, ZH-2-053, ZH-2-058, ZH-2-067, ZH-2-069, ZH-2-071, ZH-2-073, Z
  • Exemplary SuPUR probes include the compounds referred to herein as AHL-PuP-2, ZH-1-142, 20 and ZH-1-143.
  • the presently disclosed subject matter provides a method of covalently modifying a protein or peptide in a sample, wherein the method comprises contacting the sample with a SuPUR compound (e.g., a compound of Formula (I), (Ia), (Ib), (II), (II’), (III), or (III’) described hereinabove).
  • a SuPUR compound e.g., a compound of Formula (I), (Ia), (Ib), (II), (II’), (III), or (III’
  • the contacting provides a covalent modified peptide or protein, 25 wherein said covalently modified peptide or protein comprises one or more covalently modified tyrosine or covalently modified lysine residues, wherein the covalently modified tyrosine or covalently modified lysine residues comprise a structure: - 52 - Attorney Docket No.: 3436/3 PCT wherein R 1 is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.
  • R 1 is selected from phenyl and substituted phenyl.
  • the sample is a biological sample.
  • covalently 5 modifying the protein or peptide modulates a biological activity of the protein or peptide.
  • modifying the protein or peptide inhibits or activates a biological activity of the protein or peptide.
  • the protein is an enzyme and covalently modifying the enzyme with the compound inhibits the enzyme.
  • modulating the activity of a protein comprises enhancing or reducing the ability of the protein to interact with other compounds,10 such as other proteins.
  • the modulation results in reducing the protein- protein interactions of the protein comprising the reactive amino acid.
  • modulating the activity of a protein comprising a comprises inhibiting, blocking (partially or substantially completely) or disrupting a protein-RNA interaction, a protein-DNA interaction, a protein- lipid interaction, and/or a protein-metabolite interaction of the protein.
  • the presently disclosed ligands can serve as tools for the global investigation of protein function.
  • the protein or peptide is selected from ABAD, guanine nucleotide binding protein alpha stimulating activity polypeptide (GNAS), and ABHD10.
  • the protein is ABHD10 or another serine hydrolase.
  • the protein is ABAD.
  • the method comprises 20 contacting a sample comprising ABAD with a SuPUR compound as described herein.
  • the SuPUR compound is a compound of Formula (I).
  • Y and Z in the compound of Formula (I) are each selected from N and NH.
  • the SuPUR compound is a compound of Formula (Ia), (Ib), (II’), or (III’).
  • the substituted phenyl group comprises a halo, alkyl (e.g., methyl), perhaloalkyl (e.g., - CF 3 ), perhaloalkoxy (e.g., -OCF 3 ), or alkoxy (e.g., methoxy) substituent at one or both ortho positions.
  • the substituted phenyl group comprises a cyano substituent at the para position.
  • the SuPUR compound is selected from ZH-2-025, ZH-2-029, ZH-2-085, ZH-2- 093, and ZH-2-101.
  • the SuPUR compound modifies ABAD at a non-catalytic tyrosine residue.
  • the non-catalytic tyrosine residue is tyrosine-168 (Y168).
  • the covalently modified ABAD exhibits lower enzymatic (or catalytic) activity compared to a corresponding unmodified ABAD (e.g., covalently modified human ABAD exhibits reduced enzymatic (or catalytic) activity compared to unmodified human ABAD under otherwise identical 35 conditions).
  • the covalently modified ABAD exhibits decreased binding for A ⁇ compared to a corresponding unmodified ABAD.
  • the covalently modified - 53 - Attorney Docket No.: 3436/3 PCT ABAD exhibits a reduction in reactive oxygen species (ROS) induced by beta-amyloid (A ⁇ ) compared to a corresponding unmodified ABAD.
  • the protein is ABHD10.
  • the method comprises contacting a sample comprising ABHD10 with a SuPUR compound as described herein.
  • the SuPUR compound is a compound of Formula (I).
  • Y and Z in the compound of Formula (I) are each selected from N and NH.
  • the SuPUR compound is a compound of Formula (Ia), (Ib), (II’), or (III’).
  • the substituted phenyl group comprises a halo, alkyl (e.g., methyl), perhaloalkyl (e.g., - 10 CF 3 ), or perhaloalkoxy (e.g., -OCF 3 ) substituent at one or both ortho positions.
  • the SuPUR compound is selected from ZH-1-049-2, ZH-2-025, ZH-2-097, and ZH-2-103.
  • the SuPUR compound modifies ABHD10 at a non-catalytic tyrosine residue.
  • the non-catalytic tyrosine residue is tyrosine-215 (Y215).
  • the covalently modified ABHD10 exhibits lower enzymatic (or catalytic) activity 15 compared to a corresponding unmodified ABHD10 (e.g., covalently modified human ABHD10 exhibits reduced enzymatic (or catalytic) activity compared to unmodified human ABHD10 under otherwise identical conditions).
  • the sample comprises an isolated protein.
  • the sample comprises a cell lysate, a biological fluid (e.g., saliva, ascites, blood, urine, etc.) or a live cell.
  • a biological fluid e.g., saliva, ascites, blood, urine, etc.
  • a live cell e.g., a cell lysate, a biological fluid (e.g., saliva, ascites, blood, urine, etc.) or a live cell.
  • the sample comprising living cells comprises an organ, or a living organism (e.g., a subject, such as a human or other mammal). IV.
  • the presently disclosed subject matter provides a method of identifying a reactive amino acid residue of a protein, the method comprising: providing a protein sample 25 comprising isolated proteins, living cells, or a cell lysate; (b) contacting the protein sample with a compound of Formula (I) (or a sub-formula thereof, e.g., Formula (II), (II’), (III), or (III’)) for a period of time sufficient for the compound to react with at least one reactive amino acid residue (e.g., a tyrosine or lysine residue) in a protein in the protein sample, thereby forming at least one modified reactive amino acid residue; and (c) analyzing proteins in the protein sample to identify at least one modified 30 reactive amino acid residue, thereby identifying at least one reactive amino acid residue of a protein; wherein the probe compound has a structure of Formula (I): - 54 - Attorney Docket No.:
  • Y and Z are each selected from N and NH.
  • the analyzing of step (c) further comprisies tagging the at least one modified reactive amino acid residue with a compound comprising a detectable labeling group, thereby 15 forming at least one tagged reactive amino acid residue comprising said detectable labeling group.
  • the detectable labeling group comprises biotin or a biotin derivative.
  • the biotin derivative is desthiobiotin.
  • the tagging comprises reacting an alkyne group of at least one tagged reactive amino acid residue with a compound comprising both an azide moiety (or other alkyne-reactive 20 group) and a detectable labeling group (e.g., biotin or a biotin derivative).
  • the compound comprising the azide moiety and the detectable labeling group further comprises an alkylene linker, which in some embodiments, can comprise a polyether group, such as an oligomer of methylene glycol, ethylene glycol or propylene glycol (e.g., a group having the formula –(O-C 2 H 4 -) x -).
  • the tagging comprises performing a copper-catalyzed azide-alkyne cycloaddition 25 (CuAAC) coupling reaction.
  • the analyzing further comprises digesting the protein sample to provide a digested protein sample comprising a protein fragment comprising the at least one tagged reactive amino acid residue comprising the detectable group.
  • the digesting is performed with a peptidase.
  • the digesting is performed with trypsin.
  • the analyzing further comprises enriching the digested protein sample for the detectable labeling group.
  • the enriching comprises contacting the digested protein sample with a solid support comprising a binding partner of the detectable labeling group.
  • the detectable labeling group comprises biotin or a derivative thereof
  • the solid support comprises streptavidin.
  • the analyzing 35 further comprises analyzing the digested protein sample (e.g., the enriched digested protein sample) via liquid chromatography-mass spectrometry or via a gel-based assay. - 55 - Attorney Docket No.: 3436/3 PCT
  • providing the protein sample further comprises separating the protein sample into a first protein sample and a second protein sample.
  • the first protein sample can be contacted with a first probe compound (e.g., a probe compound of Formula (I)) at a first probe concentration for a first period of time and the second protein sample can be contacted 5 with a second probe compound (e.g., a second probe compound of Formula (I) having a different structure than that of the first probe compound) at the same probe concentration (i.e., at the first probe concentration) for the same time period (i.e., for the first period of time.
  • the second protein sample can be contacted with the same probe compound as the first protein sample, but at a different probe concentration (i.e., a second probe concentration) or for a different period of time.
  • analyzing proteins comprises analyzing the first and second protein samples to determine the presence and/or identity of a modified reactive amino acid residue in the first sample and the presence and/or identity of a modified reactive amino acid residue in the second sample. In some embodiments, the identities and/or amounts of identified modified reactive amino acid residues from the first and second protein samples are compared. 15 In some embodiments, the protein sample comprises living cells.
  • providing the protein sample further comprises separating the protein sample into a first protein sample and a second protein sample and culturing the first protein sample in a first cell culture medium comprising heavy isotopes prior to the contacting of step (b) and culturing the second protein sample in a second cell culture medium, wherein the second culture medium comprises a naturally occurring 20 isotope distribution prior to the contacting of step (b).
  • the first cell culture medium comprises 13 C- and/or 15 N-labeled amino acids.
  • the first cell culture medium comprises 13 C-, 15 N-labeled lysine and arginine.
  • the probe compound can comprise a detectable labeling group comprising a heavy isotope (e.g., a 13 C label) or 25 the method can comprise tagging the at least one modified amino acid residue with a detectable labeling group comprising a heavy isotope.
  • the protein sample is separated into a first and a second protein sample and one of the first and the second protein sample is cultured in the presences of an inhibitor of a protein of interest (e.g., a serine hydrolase).
  • a sample e.g., a 35 cell sample, cell lysate sample or a biological organism.
  • the sample for use with the methods described herein is obtained from cells of an animal.
  • the animal cell - 56 - Attorney Docket No.: 3436/3 PCT includes a cell from a marine invertebrate, fish, insects, amphibian, reptile, or mammal.
  • the mammalian cell is a primate, ape, equine, bovine, porcine, canine, feline, or rodent.
  • the mammal is a primate, ape, dog, cat, rabbit, ferret, or the like.
  • the rodent is a mouse, rat, hamster, gerbil, hamster, chinchilla, or guinea pig.
  • the 5 bird cell is from a canary, parakeet or parrots.
  • the reptile cell is from a turtles, lizard or snake.
  • the fish cell is from a tropical fish.
  • the fish cell is from a zebrafish (e.g. Danino rerio).
  • the worm cell is from a nematode (e.g. C. elegans).
  • the amphibian cell is from a frog.
  • the arthropod cell is from a tarantula or hermit crab. 10
  • the sample for use with the methods described herein is obtained from a mammalian cell.
  • the mammalian cell is an epithelial cell, connective tissue cell, hormone secreting cell, a nerve cell, a skeletal muscle cell, a blood cell, or an immune system cell.
  • exemplary mammalian cell lines include, but are not limited to, 293A cells, 293FT cells, 293F cells, 293H cells, HEK 293 cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, and PC12 cells.
  • the sample for use with the methods described herein is obtained from cells of a tumor cell line.
  • the sample is obtained from cells of a solid tumor cell line.
  • the solid tumor cell line is a sarcoma cell line.
  • the solid tumor cell line is a carcinoma cell line.
  • the sarcoma cell line is obtained from a cell line of alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastoma, angiosarcoma, 20 chondrosarcoma, chordoma, clear cell sarcoma of soft tissue, dedifferentiated liposarcoma, desmoid, desmoplastic small round cell tumor, embryonal rhabdomyosarcoma, epithelioid fibrosarcoma, epithelioid hemangioendothelioma, epithelioid sarcoma, esthesioneuroblastoma, Ewing sarcoma, extrarenal rhabdoid tumor, extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, giant cell tumor, hemangiopericytoma,
  • the carcinoma cell line is obtained from a cell line of adenocarcinoma, 35 squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder - 57 - Attorney Docket No.: 3436/3 PCT cancer, brain tumor, breast cancer, cervical cancer, colon cancer, cancer of Unknown Primary (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastroenterological cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach 5 cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.
  • CUP Unknown Primary
  • the sample is obtained from cells of a hematologic malignant cell line.
  • the hematologic malignant cell line is a T-cell cell line.
  • the hematologic malignant cell line is obtained from a T-cell cell line of: peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), anaplastic large cell lymphoma, 10 angioimmunoblastic lymphoma, cutaneous T-cell lymphoma, adult T-cell leukemia/lymphoma (ATLL), blastic NK-cell lymphoma, enteropathy-type T-cell lymphoma, hematosplenic gamma-delta T-cell lymphoma, lymphoblastic lymphoma, nasal NK/T-cell lymphomas, or treatment-related T-cell lymphomas.
  • PTCL-NOS peripheral T-cell lymphoma not otherwise specified
  • anaplastic large cell lymphoma 10 angioimmunoblastic lymphom
  • the hematologic malignant cell line is obtained from a B-cell cell line of: 15 acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), high-risk chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk small lymphocytic lymphoma (SLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Waldenstrom’s macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, 20 nodal marginal zone B cell lymphoma, Burkitt’s lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma
  • the sample for use with the methods described herein is obtained from a tumor cell line.
  • tumor cell lines include, but are not limited to, 600MPE, AU565, BT-20, BT-474, BT-483, BT-549, Evsa-T, Hs578T, MCF-7, MDA-MB-231, SkBr3, T-47D, HeLa, DU145, PC3, LNCaP, A549, H1299, NCI-H460, A2780, SKOV-3/Luc, Neuro2a, RKO, RKO-AS45-1, HT-29, 30 SW1417, SW948, DLD-1, SW480, Capan-1, MC/9, B72.3, B25.2, B6.2, B38.1, DMS 153, SU.86.86, SNU-182, SNU-423, SNU-449, SNU-475, SNU-387, Hs 817.T, LMH, LMH/2A, SNU-398, PLHC-1,
  • the sample for use in the methods is from any tissue or fluid from an individual.
  • Samples include, but are not limited to, tissue (e.g. connective tissue, muscle tissue, nervous tissue, or epithelial tissue), whole blood, dissociated bone marrow, bone marrow aspirate, pleural fluid, peritoneal fluid, central spinal fluid, abdominal fluid, pancreatic fluid, cerebrospinal fluid, brain fluid, 5 ascites, pericardial fluid, urine, saliva, bronchial lavage, sweat, tears, ear flow, sputum, hydrocele fluid, semen, vaginal flow, milk, amniotic fluid, and secretions of respiratory, intestinal or genitourinary tract.
  • tissue e.g. connective tissue, muscle tissue, nervous tissue, or epithelial tissue
  • whole blood e.g. connective tissue, muscle tissue, nervous tissue, or epithelial tissue
  • dissociated bone marrow e.g. connective tissue, muscle tissue, nervous tissue, or epithelial
  • the sample is a tissue sample, such as a sample obtained from a biopsy or a tumor tissue sample.
  • the sample is a blood serum sample.
  • the sample is a blood cell sample containing one or more peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the sample contains one or more circulating tumor cells (CTCs).
  • the sample contains one or more disseminated tumor cells (DTC, e.g., in a bone marrow aspirate sample).
  • the samples are obtained from the individual by any suitable means of obtaining the sample using well-known and routine clinical methods. Procedures for obtaining tissue 15 samples from an individual are well known.
  • the sample is a biological organism.
  • the biological organism is a rodent, e.g., a mouse or a rat.
  • the biological organism is a primate, e.g., a monkey.
  • the biological organism is a bacteria or a fungi.
  • the sample e.g., cell sample, cell lysate sample, or comprising isolated 25 proteins
  • the sample solution comprises a solution such as a buffer (e.g. phosphate buffered saline) or a media.
  • the media is an isotopically labeled media.
  • the sample solution is a cell solution.
  • the sample e.g., cell sample, cell lysate sample, or comprising isolated proteins
  • the sample e.g., cell sample, cell lysate sample, or comprising isolated proteins
  • the sample is further incubated in the presence of an additional compound probe prior to addition of the one or more probes.
  • the sample e.g., cell sample, cell lysate sample, or comprising isolated proteins
  • the sample is further incubated with a non-probe small molecule ligand, in which the non-probe small molecule ligand does not contain a photoreactive moiety and/or an alkyne group.
  • the 35 sample is incubated with a probe and non-probe small molecule ligand for competitive protein profiling analysis.
  • the sample is compared with a control. In some cases, a difference is observed between a set of probe protein interactions between the sample and the control. In some instances, the difference correlates to the interaction between the small molecule fragment and the proteins.
  • one or more methods are utilized for labeling a sample (e.g. cell sample, 5 cell lysate sample, or comprising isolated proteins) for analysis of probe protein interactions. In some instances, a method comprises labeling the sample (e.g. cell sample, cell lysate sample, or comprising isolated proteins) with an enriched media. In some cases, the sample (e.g.
  • the labeled sample is further compared with a non-labeled sample 10 to detect differences in probe protein interactions between the two samples. In some instances, this difference is a difference of a target protein and its interaction with a small molecule ligand in the labeled sample versus the non-labeled sample. In some instances, the difference is an increase, decrease or a lack of protein-probe interaction in the two samples.
  • the isotope-labeled method is termed SILAC, stable isotope labeling using amino acids in cell culture.
  • a method comprises incubating a sample (e.g. cell sample, cell lysate sample, or comprising isolated proteins) with a labeling group (e.g., an isotopically labeled labeling group) to tag one or more proteins of interest for further analysis.
  • a labeling group e.g., an isotopically labeled labeling group
  • the detectable labeling group comprises a biotin, a streptavidin, bead, resin, a solid support, or a combination thereof, and further comprises a linker that is optionally isotopically labeled.
  • the linker can be 20 about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more residues in length and might further comprise a cleavage site, such as a protease cleavage site (e.g., TEV cleavage site).
  • the labeling group is a biotin-linker moiety, which is optionally isotopically labeled with 13 C and 15 N atoms at one or more amino acid residue positions within the linker.
  • the biotin-linker moiety is a isotopically- labeled TEV-tag as previously described. 10 25
  • an isotopic reductive dimethylation (ReDi) method is utilized for processing a sample.
  • the ReDi labeling method involves reacting peptides with formaldehyde to form a Schiff base, which is then reduced by cyanoborohydride. This reaction dimethylates free amino groups on N-termini and lysine side chains and monomethylates N-terminal prolines.
  • the ReDi labeling method comprises methylating peptides from a first 30 processed sample with a “light” label using reagents with hydrogen atoms in their natural isotopic distribution and peptides from a second processed sample with a “heavy” label using deuterated formaldehyde and cyanoborohydride.
  • proteomic analysis e.g., mass spectrometry analysis
  • proteomic analysis based on a relative peptide abundance between the heavy and light peptide version might be used for analysis of probe-protein interactions.
  • isobaric tags for relative and absolute quantitation (iTRAQ) method is utilized for processing a sample.
  • the iTRAQ method is based on the covalent labeling of - 60 - Attorney Docket No.: 3436/3 PCT the N-terminus and side chain amines of peptides from a processed sample.
  • reagent such as 4-plex or 8-plex is used for labeling the peptides.
  • the probe-protein complex is further conjugated to a chromophore, such as a fluorophore.
  • the probe-protein complex is separated and visualized utilizing an 5 electrophoresis system, such as through a gel electrophoresis, or a capillary electrophoresis.
  • Exemplary gel electrophoresis includes agarose based gels, polyacrylamide based gels, or starch based gels.
  • the probe-protein is subjected to a native electrophoresis condition.
  • the probe-protein is subjected to a denaturing electrophoresis condition.
  • the probe-protein after harvesting is further fragmentized to generate protein 10 fragments.
  • fragmentation is generated through mechanical stress, pressure, or chemical means.
  • the protein from the probe-protein complexes is fragmented by a chemical means.
  • the chemical means is a protease.
  • Exemplary proteases include, but are not limited to, serine proteases such as chymotrypsin A, penicillin G acylase precursor, dipeptidase E, DmpA aminopeptidase, subtilisin, prolyl oligopeptidase, D-Ala-D-Ala peptidase C, 15 signal peptidase I, cytomegalovirus assemblin, Lon-A peptidase, peptidase Clp, Escherichia coli phage KIF endosialidase CIMCD self-cleaving protein, nucleoporin 145, lactoferrin, murein tetrapeptidase LD-carboxypeptidase, or rhomboid-1;
  • the fragmentation is a random fragmentation. In some instances, the fragmentation generates specific lengths of protein fragments, or the shearing occurs at particular sequence of amino acid regions.
  • the protein fragments are further analyzed by a proteomic method such as by liquid chromatography (LC) (e.g. high performance liquid chromatography), liquid chromatography- 30 mass spectrometry (LC-MS), matrix-assisted laser desorption/ionization (MALDI-TOF), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis-mass spectrometry (CE-MS), or nuclear magnetic resonance imaging (NMR).
  • LC liquid chromatography
  • LC-MS liquid chromatography- 30 mass spectrometry
  • MALDI-TOF matrix-assisted laser desorption/ionization
  • GC-MS gas chromatography-mass spectrometry
  • CE-MS capillary electrophoresis-mass spectrometry
  • NMR nuclear magnetic resonance imaging
  • the LC method is any suitable LC methods well known in the art, for separation of a sample into its individual parts. This separation occurs based on the interaction of the 35 sample with the mobile and stationary phases. Since there are many stationary/mobile phase combinations that are employed when separating a mixture, there are several different types of - 61 - Attorney Docket No.: 3436/3 PCT chromatography that are classified based on the physical states of those phases.
  • the LC is further classified as normal-phase chromatography, reverse-phase chromatography, size- exclusion chromatography, ion-exchange chromatography, affinity chromatography, displacement chromatography, partition chromatography, flash chromatography, chiral chromatography, and 5 aqueous normal-phase chromatography.
  • the LC method is a high performance liquid chromatography (HPLC) method.
  • the HPLC method is further categorized as normal-phase chromatography, reverse-phase chromatography, size-exclusion chromatography, ion-exchange chromatography, affinity chromatography, displacement chromatography, partition chromatography, 10 chiral chromatography, and aqueous normal-phase chromatography.
  • the HPLC method of the present disclosure is performed by any standard techniques well known in the art.
  • Exemplary HPLC methods include hydrophilic interaction liquid chromatography (HILIC), electrostatic repulsion-hydrophilic interaction liquid chromatography (ERLIC) and reverse phase liquid chromatography (RPLC).
  • HILIC hydrophilic interaction liquid chromatography
  • ERLIC electrostatic repulsion-hydrophilic interaction liquid chromatography
  • RPLC reverse phase liquid chromatography
  • the LC is coupled to a mass spectroscopy as a LC-MS method.
  • the LC-MS method includes ultra-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS), ultra-performance liquid chromatography-electro spray ionization tandem mass spectrometry (UPLC-ESI-MS/MS), reverse phase liquid chromatography-mass spectrometry (RPLC-MS), hydrophilic interaction liquid 20 chromatography-mass spectrometry (HILIC-MS), hydrophilic interaction liquid chromatography-triple quadrupole tandem mass spectrometry (HILIC-QQQ), electrostatic repulsion-hydrophilic interaction liquid chromatography-mass spectrometry (ERLIC-MS), liquid chromatography time-of-flight mass spectrometry (LC-QTOF-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS).
  • the LC-MS method is LC/LC-MS/MS.
  • the LC-MS methods of the present disclosure are performed by standard techniques well known in the art.
  • the GC is coupled to a mass spectroscopy as a GC-MS method.
  • the GC-MS method includes two-dimensional gas chromatography time-of-flight mass spectrometry (GC*GC-TOFMS), gas chromatography time-of-flight mass spectrometry (GC-QTOF- 30 MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS).
  • CE is coupled to a mass spectroscopy as a CE-MS method.
  • the CE-MS method includes capillary electrophoresis-negative electrospray ionization- mass spectrometry (CE-ESI-MS), capillary electrophoresis-negative electrospray ionization- quadrupole time of flight-mass spectrometry (CE-ESI-QTOF-MS) and capillary electrophoresis- 35 quadrupole time of flight-mass spectrometry (CE-QTOF-MS).
  • CE-ESI-MS capillary electrophoresis-negative electrospray ionization- mass spectrometry
  • CE-ESI-QTOF-MS capillary electrophoresis-negative electrospray ionization- quadrupole time of flight-mass spectrometry
  • CE-QTOF-MS capillary electrophoresis- 35 quadrupole time of flight-mass spectrometry
  • the NMR method includes one dimensional (1D) NMR methods, two dimensional (2D) NMR methods, solid state NMR methods and NMR chromatography.
  • 5 Exemplary 1D NMR methods include 1 Hydrogen, 13 Carbon, 15 Nitrogen, 17 Oxygen, 19 Fluorine, 3 1 Phosphorus, 39 Potassium, 23 Sodium, 33 Sulfur, 87 Strontium, 27 Aluminium, 43 Calcium, 35 Chlorine, 3 7 Chlorine, 63 Copper, 65 Copper, 57 Iron, 25 Magnesium, 199 Mercury or 67 Zinc NMR method, distortionless enhancement by polarization transfer (DEPT) method, attached proton test (APT) method and 1D- enormous natural abundance double quantum transition experiment (INADEQUATE) method.
  • DEPT polarization transfer
  • APIT attached proton test
  • IADEQUATE 1D- enormous natural abundance double quantum transition experiment
  • Exemplary 2D NMR methods include correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), 2D-INADEQUATE, 2D-adequate double quantum transfer experiment (ADEQUATE), nuclear overhauser effect spectroscopy (NOSEY), rotating-frame NOE spectroscopy (ROESY), heteronuclear multiple-quantum correlation spectroscopy (HMQC), heteronuclear single quantum coherence spectroscopy (HSQC), short range coupling and long range coupling methods.
  • Exemplary15 solid state NMR method include solid state 13 Carbon NMR, high resolution magic angle spinning (HR- MAS) and cross polarization magic angle spinning (CP-MAS) NMR methods.
  • Exemplary NMR techniques include diffusion ordered spectroscopy (DOSY), DOSY-TOCSY and DOSY-HSQC.
  • the results from the mass spectroscopy method are analyzed by an algorithm for protein identification.
  • the algorithm combines the results from the 20 mass spectroscopy method with a protein sequence database for protein identification.
  • the algorithm comprises ProLuCID algorithm, Probity, Scaffold, SEQUEST, or Mascot.
  • there can be employed conventional chemical, cellular, histochemical, biochemical, molecular biology, microbiology, recombinant DNA, and clinical techniques which are 25 known to those of skill in the art.
  • kits and articles of manufacture for use with one 35 or more methods described herein.
  • described herein is a kit for generating a protein comprising a detectable group and/or a fragment of a ligand compound described herein.
  • such kit includes a probe or ligand as described herein, small molecule fragments or libraries, and/or controls, and reagents suitable for carrying out one or more of the methods described herein.
  • the kit further comprises samples, such as a cell sample, and suitable solutions such as buffers or media.
  • the kit further comprises recombinant proteins for use 5 in one or more of the methods described herein.
  • additional components of the kit comprises a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein.
  • Suitable containers include, for example, bottles, vials, plates, syringes, and test tubes.
  • the containers are formed from a variety of 10 materials such as glass or plastic.
  • the articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, bottles, tubes, bags, containers, and any packaging material suitable for a selected formulation and intended mode of use.
  • the container(s) include probes, ligands, control compounds, and one or more reagents for use in a method 15 disclosed herein.
  • the presently disclosed kits and articles of manufacture optionally include an identifying description or label or instructions relating to its use in the methods described herein.
  • a kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.
  • a label 20 is on or associated with the container.
  • a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert.
  • a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the 25 contents, such as in the methods described herein.
  • the cells were pelleted by centrifugation at 400g for 5 min, snap-frozen using liquid nitrogen 10 and stored at ⁇ 80 °C until further use.
  • Gel-Based Chemical Proteomic Assay (rhodamine-azide). Cell pellets were lysed in PBS by sonication and fractionated (100,000g, 45 min, 4 °C) to generate soluble and membrane fractions. Protein concentrations were determined using the Bio-Rad DC protein assay and adjusted to 1 mg ml -1 in PBS. Proteome samples (48 ⁇ l aliquots) were treated with SuPUR ligands or probe at the indicated 15 concentrations (1 ⁇ l, 50 ⁇ stock in DMSO) for 1 h at room temperature.
  • Probe-labeled samples were conjugated by copper-catalyzed azide-alkyne cycloaddition (CuAAC) to rhodamine-azide (1 ⁇ l of 1.25 mM stock; final concentration of 25 ⁇ M), tris(2-carboxyethyl)phosphine (TCEP; 1 ⁇ l of fresh 50 mM stock in water, final concentration of 1 mM), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, 3 ⁇ l of a 1.7 mM 4:1 t-butanol/DMSO stock, final concentration of 100 ⁇ M) and copper sulfate 20 (CuSO 4 , 1 ⁇ l of 50 mM stock, final concentration of 1 mM).
  • CuAAC copper-catalyzed azide-alkyne cycloaddition
  • Proteome samples (48 ⁇ l aliquots) were treated with SuPUR ligand or fluorophosphonate-rhodamine (FP-Rh, 1 ⁇ l of 50 ⁇ M stock; final concentration of 1 ⁇ M). Samples were reacted for 1 h at room temperature, quenched with 16.7 ⁇ l of 4x SDS–PAGE loading buffer and ß- mercaptoethanol (ßME) and quenched samples (30 ⁇ l) analyzed by SDS–PAGE and in-gel fluorescence 30 scanning. Live cell evaluation of probes.
  • SuPUR ligand or fluorophosphonate-rhodamine FP-Rh, 1 ⁇ l of 50 ⁇ M stock; final concentration of 1 ⁇ M.
  • Samples were reacted for 1 h at room temperature, quenched with 16.7 ⁇ l of 4x SDS–PAGE loading buffer and ß- mercaptoethanol (ßME) and quenched samples (30 ⁇ l) analyzed by SDS–P
  • SuPUR-modified peptides were enriched with 15 avidin agarose and eluted using 150 ⁇ L of 50% ACN + 0.1% formic acid (3X) and stored at -80 °C until analysis.
  • FP-Rh Competition Cell soluble or membrane proteome fraction (1 mg/mL) was incubated with SuPUR compound for 1 hour at 37 °C followed by incubation with 1 ⁇ M FP-Rh (1 hour, room temperature). Results were determined by gel-based scanning and LC-MS/MS analysis. 20 LC–MS/MS analysis of samples.
  • Nano-electrospray ionization–LC–MS/MS analyses were performed using an Ultimate 3000 RSLC nanoSystem-Orbitrap Q Exactive Plus mass spectrometer (Thermo Scientific) as previously described (see Franks et al. (2017) Cell Chemical Biology 24(7):870- 880) except LC conditions were modified to use the following gradient (A, 0.1% formic acid/H 2 O; B, 80% MeCN, 0.1% formic acid in H 2 O): 0–1.48 min 1% B, 400 nL min -1 ; 1.48–2:00 min 1% B, 300 25 nL min -1 ; 2–90 min 16% B; 90–14625% B; 146–147 min 95% B; 147–153 min 95% B; 153–154 min 1% B; 154.0–154.1 min 1% B, 400 nL min -1 ; 154.1–180 min 1% B, 400 nL min -1 .
  • ABAD enzyme to the desired enzyme concentration based on the specific activity of the enzyme lot.
  • 20 ⁇ L of diluted enzyme (20 nM) was added to each well along with 2.5 ⁇ L of 10X inhibitor solution; 20 2.
  • Assay plate was incubated at RT for 30 minutes, and then 2.5 ⁇ L of a 20X estradiol/ 20X NAD + mix was added to each well for a final concentration of 50 ⁇ M estradiol and 500 ⁇ M NAD + .
  • the assay plate was incubated at 37 o C for 3 hours; 3.
  • Detection system reagents (sold under the tradename NAD(P)H-GLOTM Detection System; Promega Corporation, Madison, Wisconsin, United States of America) were prepared according to 25 manufacturer’s specifications, and 25 ⁇ L was added to each well. After incubating for 1 hour at RT, luminescence was measured using BioTek 5. WST-1 assay. 1. Plate cells in 96-well plates at an appropriate density (4,000-6,000 cells per well); 30 2. Incubate the cells in a humidified incubator at 37°C with 5% CO 2 until they adhere or reach the desired growth phase; 3. Treat cells with the experimental compounds for 48 hours; 4. After treatment, add the 10 ⁇ L WST-1 working solution to each well containing cells; 35 5.
  • Exemplary SuPUR compound ZH-1- 049-2 was active in modifying ABAD in a concentration-dependent manner. See Figure 2A.
  • ZH-1-049-2 was reactive with wild-type ABAD, but not with a mutant ABAD where tyrosine 10 168 was changed to a glycine
  • FIG 2C The presence of the tyrosine at residue 168 was important for maintaining ABAD activity.
  • Figure 2C Gel-based screening using a library of SuPUR compounds (see Figure 3A) indicated that, in addition to ZH-1-049-2 (see Figure 3B), ZH-2-025 and ZH-2-029 showed concentration-dependent labeling of ABAD. See Figure 3C and 3D.
  • ZH-2-029 and ZH-2-025 showed better reactivity for ABAD 15 compared to ZH-1-049-2.
  • the 50% inhibitory concentrations (IC 50 s) of these three compounds was determined. See Figure 3E.
  • ZH-2-025 was most effective at inhibiting the activity of ABAD, with an IC 50 of 430 nM.
  • ZH-2-029 had an IC 50 of 571 nm and ZH-1-049-2 had an IC 50 of 776 nM.
  • the initial data and subsequent modeling of ZH-2-025 in the ABAD binding pocket suggested the possibility of interactions (e.g., the formation of a halogen bond) between the ortho-chloro20 substituent in ZH-2-025 and Gly93. See Figure 4A.
  • ZH-2-025 exhibited cell toxicity, while ZH-2-029 exhibited moderate cell toxicity. See Figures 5E and 5G. ZH-2-029 could stabilize ABAD, particularly the dimer. See Figure 5H. ZH-2-029 was able to reduce ROS production. See Figure 5I. 30 Initial studies with ABHD10 showed that ZH-1-049-2 was also able to modify ABHD10 in a concentration-dependent manner. See Figure 6. Further studies using a library of SuPUR compounds (see Figure 7A) showed that ZH-2-025 had potent labeling activity for ABHD10. See Figures 7B and 7C. Further studies showed that ZH-2-097 and ZH-2-103 also showed potent ADHD10 labeling. See Figures 8A-8D.
  • SuPUR Probes of the presently disclosed subject matter can be synthesized as per Scheme 4 below. Materials were from commercial sources and were used as received. Organic solvents were concentrated via a rotary evaporator 10 (Heidolph) under reduced pressure (VARIO PC 3001) at 35 °C - 50 °C. Reactions were monitored using silica gel TLC plates (GF254, 0.25 mm) and visualized under UV (365/254 nm) light. Proton nuclear magnetic resonance (1H NMR) and carbon nuclear magnetic resonance (13C NMR) spectra were determined using Bruker AV-600 MHz, AV-500 MHz or Agilent 400 MHz instruments.
  • AHL-PuP-2 an average of about 13,000 modified sites were detected from each cell line that collectively translated to greater than 31,000 total modified sites ( ⁇ 50/50 distribution between Y and K sites) from about 4,300 proteins.
  • This chemoproteomic dataset is believed to represent the most comprehensive database of tyrosine and lysine residues in the human proteome amenable to covalent targeting. It was further discovered that ⁇ 20% of SuPUR-modified sites resided in a predicted 35 drug pocket, providing insights into which detectable sites are likely candidates for further ligand discovery efforts.
  • SuPUR chemoproteomics can capture a - 86 - Attorney Docket No.: 3436/3 PCT substantial fraction (41%) of the human purine interactome with coverage across multiple subclasses of purine-binding proteins (30-50%; see Figure 19).
  • gene ontology (GO) and KEGG pathway enrichment of proteins identified by AHL- PuP-2 was performed to investigate their associated biological process(es). The top enrichments were 5 associated with cellular metabolism and RNA processing, likely due to the role of purine as an endogenous metabolite and a building block for RNA, which can mimic substrates such as ATP, GTP, NAD + , adenosine, and other purine-based metabolites.
  • HEK293T proteomes were pretreated with SuPUR ligand (25 ⁇ M, 1 hour) followed by SuPUR probe labeling (100 ⁇ M, 1 hour), CuAAC conjugation of desthiobiotin-azide, trypsin digestion, TMT labeling, avidin 5 chromatography, and LC-MS/MS analysis of probe-modified peptides.
  • TMT-ABPP analysis indicated that the SuPUR ligand ZH-1-049-2 but not the corresponding N-delete control ZH-2-036 exhibited potent covalent binding activity towards multiple druggable sites, including ABHD10 Y215, GSTP1 Y8, ABAD Y168, GNPNAT1 Y165, and PARP2 Y455 (see Figures 14A and 14B). These competition events were due to probe binding blockade and not protein expression changes 10 as evidenced by matching unenriched TMT analyses. Subsequently, SuPUR TMT-ABPP was performed using ZH-2-087 probe and the matching N9 SuPUR ligand ZH-2-055.
  • EXAMPLE 5 20 SuPUR Fragment Ligand Binds Catalytic and Non-catalytic Sites to Disrupt Enzymatic Functions Several ZH-1-049-2-liganded sites are located within or near active sites prompting its further testing as an inhibitor of target proteins.
  • Amyloid- ⁇ (A ⁇ ) peptide-binding alcohol dehydrogenase (ABAD) and alpha/ ⁇ -hydrolase domain containing 10 (ABHD10) were selected for initial evaluation.
  • Covalent inhibitors of ABHD10, a serine hydrolase enzyme are reported but inactivate principally 25 through binding the catalytic and highly conserved active site serine residue (Cao et al. (2019) Nature Chemical Biology 15(12):1232-1240).
  • ABAD is a mitochondrial dehydrogenase involved in steroid metabolism and reported to interact with intracellular amyloid-beta resulting in neuronal dysfunction in Alzheimer’s disease (AD).
  • AD Alzheimer’s disease
  • ABAD inhibitors see Morsy & Trippier (2019) Journal of 5 Medicinal Chemistry 62(9):4252-4264).
  • ZH-1-049- 2-liganded site Y168 is a part of a highly conserved catalytic triad in ABAD and forms hydrogen bond with its cofactor NAD + .
  • NAD(P)/NAD(P)H-Glo biochemical assay for ABAD was established.
  • ZH-1-049-2 showed moderate 10 inhibitory activity, with an IC 50 of 1.45 ⁇ 0.15 ⁇ M.
  • control ZH-2-036 exhibited only weak inhibitory activity, while the reported ABAD inhibitor Frentizole showed no activity at 100 ⁇ M.
  • ABHD10 was pursued for initial discovery because development of a proteome-wide selective serine hydrolase inhibitor through targeting a non-catalytic tyrosine residue 35 has not been achieved to date.
  • - 89 Attorney Docket No.: 3436/3 PCT
  • Molecular docking revealed that the terminal phenyl group of ZH-1-049-2 extends into a solvent-exposed pocket, suggesting that substituent groups could be introduced at this position to improve binding affinity.
  • ZH-2-097 functions as a “lock” by covalently binding to Tyr215, thereby locking the serine catalytic pocket and disrupting the recruitment of its substrate (Figure 15).
  • MS-based ABPP was conducted to identify its targets in the HEK293T proteome using FP-biotin with in situ treatment.
  • the MS analysis identified 65 serine hydrolases, and ZH-2-097 demonstrated remarkable selectivity with a dose-dependent inhibition of ABHD10 ( Figure 16).
  • ZH-2-097 still maintained excellent selectivity even when considering all 1770 proteins detectable by LC-MS/MS, and it also showed significant inhibitory 35 activity against ABHD10 compared to control ZH-2-036.
  • EXAMPLE 7 Development of an ABAD Inhibitor Using SuPUR Ligand Targeting a Catalytic Tyrosine A ⁇ -binding alcohol dehydrogenase (ABAD) is a key enzyme involved in steroid metabolism, 20 and its binding to A ⁇ is thought to contribute to the neurotoxicity.
  • the inhibition of ABAD holds a promising therapeutic potential for the treatment of Alzheimer’s disease and various cancers.
  • ABAD a limited number of small molecules targeting ABAD with moderate binding affinities have been reported.
  • a potent ABAD inhibitor utilizing SuPUR ligand to selectively target a catalytic tyrosine.
  • the library was screened by gel-based competitive ABPP and NAD(P)/NAD(P)H-Glo assay, leading to identification of a potent ligand, ZH-2-029, which exhibited inhibitory activity against ABAD with an 10 IC 50 of 559 ⁇ 60 nM.
  • ZH-2-029 a potent ligand
  • the 7-positioned sulfonyl purine ZH-2-030 was synthesized.
  • Gel-ABPP revealed a micromolar binding affinity of ZH-2-030 for ABAD with an IC 50 of 2392 ⁇ 818 nM, indicating regioselectivity for ABAD.
  • ZH-2-029 To investigate the ability of ZH-2-029 to target ABAD in living cells, HEK293T cells overexpressing ABAD were treated with ZH-2-029 followed by protein labeled with AHL-PuP-2. The results indicated that ZH-2-029 exhibited promising activity against ABAD in living cells, with an IC 50 25 of 2.0 ⁇ 1.2 ⁇ M, while control compound ZH-5-057 showed an IC 50 greater than 25 ⁇ M.
  • the docking study revealed that ZH-2-029 could form hydrogen bonds with Tyr168, Ser155, and Phe201 of ABAD (see e.g., Accession No. NP_004484.1 of the GENBANK® biosequence database).
  • the binding site is positioned near the interaction surface of ABAD and A ⁇ , suggesting that ZH-2-029 may disrupt the ABAD-A ⁇ interaction.
  • the pull-down assay demonstrated 30 that ABAD-A ⁇ interaction was inhibited by ZH-2-029.
  • To further confirm the binding site of ZH-2-029 in living cells we performed SuPUR TMT- ABPP using AHL-PuP-2 in HEK293T proteome. Unenriched proteomics data showed no significant alterations in the ABAD protein profile between the compound-treated group and DMSO group. In contrast, the enriched proteomics analysis of probe modified peptides demonstrated that ZH-2-029 35 selectively binds to the Tyr168 site at 10 ⁇ M.
  • control compounds ZH-5-049-1, ZH-5-051, and ZH-5-049-2 20 exhibited no inhibitory activity across various cancer cell lines, suggesting that the purine core is essential for the inhibition of cancer cell proliferation. Consequently, SuPUR TMT-ABPP was performed to investigate the target and bind site of ZH- 2-077 in SiHa cells using AHL-PuP-2, with ZH-5-049-1 included as a control.
  • the enriched proteomics result revealed that ZH-2-077 selectively and covalently binds to Tyr237 of acetyl-CoA 25 acetyltransferase 2 (ACAT2 (see e.g., Accession No.
  • NP_005882.2 of the GENBANK® biosequence database a key enzyme involved in fatty acid and ketone body metabolism.
  • unenriched proteomics data showed no significant alterations in the ACAT2 protein profile between the compound- treated group and DMSO group.
  • Bioinformatics analyses of clinical profiles indicated that ACTA2 is highly expressed in certain 30 cancers and is closely associated with poor overall survival (OS), particularly in squamous and gastric cancers. Thus, whether compound ZH-2-077 might inhibit growth of cancer cells by targeting ACAT2 was tested.
  • SuPUR TMT-ABPP was performed in the SH-SY5Y neuroblastoma cell line in which ACAT2 is highly expressed.
  • SuPUR ligand ZH-2-077 was identified as a first-in-class covalent inhibitor of ACAT2 by selectively targeting Tyr237. ZH-2-077 further exhibited promising inhibitory activity in SiHa and NCI-N87 cancer cells, supporting its use as a lead compound for inhibiting ACAT2 and designing other ACAT2 inhibitors for cancer therapeutics. 20 Discussion of the EXAMPLES One class of abundant and structurally related metabolites with divergent cell biological functions are purines.
  • Substituted purines form the building blocks of energy cofactors (adenosine triphosphate [ATP] and guanosine triphosphate [GTP], coenzymes in oxidation-reduction reactions, secondary messengers (e.g., cyclic AMP, cyclic GMP), neurotransmitters (e.g., adenine), enzyme 25 cofactors (e.g., nicotinamide adenine dinucleotide and flavin adenine dinucleotide), inflammatory signals (e.g., urate), and nucleotides (e.g., adenosine monophosphate [AMP] and guanosine monophosphate [GMP]).
  • energy cofactors adenosine triphosphate [ATP] and guanosine triphosphate [GTP]
  • secondary messengers e.g., cyclic AMP, cyclic GMP
  • neurotransmitters e.g., adenine

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Abstract

L'invention concerne des composés comprenant des liaisons de sulfonyl-purine ou d'analogue de sulfonyl-purine destinés à être utilisés en tant qu'électrophiles dans la chimie de sulfonyl-purine (SuPUR). L'invention concerne également l'utilisation de SuPUR et/ou de la chimie SuPUR pour le marquage du protéome de résidus d'acides aminés réactifs et pour le développement de nouveaux agents thérapeutiques covalents qui modifient sélectivement les résidus d'acides aminés sélectionnés (par exemple, les résidus de tyrosine ou de lysine) dans des protéines et des peptides biologiquement actifs, tels que la déshydrogénase d'alcool liant l'amyloïde beta (Aβ) (ABAD), le polypeptide à activité de stimulation de la protéine de liaison aux nucléotides de guanine (GNAS), le domaine d'alpha/beta-hydrolase 10 (ABHD10), le glutathion S-transférase pi 1 (GSTP1), la glucosamine-phosphate N-acétyltransférase 1 (GNPNAT1), la poly(ADP-ribose) polymérase 2 (PARP2), et les protéines d'acétyl-CoA acétyltransférase 2 (ACAT2).
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Citations (3)

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US20030086938A1 (en) * 2000-02-21 2003-05-08 Jensen Martin Roland Novel methods for down-regulation of amyloid
US20210255193A1 (en) * 2017-06-23 2021-08-19 The Scripps Research Institute Lysine reactive probes and uses thereof
US20220362276A1 (en) * 2019-05-13 2022-11-17 The Trustees Of Princeton University Small Molecule Inhibitors of Viral Replication

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* Cited by examiner, † Cited by third party
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US20030086938A1 (en) * 2000-02-21 2003-05-08 Jensen Martin Roland Novel methods for down-regulation of amyloid
US20210255193A1 (en) * 2017-06-23 2021-08-19 The Scripps Research Institute Lysine reactive probes and uses thereof
US20220362276A1 (en) * 2019-05-13 2022-11-17 The Trustees Of Princeton University Small Molecule Inhibitors of Viral Replication

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