CYSTEINE BINDING COMPOSITIONS AND METHODS OF USE THEREOF

Abstract

Purine-derived covalent probes (e.g., halo or di-halo-substituted purine based covalent probes) and related ligands are described. The compounds can be used to identify reactive nucleophilic amino acid residues, such as reactive cysteine residues, in proteins and to modify the activity of proteins with reactive nucleophilic amino acid residues (e.g., reactive cysteine residues) via the formation of protein adducts comprising the ligands Modified proteins prepared from the probes and ligands are also described.

Description

TECHNICAL FIELD

The presently disclosed subject matter relates to diagnostics and therapeutics. In particular, it relates to tunable chemistry for global discovery of protein function and ligands, particularly with respect to design of purine-based probes for use in protein function analyses and the identification of related ligands that can interact with reactive amino acid residues in proteins.

BACKGROUND

Chemical proteomics is a powerful technology for ascribing function to the vast number of uncharacterized proteins in the human proteome^1,2. This proteomic method employs probes designed with reactive groups that exploit accessibility and reactivity of binding sites to covalently label active proteins with reporter tags for function assignment and inhibitor development³. Selective probes resulting from competitive screening efforts serve as enabling, and often first-in-class, tools for uncovering biochemical and cellular functions of proteins (e.g. serine hydrolases⁴, proteases⁵, kinases⁶, phosphatases⁷, and glycosidases⁸) and their roles in contributing to human physiology and disease. The basic and translational opportunities afforded by chemical proteomics has prompted exploration of new biocompatible chemistries for broader exploration of the proteome.

Covalent probes used for chemical proteomics range from highly chemoselective fluorophosphonates for catalytic serines⁹ to general thiol alkylating agents and amine-reactive esters of cysteines¹⁰ and lysines¹¹, respectively. The ability to globally measure protein functional states and selectively perturb proteins of interest has substantially augmented the basic understanding of protein function in cell and animal models^1,3. Exploration of new redox-based oxaziridine chemistry, for example, identified a conserved hyper-reactive methionine residue (M169) in redox regulation of mammalian enolase¹². Hydrazine probes revealed a novel N-terminal glyoxylyl post-translational modification on the poorly characterized protein SCRN3¹³. More recent exploration of photoaffinity probes has facilitated global evaluation of reversible small molecule-protein interactions to expand the scope of proteins available for chemical proteomic profiling¹⁴.

However, there remains an ongoing need for additional covalent probes for chemical proteomic profiling, particularly those that provide a scaffold amenable to optimization and drug development.

SUMMARY

This Summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. 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.

In some embodiments, the presently disclosed subject matter provides a method for identifying a reactive amino acid residue of a protein, the method comprising: (a) providing a protein sample comprising isolated proteins, living cells, a cell lysate, or a biological organism; (b) contacting the protein sample with a probe compound of Formula (I) for a period of time sufficient for the probe compound to react with at least one reactive amino acid in a protein in the protein sample, thereby forming at least one modified amino acid residue; and (c) analyzing proteins in the protein sample or removed from the protein sample to identify at least one modified 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):

wherein: X is a monovalent moiety comprising an alkyne moiety, a fluorophore moiety, a detectable labeling group, or a combination thereof; and R₁ and R₂ are independently selected from the group comprising H, halo, amino, alkyl, alkoxy, alkylthio, alkylamino, aryloxy, arylthiol, and arylamino, subject to the proviso that at least one of R₁ and R₂ is halo.

In some embodiments, the probe compound of Formula (I) has a structure of Formula (Ia):

or a structure of Formula (Ib):

wherein: X is a monovalent moiety comprising an alkyne moiety, a fluorophore moiety, a detectable labeling group, or a combination thereof; and R₁ and R₂ are independently selected from the group comprising H, halo, amino, alkyl, alkoxy, alkylthio, aryloxy, arylthiol, and arylamino, subject to the proviso that at least one of R₁ and R₂ is halo.

In some embodiments, the reactive amino acid residue is a cysteine residue. In some embodiments, the modified amino acid residue has a structure of Formula (IIa-i):

a structure of Formula (IIb-i):

a structure of Formula (IIa-ii):

or a structure of Formula (IIb-ii):

wherein: X is a monovalent moiety comprising an alkyne moiety, a fluorophore moiety, a detectable labeling group, or a combination thereof; R₁ is selected from the group comprising H, halo, amino, alkyl, alkoxy, alkylthio, alkylamino, aryloxy, arylthio, and arylamino; and R₂ is selected from the group comprising H, halo, amino, alkyl, alkoxy, alkylthio, alkylamino, aryloxy, arylthio, and arylamino.

In some embodiments, R₁ and R₂ are selected from H, halo, and amino or R₁ and R₂ are selected from H and halo. In some embodiments, R₁ is chloro or fluoro. In some embodiments, R₂ is chloro or fluoro. In some embodiments, X is —CH₂—C≡CH.

In some embodiments, the probe compound is selected from the group comprising 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine, 2,6-dichloro-9-(prop-2-yn-1-yl)-9H-purine, 6-chloro-7-(prop-2-yn-1-yl)-7H-purine, 6-chloro-9-(prop-2-yn-1-yl)-9H-purine, 2-chloro-7-(prop-2-yn-1-yl)-7H-purine, 2-chloro-9-(prop-2-yn-1-yl)-9H-purine, 2,6-difluoro-7-(prop-2-yn-1-yl)-7H-purine, 2,6, -difluoro-9-(prop-2-yn-1-yl)-9H-purine, 6-chloro-2-fluoro-7-(prop-2-yn-1-yl)-7H-purine, 6-chloro-2-fluoro-9-(prop-2-yn-1-yl)-9H-purine, 6-chloro-2-amino-7-(prop-2-yn-1-yl)-7H-purine, and 6-chloro-2-amino-9-(prop-2-yn-1-yl)-9H-purine.

In some embodiments, the probe compound has a structure of Formula (Ib). In some embodiments, the probe compound is 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine.

In some embodiments, the analyzing of step (c) further comprises tagging the at least one modified reactive amino acid residue with a compound comprising a detectable labeling group, thereby forming at least one tagged reactive amino acid residue comprising said detectable labeling group. In some embodiments, the detectable labeling group comprises biotin or a biotin derivative, optionally wherein the biotin derivative is desthiobiotin.

In some embodiments, the tagging comprises reacting an alkyne group in the X moiety of the at least one modified reactive amino acid residue with a compound comprising (i) an azide moiety and (ii) the detectable labeling group, optionally via a copper-catalyzed azide-alkyne cycloaddition (CuAAC) coupling reaction. In some embodiments, the analyzing further comprises digesting proteins with trypsin to provide a digested protein sample comprising a protein fragment comprising the at least one tagged reactive amino acid moiety comprising the detectable group. In some embodiments, the analyzing further comprises enriching the digested protein sample for the detectable labeling group, optionally wherein the enriching comprises contacting the digested protein sample with a solid support comprising a binding partner of the detectable labeling group. In some embodiments, the analyzing further comprises analyzing the enriched digested protein sample via liquid chromatography-mass spectrometry (LC-MS).

In some embodiments, the protein sample is a biological organism, optionally a mammal; wherein contacting the protein sample with the probe compound of Formula (I) comprises administering the probe compound of Formula (I) to the biological organism, optionally via oral administration or injection; and wherein prior to analyzing the proteins, tissues are removed from the biological organism and homogenized.

In some embodiments, providing the protein sample further comprises separating the protein sample into a first protein sample and a second protein sample; contacting the protein sample with a probe compound of Formula (I) comprises contacting the first protein sample with a first probe compound of Formula (I) at a first probe concentration for a first period of time and contacting the second protein sample with one of the group consisting of: (b1) a second probe compound of Formula (I) at the first probe concentration for the first period of time, (b2) the first probe compound of Formula (I) at a second probe concentration for the first period of time, and (b3) the first probe compound of Formula (I) at the first probe concentration for a second period of time; thereby forming at least one modified reactive amino acid residue in said first and/or said second protein sample; and 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 protein sample comprises living cells and wherein 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), optionally wherein the first cell culture medium comprises ¹³C- and/or ¹⁵N-labeled amino acids, further optionally wherein the first cell culture medium comprises ¹³C-¹⁵N-labeled lysine and arginine; and culturing the second protein sample in a second cell culture medium, wherein said second cell culture medium comprises a naturally occurring isotope distribution, prior to the contacting of step (b). In some embodiments, one of the first and the second protein sample is cultured in the presence of an inhibitor of an enzyme known or suspected of being present in said first or second protein sample.

In some embodiments, the probe compound of Formula (I) comprises a detectable labeling group comprising a heavy isotope or wherein the analyzing of step (c) further comprises tagging the at least one modified amino acid residue with a compound comprising a detectable labeling group comprising a heavy isotope, optionally wherein the heavy isotope is carbon-13.

In some embodiments, the presently disclosed subject matter provides a probe compound for detecting a reactive amino acid residue, optionally a reactive cysteine residue, in a protein, wherein the probe compound is selected from the group comprising 2,6-difluoro-7-(prop-2-yn-1-yl)-7H-purine, 2,6-difluoro-9-(prop-2-yn-1-yl)-9H-purine, and 6-chloro-2-fluoro-7-(prop-2-yn-1-yl)-7H-purine.

In some embodiments, the presently disclosed subject matter provides a compound having the structure of Formula (III):

wherein: Z is selected from the group comprising cycloalkyl, acyl, substituted acyl, —S(═O)₂—R₅, —S(═O)₂—N(R₆)₂, —S(═O)₂—O—R₇, and

R₃ and R₄ are independently selected from H, halo, alkyl, alkoxy, alkylamino, alkylthio, aryloxy, arylamino, and arylthiol, subject to the proviso that at least one of R₃ and R₄ is halo, optionally chloro or fluoro; R₅ is heterocyclyl, substituted heterocyclyl, aryl or substituted aryl; each R₆ is selected from H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl, or wherein the two R₆ together form an alkylene group; and R₇ is selected from alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl and substituted aryl.

In some embodiments, the compound of Formula (III) has a structure of Formula (IIIa):

or a structure of Formula (IIIb):

wherein: Z is selected from the group comprising cycloalkyl, acyl, substituted acyl, —S(═O)₂—R₅, —S(—O)₂—N(R₆)₂, —S(═O)₂—O—R₇, and

R₃ and R₄ are independently selected from H, halo, alkyl, alkoxy, alkylamino, alkylthio, aryloxy, arylamino, and arylthiol, subject to the proviso that at least one of R₃ and R₄ is halo, optionally chloro or fluoro; R₅ is heterocyclyl, substituted heterocyclyl, aryl or substituted aryl; each R₆ is selected from H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl, or wherein the two R₆ together form an alkylene group; and R₇ is selected from alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl and substituted aryl.

In some embodiments, R₃ is selected from chloro, methyl, —SH—(CH₂)₃CH₃; —NH(CH₂)₃CH₃; and —O—(C₆H₄)CH₃. In some embodiments, R₄ is chloro or fluoro. In some embodiment, Z is acetyl, n-hexanoyl, n-dodecanoyl; cyclohexyl, —S(═O)₂—R₅, —S(═O)₂—N(R₆)₂, —S(═O)₂—O—R₇, and

wherein R₅ is heterocyclyl or substituted phenyl; optionally wherein the substituted phenyl is alkoxy- or halo-substituted phenyl; each R₆ is selected from alkyl and aralkyl, optionally methyl, ethyl or benzyl; and R₇ is alkyl, optionally methyl. In some embodiments, Z is selected from

In some embodiments, the compound is selected from the group comprising 4-((2,6-dichloro-7H-purin-7-yl)sulfonyl)morpholine, 4-((2,6-dichloro-9H-purin-9-yl)sulfonyl)morpholine, 2,6-dichloro-7-((4-fluorophenyl)sulfonyl)-7H-purine, 2,6-dichloro-9-((fluorophenyl)sulfonyl)-9H-purine, 2,6-dichloro-7-((4-methoxyphenyl)sulfonyl)-7H-purine, 2,6-dichloro-9-((4-methoxyphenyl)sulfonyl)-9H-purine, 2,6-dichloro-7-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl)-7H-purine, 2,6-dichloro-9-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl)-9H-purine, 1-(2,6-dichloro-9H-purin-9-yl)dodecan-1-one, 1-(2,6-dichloro-7H-purin-7-yl)hexan-1-one, 1-(2,6-dichloro-9H-purin-9-yl)hexan-1-one, 1-(6-chloro-2-fluoro-7H-purin-7-yl)hexan-1-one, 1-(6-chloro-2-fluoro-9H-purin-9-yl)hexan-1-one, 1-(2-chloro-6-methyl-9H-purin-9-yl)hexan-1-one, 2-chloro-9-cyclohexyl-6-methyl-9H-purine, 9-cyclohexyl-2-fluoro-6-methyl-9H-purine, 1-(6-(butylthio)-2-fluoro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylthio)-2-chloro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylthio)-2-chloro-7H-purin-7-yl)ethan-1-one, 1-(6-(butylthio)-2-fluoro-7H-purin-7-yl)ethan-1-one, 1-(2-chloro-6-(p-tolyloxy)-9H-purin-9-yl)ethan-1-one, 1-(2-fluoro-6-(p-tolyloxy)-9H-purin-9-yl)ethan-1-one, 1-(2-chloro-6-(p-tolyloxy)-7H-purin-7-yl)ethan-1-one, 1-(2-fluoro-6-(p-tolyloxy)-7H-purin-7-yl)ethan-1-one, 1-(6-(butylamino)-2-chloro-7H-purin-7-yl)ethan-1-one, 1-(6-(butylamino)-2-fluoro-7H-purin-7-yl)ethan-1-one, 1-(6-(butylamino)-2-chloro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylamino)-2-fluoro-9H-purin-9-yl)ethan-1-one, 2,6-dichloro-N,N-diethyl-7H-purine-7-sulfonamide, 2,6-dichloro-N,N-diethyl-9H-purine-9-sulfonamide, N-benzyl-2,6-dichloro-N-methyl-9H-purine-9-sulfonamide, N-benzyl-2,6-dichloro-N-methyl-7H-purine-7H-sulfonamide, benzyl 2,6-dichloro-7H-purine-7-sulfonate, benzyl 2,6-dichloro-9H-purine-9-sulfonate, methyl 2,6-dichloro-9H-purine-9-sulfonate, and methyl 2,6-dichloro-7H-purine-7-sulfonate.

In some embodiments, the presently disclosed subject matter provides a compound where the compound is 2,6-dichloro-7-(4-nitrobenzyl)-7H-purine.

In some embodiments, the presently disclosed subject matter provides a modified cysteine-containing protein comprising a modified cysteine residue wherein the modified cysteine residue is formed by the reaction of a cysteine residue with a non-naturally occurring purine-based compound wherein said non-naturally occurring purine-based compound is a compound having a structure of Formula (I):

or a compound having a structure of Formula (III′):

wherein: X is a monovalent moiety comprising an alkyne moiety, a fluorophore moiety, a detectable labeling group, or a combination thereof; Z′ is selected from the group comprising alkyl, optionally —CH₂—CH═CH₂, substituted alkyl, cycloalkyl, heterocycloalkyl, acyl, substituted acyl, aralkyl, substituted aralkyl, —S(═O)₂—R₅′, —S(═O)₂—N(R₆)₂, and —S(═O)₂—O—R₇; R₁ and R₂ are independently selected from the group comprising H, halo, hydroxyl, thiol, amino, alkyl, alkoxy, alkylamino, alkylthio, aryloxy, arylamino, and arylthio, subject to the proviso that at least one of R₁ and R₂ is halo; R₃′ and R₄′ are independently selected from H, halo, alkyl, alkylamino, alkylthio, alkoxy, aryloxy, arylamino, and arylthiol, subject to the proviso that at least one of R₃′ and R₄′ is halo; R₅′ is heterocyclyl, substituted heterocyclyl, aryl or substituted aryl; each R₆ is selected from H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl, or wherein the two R₆ together form an alkylene group; and R₇ is selected from alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl and substituted aryl.

In some embodiments, the modified cysteine-containing protein comprises at least one modified cysteine residue comprising a structure of Formula (II-i):

a structure of Formula (II-ii):

a structure of Formula (IV′-i):

or a structure of Formula (IV′-ii):

wherein: X is a monovalent moiety comprising an alkyne moiety, a fluorophore moiety, a detectable labeling group, or a combination thereof; Z′ is selected from the group comprising alkyl, optionally —CH₂—CH═CH₂, substituted alkyl, cycloalkyl, heterocycloalkyl, acyl, substituted acyl, aralkyl, substituted aralkyl, —S(═O)₂—R₅′, —S(═O)₂—N(R₆)₂, and —S(═O)₂—O—R₇; R₁ is selected from the group comprising H, halo, hydroxyl, thiol, amino, alkyl, alkoxy, alkylamino, alkylthio, aryloxy, arylamino, and arylthio; R₂ is selected from the group comprising H, halo, hydroxyl, thiol, amino, alkyl, alkoxy, alkylamino, alkylthio, aryloxy, arylamino, and arylthio; R₃′ is selected from the group comprising H, halo, alkyl, alkylamino, alkylthio, alkoxy, aryloxy, arylamino, and arylthiol; R₄′ is selected from the group comprising H, halo, alkyl, alkylamino, alkylthio, alkoxy, aryloxy, arylamino, and arylthiol; R₅′ is heterocyclyl, substituted heterocyclyl, aryl or substituted aryl; each R₆ is selected from H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl, or wherein the two R₆ together form an alkylene group; and R₇ is selected from alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl and substituted aryl.

In some embodiments, the modified cysteine-containing protein is selenocysteine elongation factor (eEF-Sec) modified at cysteine 442, macrophage migration inhibitory factor modified at cysteine 81; or serine/threonine protein kinase 38-like modified at cysteine 235.

In some embodiments, the presently disclosed subject matter provides a method for modulating an activity of a protein comprising a reactive cysteine residue, wherein the method comprising contacting a protein comprising a reactive cysteine residue with a compound having a structure of Formula (III′):

wherein: Z′ is selected from the group comprising alkyl, optionally —CH₂—CH═CH₂, substituted alkyl, cycloalkyl, heterocycloalkyl, acyl, substituted acyl, aralkyl, substituted aralkyl, —S(═O)₂—R₅′, —S(═O)₂—N(R₆)₂, and —S(═O)₂—O—R₇; R₃′ and R₄′ are independently selected from H, halo, alkyl, alkylamino, alkylthio, alkoxy, aryloxy, arylamino, and arylthio, subject to the proviso that at least one of R₃′ and R₄′ is halo; R₅′ is heterocyclyl, substituted heterocyclyl, aryl or substituted aryl; each R₆ is selected from H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl, or wherein the two R₆ together form an alkylene group; and R₇ is selected from alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl and substituted aryl. In some embodiments, the compound having a structure of Formula (III′) is a compound having a structure of Formula (IIIa′):

or a structure of Formula (IIIb′):

wherein Z′, R₃′, and R₄′ are as defined for Formula (III′).

In some embodiments, R₃′ is selected from chloro, fluoro, methyl, n-butylthio, n-butylamino, or —O—(C₆H₄)—OMe. In some embodiments, Z′ is selected from —CH₂—CH═CH₂, C₂-C₁₂ acyl, cyclohexyl, benzyl, —CH₂—(C₆H₄)—NO₂, —S(═O)₂—R₅′, and

wherein R′₅ is selected from morpholinyl, 4-halophenyl, and 4-alkoxyphenyl. In some embodiments, both R₃′ and R₄′ are chloro.

In some embodiments, the compound of Formula (III′) is selected from the group comprising 4-((2,6-dichloro-7H-purin-7-yl)sulfonyl)morpholine, 4-((2,6-dichloro-9H-purin-9-yl)sulfonyl)morpholine, 2,6-dichloro-7-((4-fluorophenyl)sulfonyl)-7H-purine, 2,6-dichloro-9-((4-fluorophenyl)sulfonyl)-9H-purine, 2,6-dichloro-7-((4-methoxyphenyl)sulfonyl)-7H-purine, 2,6-dichloro-9-((4-methoxyphenyl)sulfonyl)-9H-purine, 2,6-dichloro-7-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl)-7H-purine, 2,6-dichloro-9-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl)-9H-purine, 2,6-dichloro-7-(4-nitrobenzyl)-7H-purine, 1-(2,6-dichloro-9H-purin-9-yl)dodecan-1-one, 1-(2,6-dichloro-7H-purin-7-yl)hexan-1-one, 1-(2,6-dichloro-9H-purin-9-yl)hexan-1-one, 1-(6-chloro-2-fluoro-7H-purin-7-yl)hexan-1-one, 1-(6-chloro-2-fluoro-9H-purin-9-yl)hexan-1-one, 1-(2-chloro-6-methyl-9H-purin-9-yl)hexan-1-one, 2-chloro-9-cyclohexyl-6-methyl-9H-purine, 9-cyclohexyl-2-fluoro-6-methyl-9H-purine, 1-(6-(butylthio)-2-fluoro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylthio)-2-chloro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylthio)-2-chloro-7H-purin-7-yl)ethan-1-one, 1-(6-(butylthio)-2-fluoro-7H-purin-7-yl)ethan-1-one, 1-(2-chloro-6-(p-tolyloxy)-9H-purin-9-yl)ethan-1-one, 1-(2-fluoro-6-(p-tolyloxy)-9H-purin-9-yl)ethan-1-one, 1-(2-chloro-6-(p-tolyloxy)-7H-purin-7-yl)ethan-1-one, 1-(2-fluoro-6-(p-tolyloxy)-7H-purin-7-yl)ethan-1-one, 1-(6-(butylamino)-2-chloro-7H-purin-7-yl)ethan-1-one, 1-(6-(butylamino)-2-fluoro-7H-purin-7-yl)ethan-1-one, 7-allyl-2,6-dichloro-7H-purine, 9-allyl-2,6-dichloro-9H-purine, 2,6-dichloro-7-benzyl-7H-purine, 2,6-dichloro-9-benzyl-9H-purine, 2,6-dichloro-7-(4-nitrobenzyl)-7H-purine, 2,6-dichloro-9-(4-nitrobenzyl-9H-purine, 2-(2,6-dichloro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol, 1-(6-(butylamino)-2-chloro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylamino)-2-fluoro-9H-purin-9-yl)ethan-1-one, 2,6-dichloro-N,N-diethyl-7H-purine-7-sulfonamide, 2,6-dichloro-N,N-diethyl-9H-purine-9-sulfonamide, N-benzyl-2,6-dichloro-N-methyl-9H-purine-9-sulfonamide, N-benzyl-2,6-dichloro-N-methyl-7H-purine-7H-sulfonamide, benzyl 2,6-dichloro-7H-purine-7-sulfonate, benzyl 2,6-dichloro-9H-purine-9-sulfonate, methyl 2,6-dichloro-9H-purine-9-sulfonate, and methyl 2,6-dichloro-7H-purine-7-sulfonate.

In some embodiments, modulating an activity of a protein comprising a reactive cysteine residue comprises inhibiting an activity of the protein comprising a reactive cysteine residue. In some embodiments, modulating an activity of a protein comprising a reactive cysteine residue comprises activating an activity of the protein comprising a reactive cysteine residue. In some embodiments, modulating an activity of a protein comprising a reactive cysteine residue comprises blocking a protein-protein interaction of the protein comprising a reactive cysteine residue. In some embodiments, modulating an activity of a protein comprising a reactive cysteine residue comprises disrupting a protein-RNA interaction of the protein comprising a reactive cysteine residue. In some embodiments, modulating an activity of a protein comprising a reactive cysteine residue comprises disrupting a protein-DNA interaction of the protein comprising a reactive cysteine residue. In some embodiments, modulating an activity of a protein comprising a reactive cysteine residue comprises disrupting a protein-lipid interaction of the protein comprising a reactive cysteine residue. In some embodiments, modulating the activity of a protein comprising a reactive cysteine residue comprises disrupting a protein-metabolite interaction of the protein comprising a reactive cysteine residue. In some embodiments, modulating an activity of a protein comprising a reactive cysteine residue comprises disrupting subcellular localization of the protein comprising a reactive cysteine residue. In some embodiments, modulating an activity of a protein comprising a reactive cysteine residue comprises triggering recruitment of an E3 ligase for targeted degradation of the protein comprising a reactive cysteine residue.

Accordingly, it is an object of the presently disclosed subject matter to provide methods of identifying reactive amino acid residues in proteins and methods of modulating the activity of proteins comprising reactive cysteine residues, as well as to provide covalent probes and related compounds and modified proteins. This and other objects are achieved in whole or in part by the presently disclosed subject matter.

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.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic diagram showing the design of an activity-based protein profiling (ABPP) probe for the investigation of enzymes, comprising an enzyme-recognition moiety attached to or substituted by a reactive group (i.e., a group that can react with an enzyme or other protein being investigated) and a tag (e.g., which can be reacted with a detectable moiety).

FIG. 1B is a schematic drawing of an exemplary general structure of purine-derived, activity-based protein profiling (ABPP) probes of the presently disclosed subject matter, where E groups, i.e., reactive/leaving groups, are substituted on the more electron-deficient pyrimidine ring of the purine scaffold and a tag is attached to the more electron-rich imidazole ring of the purine scaffold. The tag can comprise a detectable group or be a group that can be derivatized with a detectable group.

FIG. 1C is a schematic diagram showing the reaction of a nucleophilic group (Nu:) of a reactive amino acid residue of an enzyme (Enz) or other protein with an exemplary purine-based probe compound of the presently disclosed subject matter. Reaction with the nucleophilic group results in covalent modification of the enzyme or other protein and the loss of the chloro leaving group at carbon-6 of the purine-based probe. The propargyl group on the imidazole ring of the purine scaffold can be used for later derivatization of the modified enzyme or other protein with a detectable group (e.g., a fluorophore or specific binding partner). When the R group at carbon-2 of the purine-based probe is a potential leaving group (e.g., a halo atom), the nucleophilic group of the reactive amino acid residue of the enzyme can alternatively react to form a covalent bond at carbon-2.

FIG. 2A is a schematic diagram showing the synthesis of exemplary activity-based probes (ABPs) of the presently disclosed subject matter, i.e., 6-chloro-9-(prop-2-yn-1-yl)-9H-purine (Pu-4) and 6-chloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-3). The major product of the reaction of 6-chloropurine and propargyl bromide is the N9-substituted purine.

FIG. 2B is a schematic diagram showing the structures of exemplary activity-based probes (ABP) of the presently disclosed subject matter comprising one or two chloro substituents on the pyrimidine ring and a propargyl group attached at one of the two nitrogen atoms of the imidazole group of the purine core structure. Ratios are provided for the major (N9-substituted) and minor (N7-substituted) products of the reactions of the chloro- or dichloropurine starting material with propargyl bromide.

FIG. 3A is a schematic drawing showing the workflow for a solution-based activity assay of purine probes with small molecule mimetics of amino acid residues, e.g., butanethiol. Analysis of the time course of the reaction is performed via high performance liquid chromatography (HPLC).

FIG. 3B is a series of graphs showing the solution-based reactivity (% starting material consumed versus time) of two exemplary probes, i.e., 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine (AHL125 or Pu-1; data shown by “+”s) and 2,6-dichloro-9-(prop-2-yn-1-yl)-9H-purine (AHL128 or Pu-2, data shown by “x”s) for different small molecule amino acid residue mimetics. The graph at the top left is the reactivity data for the cysteine mimetic butanethiol; the graph at the top right is the reactivity data for the glutamine/asparagine mimetic propionamide; the graph at the middle left is the reactivity data for the aspartic acid/glutamic acid mimetic butyric acid; the graph at the middle right is the reactivity data for the tyrosine mimetic p-cresol; the graph at the bottom left is the reactivity data for the lysine mimetic butylamine.

FIG. 3C is a series of graphs showing the solution-based reactivity (% starting material consumed versus time) of ten exemplary probes of the presently disclosed subject matter for the cysteine residue mimetic butanethiol. The graph at the top left shows data for Pu-1 (filled circles) and Pu-2 (unfilled circles). The graph at the middle left shows data for Pu-3 (filled squares) and Pu-4 (unfilled squares). The graph at the bottom left shows data for Pu-5 (filled triangles) and Pu-6 (unfilled triangles). The graph at the top right shows data for Pu-7 (filled circles) and Pu-8 (unfilled circles). The graph at the middle right shows data for Pu-9 (filled circles) and Pu-10 (unfilled circles). The structures for the mono- and di-chloro purine-based probes Pu-1 to Pu-6 are shown in FIG. 2B. Pu-7 and Pu-8 are the 2-amino-6-chloro-7-(prop-2-yn-1-yl)-7H-purine and 2-amino-6-chloro-9-(prop-2-yn-1-yl)-9H-purine, respectively. Pu-9 and Pu-10 are 2,6-difluoro-7-(prop-2-yn-1-yl)-7H-purine and 2,6-difluoro-9-(prop-2-yn-1-yl)-9H-purine, respectively.

FIG. 3D is a graph comparing the solution-based reactivity (% starting material consumed versus time) of two exemplary purine-based protein ligands, 2,6-dichloro-7-benzyl-7H-purine (Pi-1, filled circles) and 2,6-dichloro-9-benzyl-9H-purine (Pi-2, unfilled circles) for the cysteine residue mimetic butanethiol.

FIG. 3E is a graph comparing the solution-based reactivity (% starting material consumed versus time) of two exemplary purine-based protein ligands, 7-allyl-2,6-dichloro-7H-purine (Pi-3, filled squares) and 9-allyl-2,6-dichloro-9H-purine (Pi-4, unfilled squares) for the cysteine residue mimetic butanethiol.

FIG. 3F is a graph comparing the solution-based reactivity (% starting material consumed versus time) of two exemplary purine-based protein probes, 6-butylthio-2-chloro-7-(prop-2-yn-1-yl)-7H-purine (Pa-1, data shown in circles) and 6-butylthio-2-chloro-9-(prop-2-yn-1-yl)-9H-purine (Pa-4, data shown in squares) for the cysteine residue mimetic butanethiol.

FIG. 3G is a graph comparing the solution-based reactivity (% starting material consumed versus time) of two exemplary purine-based protein probes, 2-butylthio-6-chloro-9-(prop-2-yn-1-yl)-9H-purine (Pa-3, data shown in triangles) and 6-butylthio-2-chloro-9-(prop-2-yn-1-yl)-9H-purine (Pa-4, data shown in squares) for the cysteine residue mimetic butanethiol.

FIG. 4A is a schematic diagram showing the workflow for an assay for the detection of reactive amino acid residues in a protein sample using a purine-derived activity-based probe (ABP) of the presently disclosed subject matter. Modification of proteins comprising reactive amino acid residues in a cell or cell lysate sample with the probe is followed by reaction of an alkyne group via copper-catalyzed azide-alkyne cycloaddition (CuAAC) to provide a triazole adduct conjugated to a detectable moiety for detection of the adduct via in-gel fluorescence detection using a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the presently disclosed subject matter.

FIG. 4B is a series of photographic images of fluorescent gel assays showing the activity of purine-based probes in live cells. The image at the left compares the activity of several exemplary probes, Pu-1 through Pu-10, the structures of which are described in FIG. 2B or the brief description for FIG. 3C. The image in the center shows the concentration dependence of the activity of Pu-1 (AHL125) and Pu-2 (AHL128). The image at the right shows the time dependence of the activity of Pu-1 and Pu-2.

FIG. 4C is a series of photographic images of fluorescent gel assays showing the activity of purine-based probes in cell lysates. The image at the left compares the activity of several exemplary probes, Pu-1 through Pu-10, the structures of which are described in FIG. 2B or the brief description for FIG. 3C. The image in the center shows the concentration dependence of the activity of Pu-1 (AHL125). The image at the right shows the time dependence of the activity of Pu-1 and Pu-2 (AHL128).

FIG. 5A is a composite image of a fluorescent gel showing the in vivo activity in lung tissue of three exemplary probes of the presently disclosed subject matter, 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-1), 2,6-dichloro-9-(prop-2-yn-1-yl)-9H-purine (Pu-2), and 2-chloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-5), administered to mice via intraperitoneal injection (IP) or oral gavage (OG) at two different doses.

FIG. 5B is a composite image of a fluorescent gel showing the in vivo activity in liver tissue of three exemplary probes of the presently disclosed subject matter, 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-1), 2,6-dichloro-9-(prop-2-yn-1-yl)-9H-purine (Pu-2), and 2-chloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-5), administered to mice via intraperitoneal injection (IP) or oral gavage (OG) at two different doses.

FIG. 5C is a composite image of a fluorescent gel showing the in vivo activity in spleen tissue of three exemplary probes of the presently disclosed subject matter, 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-1), 2,6-dichloro-9-(prop-2-yn-1-yl)-9H-purine (Pu-2), and 2-chloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-5), administered to mice via intraperitoneal injection (IP) or oral gavage (OG) at two different doses.

FIG. 5D is a composite image of a fluorescent gel showing the in vivo activity in heart tissue of three exemplary probes of the presently disclosed subject matter, 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-1), 2,6-dichloro-9-(prop-2-yn-1-yl)-9H-purine (Pu-2), and 2-chloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-5), administered to mice via intraperitoneal injection (IP) or oral gavage (OG) at two different doses.

FIG. 5E is a composite image of a fluorescent gel showing the in vivo activity in brain tissue of three exemplary probes of the presently disclosed subject matter, 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-1), 2,6-dichloro-9-(prop-2-yn-1-yl)-9H-purine (Pu-2), and 2-chloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-5), administered to mice via intraperitoneal injection (IP) or oral gavage (OG) at two different doses.

FIG. 5F is a composite image of a fluorescent gel showing the in vivo activity in kidney tissue of three exemplary probes of the presently disclosed subject matter, 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-1), 2,6-dichloro-9-(prop-2-yn-1-yl)-9H-purine (Pu-2), and 2-chloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-5), administered to mice via intraperitoneal injection (IP) or via oral gavage (OG) at two different doses in kidney tissue.

FIG. 5G is a composite image of a fluorescent gel showing the in vivo activity in white adipose tissue (WAT) of three exemplary probes of the presently disclosed subject matter, 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-1), 2,6-dichloro-9-(prop-2-yn-1-yl)-9H-purine (Pu-2), 2-chloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-5), administered to mice via intraperitoneal injection (IP) or via oral gavage (OG) at two different doses.

FIG. 6 is a pair of photographic images of the fluorescent gel-based analysis of (left) the activity of 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-1) in vivo in mice in different tissues after two hours and (right) the activity of 2,6-dichloro-9-(prop-2-yn-1-yl)-9H-purine (Pu-2) in vivo in mice in different tissues after four hours.

FIG. 7 is a schematic diagram showing the workflow of an assay for the detection of reactive amino acid residues in a protein sample using a purine-derived activity-based probe (ABP) according to the presently disclosed subject matter. The work flow is the same as that shown in FIG. 4A, except that after formation of the triazole adduct, the modified proteins are digested with trypsin, the digested sample is enriched for the modified fragments, and the modified fragments are analyzed via liquid chromatography-tandem mass spectroscopy (LC-MS/MS).

FIG. 8 is a schematic diagram showing the workflow of a fluorescent gel-based competition assay using an activity-based probe (ABP) of the presently disclosed subject matter and a competitive inhibitor.

FIG. 9 is a schematic diagram showing the structures of exemplary purine-based ligands of the presently disclosed subject matter.

FIG. 10A is a schematic diagram of the four binding domains (D1, D2, D3, and D4) of selenocysteine elongation factor (eEF-Sec) and the binding site (Cysteine 442) of an exemplary probe of the presently disclosed subject matter, i.e., 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine, also referred to herein as Pu-1, AHL-Pu-1, and AHL125.

FIG. 10B is a pair of photographic images of the fluorescent gel-based analysis of the time dependent reaction of (left) 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine (AHL-Pu-1) or (right) 2,6-dichloro-9-(prop-2-yn-1-yl)-9H-purine (AHL-Pu-2) with wild-type selenocysteine elongation factor (eEF-Sec; WT) in live cells. The arrow pointing to the band at about 75 kilodalton (kDa) shows the band for probe-modified eEF-Sec. For comparison, the probes were contacted with cells overexpressing a mutant eEF-SEC where the cysteine at amino acid 442 is changed to alanine (Mutant) and cells not transfected with or overexpressing any eEF-Sec (Mock). Reaction times were varied from 0.5 to 2 hours.

FIG. 10C is a photographic image of the fluorescent gel-based analysis of the concentration dependence of the reaction of 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine (AHL-Pu-1) with wild-type selenocysteine elongation factor (WT) or a mutant eEF-Sec where the cysteine at amino acid 442 is changed to alanine (CS) overexpressed in human embryonic kidney (HEK) cell proteomes. Reaction time was varied from 30 minutes to 120 minutes. The arrow points to the band for the probe-modified eEF-Sec at about 75 kilodaltons. The concentration of AHL-Pu-1 was varied from 1 micromolar (μM) to 50 μM. “Mock” refers to cells not transfected with WT or mutant eEF-Sec.

FIG. 10D is a photographic image of the fluorescent gel-based analysis of a competition assay where purine ligands (2,6-dichloro-7-(4-nitrobenzyl)-7H-purine (Pi-5), 4-((2,6-dichloro-9H-purin-9-yl)sulfonyl)morpholine (Pi-8), or 2,6-dichloro-7-benzyl-7H-purine (Pi-1); 4 hr treatment) block 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine (AHL-Pu-1) probe labeling (25 μM; 1 hr) of selenocysteine elongation factor (eEF-Sec) in a concentration dependent manner in live cells. Concentrations of the purine ligands was varied from 0.5 micromolar (μM) to 25 μM.

FIG. 10E is a photographic image of the fluorescent gel-based analysis of a competition assay where purine ligands (2,6-dichloro-7-(4-nitrobenzyl)-7H-purine (Pi-5) or 2,6-dichloro-7-benzyl-7H-purine (Pi-1); 4 hr treatment) block 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine (AHL-Pu-1) probe labeling (25 μM; 1 hr) of selenocysteine elongation factor (eEF-Sec) in a concentration dependent manner in live cells. Concentrations of the purine ligands was varied from 0.05 micromolar (μM) to 25 μM.

FIG. 11 is a graph showing the functional protein domains that are statistically significantly enriched by Pu-1 and Pu-2 purine probes.

FIG. 12 is a graph showing the subcellular location analysis of Pu-1- and Pu-2-modified proteins from live cell studies.

DETAILED DESCRIPTION

The presently disclosed subject matter will now be described more fully hereinafter with reference to the accompanying Figures and Examples, in which representative embodiments are shown. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art. Certain components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the presently disclosed subject matter (in some cases schematically).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently described subject matter belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Throughout the specification and claims, a given chemical formula or name shall encompass all active optical and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist.

I. Definitions

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the presently disclosed subject matter.

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art.

In describing the presently disclosed subject matter, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques.

Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the presently disclosed and claimed subject matter.

Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including in the claims. For example, the phrase “a protein” refers to one or more proteins, including a plurality of the same protein. Similarly, 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, 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.

Unless otherwise indicated, 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 term “about”, as used herein when referring to a measurable value such as an amount of mass, weight, time, volume, concentration, or percentage, is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods and/or employ the disclosed compositions. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

A disease or disorder is “alleviated” if the severity of a symptom of the disease, condition, or disorder, or the frequency at which such a symptom is experienced by a subject, or both, are reduced.

As used herein, the term “and/or” when used in the context of a list of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.

The terms “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 compound for an additional therapeutic use for a particular injury, disease, or disorder being treated. Such a compound, for example, 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.

As used herein, the term “adjuvant” refers to a substance that elicits an enhanced immune response when used in combination with a specific antigen.

As use herein, the terms “administration of” and/or “administering” a compound should be understood to refer to providing a compound of the presently disclosed subject matter to a subject in need of treatment.

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 matter.

As used herein, the phrase “consisting essentially of” limits the scope of the related disclosure or claim to the specified materials and/or steps, plus those that do not materially affect the basic and novel characteristic(s) of the disclosed and/or claimed subject matter. For example, a pharmaceutical composition can “consist essentially of” a pharmaceutically active agent or a plurality of 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. It is noted, however, that carriers, excipients, and/or other inactive agents can and likely would be present in such a pharmaceutical composition and are encompassed within the nature of the phrase “consisting essentially of”.

As used herein, the phrase “consisting of” excludes any element, step, or ingredient not specifically recited. It is noted that, when the phrase “consists of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

With respect to the terms “comprising”, “consisting of”, and “consisting essentially of”, where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms. For example, a composition 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.

The term “aqueous solution” as used herein 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.

The term “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”, as used herein, refers to a molecule capable of binding to another molecule.

The term “biocompatible”, as used herein, refers to a material that does not elicit a substantial detrimental response in the host.

As used herein, the terms “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.

The term “biological sample”, as used herein, refers to samples obtained from a subject, to including but not limited to skin, hair, tissue, blood, plasma, cells, sweat, and urine.

A “coding region” of a gene comprises the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.

“Complementary” as used herein refers to the broad concept of subunit sequence complementarity between two nucleic acids (e.g., two DNA molecules). When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other at a given position, the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other when a substantial number (in some embodiments at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides that can base pair with each other (e.g., A:T and G:C nucleotide pairs). Thus, it is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. By way of example and not limitation, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, in some embodiments at least about 50%, in some embodiments at least about 75%, in some embodiments at least about 90%, and in some embodiments at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In some embodiments, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

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 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.

As used herein, the terms “condition”, “disease condition”, “disease”, “disease state”, and “disorder” refer to physiological states in which diseased cells or cells of interest can be targeted with the compositions of the presently disclosed subject matter. In some embodiments, a disease is leukemia, which in some embodiments is Acute Myeloid Leukemia (AML).

As used herein, the term “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 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.

In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

As used herein, 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. In the context of administering compounds in the form of a combination, such as multiple compounds, 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. Thus, 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 relative to the second treatment to which it is being compared.

“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of an mRNA corresponding to or derived from that gene produces the protein in a cell or other biological system and/or an in vitro or ex vivo system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence (with the exception of uracil bases presented in the latter) and is usually provided in Sequence Listing, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

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.

In some embodiments, the terms “fragment”, “segment”, or “subsequence” as used herein refers 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. Thus, in some embodiments, the terms “fragment”, “segment”, and “subsequence” are used interchangeably herein. In some embodiments, the term “fragment” refers to a compound (e.g., a small molecule compound, such as a small molecule comprising a purine scaffold) that can react with a reactive amino acid residue (e.g., a reactive cysteine) to form an adduct comprising a modified amino acid residue. Thus, in some embodiments, the terms “fragment” and “ligand” are used interchangeably. In some embodiments, the term “fragment” refers to that portion of a ligand that remains covalently attached to the reactive amino acid residue.

As used herein, a “ligand” is a compound (e.g., a purine-based compound) that specifically binds to a target compound or molecule, such as a reactive nucleophilic amino acid residue in a protein. In some embodiments, the ligand can bind to the target covalently. A ligand “specifically binds to” or “is specifically reactive with” a compound (e.g., a reactive amino acid residue) when the ligand functions in a binding reaction which is determinative of the presence of the compound in a sample of heterogeneous compounds.

As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property by which it can be characterized. A functional enzyme, for example, is one that exhibits the characteristic catalytic activity by which the enzyme can be characterized.

As used herein “injecting”, “applying”, and administering” 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, sublingual, vaginal, ophthalmic, pulmonary, vaginal, and rectal approaches.

As used herein, the term “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.

As used herein, the term “linker” refers to a molecule that joins two other molecules either 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 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. Such assays are coupled with processes or systems to store and process information and to help quantify levels, signals, etc. and to digitize the information for use in comparing levels.

The term “otherwise identical sample”, as used herein, refers to a sample similar to a first sample, that is, it is obtained in the same manner from the same subject from the same tissue or fluid, or it refers a similar sample obtained from a different subject. The term “otherwise identical sample from an unaffected subject” refers to a sample obtained from a subject not known to have the disease or disorder being examined. The sample may of course be a standard sample. By analogy, the term “otherwise identical” can also be used regarding regions or tissues in a subject or in an unaffected subject.

As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, 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 a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.

The term “pharmaceutical composition” refers to a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, without limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.

“Pharmaceutically acceptable” means physiologically tolerable, for either human or veterinary application. Similarly, “pharmaceutical compositions” include formulations for human and veterinary use.

As used herein, 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.

As used herein, the term “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.

“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.

As used herein, the term “mass spectrometry” (MS) refers to a technique for the identification 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,” 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. In some embodiments, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) can be used in this context. In some embodiments, intact protein molecules can be ionized by the above techniques, and then introduced to a mass analyzer. Alternatively, 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.

As used herein, the term “mass spectrometer” is used to refer an apparatus for performing mass 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 matter. For example, whole protein mass spectroscopy analysis can be conducted using time-of-flight (TOF) or Fourier transform ion cyclotron resonance (FT-ICR) instruments. For peptide mass analysis, MALDI time-of-flight instruments can be employed, as they permit the acquisition of peptide mass fingerprints (PMFs) at high pace. Multiple stage quadrupole-time-of-flight and the quadrupole ion trap instruments can also be used.

The terms “high throughput protein identification,” “proteomics” and other related terms are used herein to refer to the processes of identification of a large number or (in some cases, all) proteins in a certain protein complement. Post-translational protein modifications and quantitative information can also be assessed by such methods. One example of “high throughput protein identification” is a gel-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 tryptic peptides are extracted from the gel, further fractionated by liquid chromatography and analyzed by mass spectrometry. In another approach, a sample can be fractionated without using the gels, for example, by protein extraction followed by liquid chromatography. The proteins can then be digested in-solution, and the proteolytic fragments further fractionated by liquid chromatography and analyzed by mass spectrometry.

As used herein, the term “Western blot,” which can be also referred to as “immunoblot”, and related terms refer to an analytical technique used to detect specific proteins in a sample. The technique 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.

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 quantitative proteomics. SILAC comprises metabolic incorporation of a given “light” or “heavy” form of the amino acid into the proteins. For example, SILAC comprises the incorporation of amino acids with substituted stable isotopic nuclei (e.g. deuterium, ¹³C, ¹⁵N). In an illustrative 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, ¹²C and ¹³C labeled L-lysine, respectively). When 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.

The term “prevent”, as used herein, means to stop something from happening, or taking advance measures against something possible or probable from happening. In the context of medicine, “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 preventative treatment is administered for the purpose of decreasing the risk of developing pathology associated with developing the condition, disease, or disorder.

The term “protein” typically refers to large polypeptides. Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.

As used herein, the term “purified” and like terms relate to an enrichment of a molecule or 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 embodiments greater than 98% pure.

As used herein, 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 does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.

The term “subject” as used herein 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. Accordingly, the term “subject” is intended to encompass in some embodiments any member of the Kingdom Animalia including, but not limited to the phylum Chordata (e.g., members of Classes Osteichythyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Ayes (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. Thus, in some embodiments the presently disclosed subject matter concerns mammals and birds. More particularly provided are compositions and methods derived from 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, rats, and rabbits), marsupials, and horses. Also provided is the use of the disclosed methods and compositions on birds, including those kinds of birds that are endangered, kept in zoos, as well as fowl, and more particularly 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. Thus, also provided is the use of the disclosed methods and compositions on livestock, including but not limited to domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.

A “sample”, as used herein, 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. A sample can also be obtained from cell or tissue culture.

The term “standard”, as used herein, 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 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.

As used herein, 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. Typically, a compound is substantially 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 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.

The term “symptom”, as used herein, refers to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease. In contrast, a “sign” is objective evidence of disease. For example, 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 sufficient to provide a beneficial effect to the subject to which the compound is administered.

As used herein, 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.

The terms “treatment” and “treating” as used herein refer to both therapeutic treatment and 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 whom the condition is to be prevented.

As used herein, the terms “vector”, “cloning vector”, and “expression vector” refer to a vehicle by which a polynucleotide sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transduce and/or transform the host cell in order to promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc.

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 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.

As used herein the term “alkyl” refers to C₁-20 inclusive, linear (i.e., “straight-chain”), branched, 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 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. In some embodiments, the 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.

Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more alkyl group substituents, which can be the same or different. The term “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. There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), or aryl.

Thus, as used herein, the term “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, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

The term “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. The term “aryl” specifically encompasses heterocyclic aromatic compounds. The aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone, among others. In particular embodiments, the term “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.

The 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″, wherein R′ and R″ can each be independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl.

Thus, as used herein, the term “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, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

Specific examples of 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.

The term “heteroaryl” refers to aryl groups wherein at least one atom of the backbone of the aromatic ring or rings is an atom other than carbon. Thus, heteroaryl groups have one or more non-carbon atoms selected from the group including, but not limited to, nitrogen, oxygen, and sulfur.

As used herein, the term “acyl” refers to an organic carboxylic acid group wherein the —OH of the carboxyl group has been replaced with another substituent (i.e., as represented by RC(═O)—, wherein R is an alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl or substituted aryl group as defined herein). As such, the term “acyl” specifically includes arylacyl groups, such as an acetylfuran and a phenacyl group. Specific examples of 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 group substituent as defined herein, oxo, and/or alkylene. 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, camphane, and noradamantyl.

The terms “heterocycle”, “heterocyclyl” “heterocycloalkyl” or “heterocyclic” refer 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). Examples of heterocycles include, but are not limited to, tetrahydrofuran, tetrahydropyran, morpholine, dioxane, piperidine, piperazine, and pyrrolidine. Additional examples of 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 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.

Exemplary alkylene groups include methylene (—CH₂—); ethylene (—CH₂—CH₂—); propylene (—(CH₂)₃—); cyclohexylene (—C₆H₁₀—); —CH═CH—CH═CH—; —CH═CH—CH₂—; —(CH₂)_q—N(R)—(CH₂)_r—, wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (—O—CH₂—O—); and ethylenedioxyl (—O—(CH₂)₂—O—). An 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. The term “alkoxyl” as used herein can refer to, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, t-butoxyl, and pentoxyl. The term “oxyalkyl” can be used interchangably with “alkoxyl”.

The terms “aryloxy” and “aryloxyl” refer to an aryl-O-group, wherein aryl is as previously described. The term “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 include substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl. In some embodiments, 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 selected from the group including H and substituted and unsubstituted alkyl, cycloalkyl, heterocycle, aralkyl, aryl, and heteroaryl. In some embodiments, the amino group is —NH₂.

The terms “alkylamino” and “aminoalkyl” refer to a —NHR group where R is alkyl or substituted alkyl. The term “arylamino” refers to a —NHR group where R is aryl or substituted aryl.

The term “carbonyl” refers to the —(C═O)— or a double bonded oxygen substituent attached to a carbon atom of a previously named parent group.

The terms “carboxylate” and “carboxylic acid” can refer to the groups —C(═O)—O— and —C(═O)—OH, respectively. In some embodiments, “carboxylate” can refer to either the —C(═O)—O⁻ or —C(═O)—OH group. In some embodiments, the term “carboxyl” can also be used to refer to a carboxylate or carboxylic acid group.

The terms “sulfonyl”, “sulfone”, and “sulphone” as used herein refer to the —S(═O)₂— or —S(═O)₂R group, wherein R is alkyl, substituted alkyl, cycloalkyl, heterocycloalkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl.

The term “sulfonamide” refers to the —S(═O)₂—N(R)₂ group, wherein each R is independently selected from H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl, or wherein the two R together can for a ring with the nitrogen atom (e.g., wherein the two R are together an alkylene group, such as a butylene or pentylene group).

The term “sulfonate” as used herein refers to a —S(═O)₂—O—R group, wherein R is selected from alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl.

The terms “halo”, “halide”, or “halogen” as used herein refer to fluoro, chloro, bromo, and iodo groups.

The term “perhaloalkyl” refers to an alkyl group wherein all of the hydrogen atoms are replaced by halo. Thus, for example, 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, —CF₃.

The terms “hydroxyl” and “hydroxy” refer to the —OH group.

The term “oxo” refers to a compound described previously herein wherein a carbon atom is replaced by an oxygen atom.

The term “thio” refers to the —S— or —SH group.

The terms “alkylthio” and “thioalkyl” refer to a —SR group where R is alkyl or substituted alkyl. The term “arylthiol” refers to a —SR group where R is aryl or substituted aryl.

The term “cyano” refers to the —CN group.

The term “nitro” refers to the —NO₂ group.

A line crossed by a wavy line, e.g., in the structure:

indicates the site where the indicated substituent can bond to another group.

II. General Considerations

Covalent probes serve as invaluable tools for the global investigation of protein function and ligand binding capacity. While several probes have been deployed for the interrogation of nucleophilic residues such as cysteine (IA-Alkyne), lysine (NaTFBS-Alkyne), and methionine, a large fraction of the human proteome still remains inaccessible to pharmacological modulation.

Activity-based protein profiling (ABPP) utilizes active-site directed chemical probes to measure the functional state of large numbers of enzymes in native biological systems (e.g. cells or tissues). Activity-based probes consist of a reactive group for targeting a specific enzyme class and a reporter tag for detection by in-gel fluorescence scanning or by avidin-enrichment coupled with liquid chromatography mass spectrometry (LCMS), respectively. See FIG. 1A. For ligand (e.g., inhibitor) discovery, the potency and selectivity of small molecules can be profiled against many enzymes in parallel by performing competitive ABPP in complex proteomes, where ligands compete for probe labeling of enzyme targets.

Purines are essential components of DNA and RNA and have been fine-tuned by nature for biological activity. Purines have historically been explored as a scaffold for the development of inhibitors but their application in chemical biology as probes to discover new target proteins and druggable sites has been limited. In one aspect, the presently disclosed subject matter relates to purine-derived chemical probes and their use as chemoproteomic tools for activity-based profiling of the proteome (e.g., the human proteome).

The chemical structure of purine with the atoms numbered, is shown at the top of Scheme 1, above. Scheme 1 further shows structures of the four purine tautomers, the main two tautomers, i.e., 9H-purine and 7H-purine, and the two minor tautomers, i.e., 3H-purine and 1H-purine. Chemically, the reactions of purine reflect the interplay between the constituent pyrimidine and imidazole rings of the purine scaffold. The general structure of purine-based probes of the presently disclosed subject matter is shown in FIG. 1B. Electron localization within the nitrogen atoms of the pyrimidine ring can render the C2 and particularly the C6 site amenable to nucleophilic attack by protein residues. Thus, one aspect of the presently disclosed purine-based probes (and ligands) is the addition of an electrophilic “warhead” (“E” in the structure of FIG. 1B) on the pyrimidine ring that can serve as an effective leaving group during nucleophilic attack by a nucleophilic group on a side chain of a protein residue. Meanwhile, the more electron rich imidazole ring can provide for facile derivatization, e.g., to attach detectable tags or taggable groups. Alternatively, the electron rich imidazole ring can serve for derivatization of the purine scaffold to provide a wide variety of covalent protein modulators (e.g., inhibitors or activators), also referred to herein as “ligands”. FIG. 1C shows the mechanism whereby a covalent enzyme/probe adduct is formed when the probe is contacted with an enzyme having a reactive nucleophilic residue. After formation (e.g., in a cell lysate, a live cell, a tissue, a living organism, or another sample comprising one or more proteins), the covalent enzyme/probe adduct can be analyzed in-gel and/or by LC-MS/MS. The presently disclosed probes and related ligands/modulators can be used for target protein discovery, competitive ABPP, and inverse drug discovery.

In some embodiments, the presently disclosed subject matter provides small molecule probes that interact with reactive nucleophilic residues on proteins or peptides, such as a reactive cysteine residue of a cysteine-containing protein, as well as methods of identifying a protein or peptide that contains such a reactive residue (e.g., a druggable cysteine residue). In some instances, also described herein, are methods of profiling a small molecule purine-based ligand that interacts with one or more cysteine-containing protein comprising one or more reactive cysteine.

In some embodiments, the presently disclosed subject matter provides a method for identifying a reactive amino acid residue of a protein. In some embodiments, the method comprises: (a) providing a protein sample; (b) contacting the protein sample with a purine-based probe compound (e.g., a halo-substituted purine-based probe compound) for a period of time sufficient for the probe compound to react with at least one reactive amino acid in the protein sample, thereby forming at least one modified amino acid residue; and (c) analyzing proteins in or from the protein sample to identify the at least one modified amino acid residue, thereby identifying at least one reactive amino acid residue of a protein. In some embodiments, the protein sample comprises isolated proteins, living cells, a cell lysate or a biological organism (e.g., a mammal or other animal, a plant, a bacteria, etc.). In some embodiments, the probe compound has a structure of Formula (I):

wherein: X is a monovalent moiety comprising an alkyne moiety, a fluorophore moiety, a detectable labeling group, or a combination thereof; and R₁ and R₂ are independently selected from the group comprising H, halo, amino, alkyl (e.g., C₁-C₆ alkyl), alkoxy (e.g., C₁-C₆ alkoxy), alkylthio (e.g., C₁-C₆ alkylthio), alkylamino (e.g., C₁-C₆ alkylamino), aryloxy, arylthiol, and arylamino, subject to the proviso that at least one of R₁ and R₂ is halo. In some embodiments, R₁ and R₂ are independently selected from H, halo and amino, subject to the proviso that at least one of R₁ and R₂ is halo. In some embodiments, at least one of R₁ and R₂ is chloro or fluoro.

In some embodiments, X is attached at one of the nitrogen atoms of the imidazole ring (i.e., N9 or N7). Thus, in some embodiments, the probe compound of Formula (I) has a structure of Formula (Ia):

or a structure of Formula (Ib):

wherein X is a monovalent moiety comprising an alkyne moiety, a fluorophore moiety, a detectable labeling group, or a combination thereof; and R₁ and R₂ are independently selected from the group comprising H, halo, amino, alkyl, alkoxy, alkylthio, alkylamino, aryloxy, arylthiol, and arylamino, subject to the proviso that at least one of R₁ and R₂ is halo. In some embodiments, R₁ and R₂ are independently selected from the group consisting of H, halo, and amino, subject to the proviso that at least one of R₁ and R₂ is halo.

In some embodiments, the reactive amino acid residue is selected from the group comprising cysteine, lysine, glutamic acid, arginine, aspartic acid, glutamine, tyrosine, histidine, asparagine, methionine, threonine, tryptophan, and serine. In some embodiments, the reactive amino acid residue is selected from cysteine, lysine, glutamic acid, arginine, and aspartic acid. In some embodiments, the reactive amino acid residue is selected from cysteine, aspartic acid, glutamic acid, tyrosine, lysine, and glutamine. In some embodiments, the reactive amino acid residue is cysteine.

In some embodiments, the reactive amino acid residue is cysteine and the modified amino acid residue has a structure of Formula (IIa-i):

a structure of Formula (IIb-i):

a structure of Formula (IIa-ii):

or a structure of Formula (IIb-ii):

wherein X is a monovalent moiety comprising an alkyne moiety, a fluorophore moiety, a detectable labeling group, or a combination thereof; R₁ is selected from the group comprising H, halo, amino, alkyl, alkyoxy, alkylthio, alkylamino, aryloxy, arylthio, and arylamino; and R₂ is selected from the group comprising H, halo, amino, alkyl, alkoxy, alkylthio, alkylamino, aryloxy, arylthiol, and arylamino. In some embodiments, R₁ is H, halo or amino. In some embodiments, R₂ is H, halo or amino. In some embodiments, the modified amino acid residue has a structure of Formula (IIa-i) or Formula (IIb-i). In some embodiments, the modified amino acid residue has a structure of Formula (IIb-i).

In some embodiments, the compound of Formula (I), (Ia), or (Ib) is a compound where R₁ is halo. In some embodiments, R₁ is chloro or fluoro. In some embodiments, R₁ is chloro.

In some embodiments, the compound of Formula (I), (Ia), or (Ib) is a compound where R₂ is halo. In some embodiments, R₂ is chloro or fluoro. In some embodiments, R₂ is chloro.

In some embodiments, both of R₁ and R₂ are halo. In some embodiments, R₁ and R₂ are each chloro. In some embodiments, R₁ and R₂ are each fluoro. In some embodiments, R₁ is chloro and R₂ is fluoro.

In some embodiments, X comprises a fluorophore or a detectable labeling group such as described hereinbelow. In some embodiments, X is a monovalent moiety comprising an alkyne group (i.e., a carbon-carbon triple bond). For example, in some embodiments, X comprises or consists of —C≡CH, -alkylene-C≡CH, —C(═O)-alkylene-C≡CH, or —C(═O)—NH-alkylene-C≡CH (e.g., C(═O)—NH—CH₂—C≡CH). In some embodiments, the alkylene group is a C₁-C₅ alkylene group. In some embodiments, the alkylene group is methylene. In some embodiments, X is a propargyl group, i.e., —CH₂—C≡CH.

In some embodiments, the probe compound is selected from the group comprising 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine (also referred to herein as AHL125, AHL-Pu-1, or Pu-1), 2,6-dichloro-9-(prop-2-yn-1-yl)-9H-purine (also referred to herein as AHL128, AHL-Pu-2, or Pu-2), 6-chloro-7-(prop-2-yn-1-yl)-7H-purine (also referred to herein as AHL-Pu-3 or Pu-3), 6-chloro-9-(prop-2-yn-1-yl)-9H-purine (also referred to herein as AHL-Pu-4 or Pu-4), 2-chloro-7-(prop-2-yn-1-yl)-7H-purine (also referred to herein as AHL-Pu-5 or Pu-5), 2-chloro-9-(prop-2-yn-1-yl)-9H-purine (also referred to herein as AHL-Pu-6 or Pu-6), 2,6-difluoro-7-(prop-2-yn-1-yl)-7H-purine (also referred to herein as AHL-Pu-11 or Pu-11), 2,6,-difluoro-9-(prop-2-yn-1-yl)-9H-purine (also referred to herein as AHL-Pu-12 or Pu-12), 6-chloro-2-fluoro-7-(prop-2-yn-1-yl)-7H-purine (also referred to herein as AHL-Pu-9 or Pu-9), 6-chloro-2-fluoro-9-(prop-2-yn-1-yl)-9H-purine (also referred to herein as AHL-Pu-10 or Pu-10), 6-chloro-2-amino-7-(prop-2-yn-1-yl)-7H-purine (also referred to herein as AHL-Pu-7 or Pu-7), and 6-chloro-2-amino-9-(prop-2-yn-1-yl)-9H-purine (also referred to herein as AHL-Pu-8 or Pu-8). In some embodiments, the probe is selected from Pu-1, Pu-2, Pu-9, and Pu-10.

In some embodiments, the N7-substituted probe is more reactive. Thus, in some embodiments, the probe compound has a structure of Formula (Ib), as shown above. In some embodiments, the purity of the probe compound having a structure of Formula (Ib) is about 90% or more (e.g., about 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% or more), e.g., by HPLC. Thus, the N7-substituted probe can be provided substantially as a single regioisomer. In some embodiments, the probe compound is 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine, 6-chloro-2-fluoro(7-prop-2-yn-1-yl)-7H-purine or 2,6-difluoro-7-(prop-2-yn-1-yl)-7H-purine. In some embodiments, the probe compound is 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine or 6-chloro-2-fluoro(7-prop-2-yn-1-yl)-7H-purine. In some embodiments, the probe compound is 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine.

In some embodiments, one of R₁ and R₂ is halo (e.g., chloro or fluoro) and the other of R₁ and R₂ is alkoxy, alkylthio or alkylamino. In some embodiments, one of R₁ and R₂ is alkylthio and the other of R₁ and R₂ is chloro or fluoro. Thus, in some embodiments, the probe compound is a “adduct” compound (i.e., a probe compound where one of R₁ and R₂ is replaced by a group from a small molecule amino acid mimetic), such as shown in Scheme 8, below in Example 4. In some embodiments, the compound is selected from the group comprising 6-(butylthio)-2-chloro-7-(prop-2-yn-1-yl)-7H-purine (Pa-1), 2-(butylthio)-6-chloro-7-(prop-2-yn-1-yl)-7H-purine (Pa-2), 2-(butylthio)-6-chloro-9-(prop-2-yn-1-yl)-9H-purine (Pa-3), 6-(butylthio)-2-chloro-9-(prop-2-yn-1-yl)-9H-purine (Pa-4), 6-(butylthio)-2-fluoro-7-(prop-2-yn-1-yl)-7H-purine (Pa-5), and 6-(butylthio)-2-fluoro-9-(prop-2-yn-1-yl)-9H-purine (Pa-6). In some embodiments, the compound is Pa-3, Pa-4, or Pa-6.

In some embodiments, e.g., when X comprises an alkyne group, the analyzing of step (c) further comprises tagging the at least one modified reactive amino acid (e.g., cysteine) residue with a compound comprising detectable labeling group, thereby forming at least one tagged reactive amino acid (e.g., cysteine) residue comprising said detectable labeling group. In some embodiments, the detectable labeling group comprises biotin or a biotin derivative. In some embodiments, the biotin derivative is desthiobiotin.

In some embodiments, the tagging comprises reacting an alkyne group in a X moiety of at least one modified reactive amino acid (e.g., cysteine) residue with a compound comprising both an azide moiety (or other alkyne-reactive group) and a detectable labeling group (e.g., biotin or a biotin derivative). In some embodiments, 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—C₂H₄—)_x—). In some embodiments, the tagging comprises performing a copper-catalyzed azide-alkyne cycloaddition (CuAAC) coupling reaction.

In some embodiments, 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 (e.g., cysteine residue) moiety comprising the detectable group. In some embodiments, the digesting is performed with a peptidase. In some embodiments, the digesting is performed with trypsin.

In some embodiments, the analyzing further comprises enriching the digested protein sample for the detectable labeling group. For example, in some embodiments, the enriching comprises contacting the digested protein sample with a solid support comprising a binding partner of the detectable labeling group. In some embodiments, when the detectable labeling group comprises biotin or a derivative thereof, the solid support comprises streptavidin. In some embodiments, the analyzing 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.

In some embodiments, the protein sample is a biological organism and the presently disclosed method can be used to detect reactive amino acid residues of proteins in vivo. When the protein sample is a biological organism (i.e., a living biological organism), such as an animal, contacting the protein sample with the probe compound of Formula (I) comprises administering the probe compound of Formula (I) to the biological organism via a suitable route of administration. The administration can be systemic or localized (e.g., to a site of disease, such as a tumor). In some embodiments, the administration is oral administration or injection, e.g., i.v. or i.p. injection. In some embodiments, prior to analyzing the proteins, a tissue sample is removed from the biological organism and homogenized. Alternatively, a biological fluid sample (e.g., blood or saliva) can be collected and the proteins therein can be analyzed for detection of a modified amino acid residue.

In some embodiments, providing the protein sample further comprises separating the protein sample (e.g., a cell or cell lysate sample) into a first protein sample and a second protein sample. Then, in the contacting step, the first protein sample can be contacted with a first probe compound of Formula (I) at a first probe concentration for a first period of time and the second protein sample can be contacted with a second probe compound of Formula (I) (i.e., a probe compound of Formula (I) having a different structure than that of the first probe compound of Formula (I)) 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. Alternatively, 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. In some embodiments, analyzing proteins comprises analyzing the first and second protein samples to determine the presence and/or identity of a modified reactive amino acid residue (e.g., a modified reactive cysteine residue) in the first sample and the presence and/or identity of a modified reactive amino acid residue (e.g., a modified reactive cysteine residue) in the second sample. In some embodiments, the identities and/or amounts of identified modified reactive amino acid residues (e.g., the modified reactive cysteine residues) from the first and second protein samples are compared.

In some embodiments, the protein sample comprises living cells. In some embodiments, 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 isotope distribution prior to the contacting of step (b). In some embodiments, the first cell culture medium comprises ¹³C- and/or ¹⁵N-labeled amino acids. In some embodiments, the first cell culture medium comprises ¹³C-,¹⁵N-labeled lysine and arginine.

In some embodiments, e.g., if the protein sample does not comprise living cells, the probe compound of Formula (I) can comprise a detectable labeling group comprising a heavy isotope (e.g., a ¹³C label) or the method can comprise tagging the at least one modified amino acid residue with a detectable labeling group comprising a heavy isotope.

In some embodiments, 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 presence of a compound or biomolecule that interacts with a protein present in or suspected of being present in the protein sample. In some embodiments, the compound or biomolecule that interacts with a protein present in or suspected of being present in the protein sample is an inhibitor or activator of an enzyme present in or suspected of being present in the protein sample. In some embodiments, one of the first and the second protein sample can be cultured in the presence of a ligand of the presently disclosed subject matter.

III. Probes

In some embodiments, the presently disclosed subject matter provides a purine-based probe compound that comprises an electrophilic moiety (e.g., attached to a carbon on the pyrimidine ring of a purine scaffold) that can be displaced by a nucleophilic group in a side chain of an amino acid residue of a protein. The purine-based probe can also comprise a detectable group or a group (e.g., an alkyne group) that can be derivatized with a detectable group (e.g., a fluorophore or an antigen). In some embodiments, the purine-based probe reacts with a cysteine residue or other nucleophilic amino acid residue to form a covalent bond (e.g., a thio ether). Typically, the probe is a non-naturally occurring molecule, or forms a non-naturally occurring product (i.e., a “modified” protein) after reaction with the nucleophilic amino acid residue.

In some embodiments, the purine-based probe compound is a compound of one of Formulas (I), (Ia) or (Ib). Thus, in some embodiments, the probe compound has a structure of Formula (I):

wherein X is a monovalent moiety comprising an alkyne moiety, a fluorophore moiety, a detectable labeling group, or a combination thereof; and R₁ and R₂ are independently selected from the group comprising H, halo, amino, alkyl (e.g., C₁-C₆ alkyl) alkoxy (e.g., C₁-C₆ alkoxy), alkylthio (e.g., C₁-C₆ alkylthio), alkylamino (e.g., C₁-C₆ alkylamino), aryloxy, arylthiol, and arylamino, subject to the proviso that at least one of R₁ and R₂ is halo. In some embodiments, R₁ and R₂ are independently selected from H, halo and amino, subject to the proviso that at least one of R₁ and R₂ is halo. In some embodiments, at least one of R₁ and R₂ is chloro or fluoro.

In some embodiments, X comprises a fluorophore or a detectable labeling group. The fluorophore of X can be any suitable fluorophore. In some embodiments, the fluorophore is selected from the group including, but not limited to, rhodamine, rhodol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, aminorhodol, carboxyrhodol, chlororhodol, methylrhodol, sulforhodol; aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, thiorhodamine, cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7, oxadiazole derivatives, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, pyren derivatives, cascade blue, oxazine derivatives, Nile red, Nile blue, cresyl violet, oxazine 170, acridine derivatives, proflavin, acridine orange, acridine yellow, arylmethine derivatives, auramine, crystal violet, malachite green, tetrapyrrole derivatives, porphin, phtalocyanine, bilirubin 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-p-touidinyl-6-naphthalene sulfonate, 3-phenyl-7-isocyanatocoumarin, N-(p-(2-benzoxazolyl)phenyl)maleimide, stilbenes, pyrenes, 6-FAM (Fluorescein), 6-FAM (NHS Ester), 5(6)-FAM, 5-FAM, Fluorescein dT, 5-TAMRA-cadavarine, 2-aminoacridone, HEX, JOE (NHS Ester), MAX, TET, ROX, and TAMRA.

In some embodiments, X comprises a fluorophore moiety. In some cases, the fluorophore of X is obtained from a compound library. In some cases, the compound library comprises ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library, FRGx from AnalytiCon, TCI-Frag from AnCoreX, Bio Building Blocks from ASINEX, BioFocus 3D from Charles River, Fragments of Life (FOL) from Emerald Bio, Enamine Fragment Library, IOTA Diverse 1500, BIONET fragments library, Life Chemicals Fragments Collection, OTAVA fragment library, Prestwick fragment library, Selcia fragment library, TimTec fragment-based library, Allium from Vitas-M Laboratory, or Zenobia fragment library.

In some embodiments, the detectable labeling group is selected from the group comprising a member of a specific binding pair (e.g., biotin:streptavidin, antigen-antibody, nucleic acid:nucleic acid), a bead, a resin, a solid support, or a combination thereof. In some embodiments, the detectable labeling group is a biotin moiety, a streptavidin moiety, bead, resin, a solid support, or a combination thereof. In some embodiments, the detectable labeling group comprises biotin or a derivative thereof (e.g., desthiobiotin). In some embodiments, the detectable labeling group comprises a heavy isotope (i.e., ¹³C).

In some embodiments, X is a monovalent moiety comprising an alkyne group (i.e., a carbon-carbon triple bond). For example, in some embodiments, X comprises or consists of —C≡CH, -alkylene-C≡CH, —C(═O)-alkylene-C≡CH, or —C(═O)—NH-alkylene-C≡CH (e.g., C(═O)—NH—CH₂—C≡CH). In some embodiments, the alkylene group is a C₁-C₅ alkylene group. In some embodiments, the alkylene group is methylene. In some embodiments, X is a propargyl group, i.e., —CH₂—C≡CH.

In some embodiments, one of R₁ and R₂ is halo (e.g., chloro or fluoro) and the other of R₁ and R₂ is alkoxy, alkylthio or alkylamino. In some embodiments, one of R₁ and R₂ is alkylthio and the other of R₁ and R₂ is chloro or fluoro. In some embodiments, the compound is a compound shown in Scheme 8, below in Example 4, i.e., 6-(butylthio)-2-chloro-7-(prop-2-yn-1-yl)-7H-purine (Pa-1), 2-(butylthio)-6-chloro-7-(prop-2-yn-1-yl)-7H-purine (Pa-2), 2-(butylthio)-6-chloro-9-(prop-2-yn-1-yl)-9H-purine (Pa-3), 6-(butylthio)-2-chloro-9-(prop-2-yn-1-yl)-9H-purine (Pa-4), 6-(butylthio)-2-fluoro-7-(prop-2-yn-1-yl)-7H-purine (Pa-5), or 6-(butylthio)-2-fluoro-9-(prop-2-yn-1-yl)-9H-purine (Pa-6). In some embodiments, the compound is Pa-2, Pa-4, or Pa-6.

In some embodiments, the probe is selected from the group comprising 2,6-difluoro-7-(prop-2-yn-1-yl)-7H-purine, 2,6-difluoro-9-(prop-2-yn-1-yl)-9H-purine, and 6-chloro-2-fluoro-7-(prop-2-yn-1-yl)-7H-purine.

In some embodiments, the compound of Formula (III) is not one of the compounds selected from the group comprising 7-allyl-2,6-dichloro-7H-purine, 9-allyl-2,6-dichloro-9H-purine, 2,6-dichloro-7-benzyl-7H-purine, 2,6-dichloro-9-benzyl-9H-purine, 2,6-dichloro-9-(4-nitrobenzyl-9H-purine, and 2-(2,6-dichloro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol.

In some embodiments, the N7-substituted regioisomer of the probe (i.e., the compound of Formula (Ib)) is provided with a purity of at least about 90% or more (e.g., about 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% or more).

In some embodiments, e.g., as the presently disclosed probe compounds can be used to detect reactive amino acid residues in proteins in biological organisms, such as animals, the probe compound can be provided as a pharmaceutically acceptable salt or in a pharmaceutically acceptable carrier or formulation, such as a pharmaceutically acceptable carrier or formulation.

IV. Ligands

Small molecules can serve as versatile ligands for perturbing the functions of proteins in biological systems. In some instances, a plurality of human proteins lack selective chemical ligands. In some cases, several classes of proteins are further considered as undruggable. Covalent purine-based ligands (also referred to herein as “fragments”) offer a strategy to expand the landscape of proteins amenable to targeting by small molecules. In some instances, covalent ligands combine features of recognition and reactivity, thereby providing for the targeting of sites on proteins that are difficult to address by reversible binding interactions alone.

In some embodiments, a ligand of the presently disclosed subject matter can compete with a probe compound described herein for binding with a reactive amino acid residue (e.g., a reactive cysteine residue). Often, the presently disclosed ligands are non-naturally occurring, and/or form non-naturally occurring products (modified proteins) after reaction with the nucleophilic group (e.g., the thiol group) of an amino acid residue (e.g., a cysteine residue).

In some embodiments, the ligand can modify one or more activity of the protein. For example, covalent attachment of a ligand to an enzyme can inhibit or activate an enzyme. In some embodiments, covalent attachment of a ligand to a protein can disrupt one or more protein-protein interactions of the modified protein. In some embodiments, covalent attachment of a ligand can disrupt protein-RNA interactions of the modified protein. In some embodiments, covalent attachment of a ligand can disrupt protein-DNA interactions of the modified protein. In some embodiments, covalent attachment of a ligand can disrupt protein-lipid interactions of the modified protein. In some embodiments, covalent attachment of a ligand can disrupt protein-metabolite interactions of the modified protein. In some embodiments, covalent attachment of a ligand can disrupt subcellular localization of the modified protein. In some embodiments, covalent attachment of a ligand can recruit an E3 ligase for targeted degradation of the modified protein. For instance, without being bount to any one theory, it is believed that covalent modification of a target protein with the probe can result in a protein-purine adduct that can be recognized by an E3 ligase, leading to binding, attachment of a polyubiquitin signal, and degradation of the target protein by the ubiquitin-proteosome system.

In some embodiments, the presently disclosed subject matter provides a purine-based compound that can form a covalent bond with a nucleophilic group of a side chain of a reactive amino acid residue (e.g., a reactive cysteine residue). In some embodiments, the presently disclosed subject matter provides a compound having a structure of Formula (III):

wherein Z is selected from the group comprising alkyl (e.g., C₁-C₆ alkyl), substituted alkyl, cycloalkyl (e.g., C₃-C₆ cycloalkyl), acyl (e.g., C₂-C₂₄ acyl or C₂-C₁₂ acyl), substituted acyl, aralkyl (e.g., benzyl, ethylphenyl, methylnaphthyl), substituted aralkyl (e.g., substituted benzyl), sulfonyl (i.e., —S(═O)₂—R₅), sulfonamide (i.e., —S(═O)₂—N(R₆)₂), and sulfonate (i.e., —S(═O)₂—O—R₇); R₃ and R₄ are independently selected from H, halo, alkyl (e.g., C₁-C₆ alkyl), alkoxy (e.g., C₁-C₆ alkoxy), alkylamino (e.g., C₁-C₆ alkylamino), alkylthio (C₁-C₆ alkylthio), aryloxy, arylamino, and arylthiol, subject to the proviso that at least one of R₃ and R₄ is halo, optionally chloro or fluoro; R₅ is heterocyclyl, substituted heterocyclyl, aryl or substituted aryl; each R₆ is selected from H, alkyl (e.g., C₁-C₆ alkyl), substituted alkyl (e.g., substituted C₁-C₆ alkyl), aralkyl, substituted aralkyl, aryl, and substituted aryl, or wherein the two R₆ together form an alkylene group (e.g., butylene or pentylene); and R₇ is selected from alkyl (e.g., C₁-C₆ alkyl), substituted alkyl, aralkyl, substituted aralkyl, aryl and substituted aryl. In some embodiments, Z is selected from the group comprising cycloalkyl, acyl, —S(═O)₂—R₅, —S(═O)₂—N(R₆)₂, —S(═O)₂—O—R₇, and

In some embodiments, the compound of Formula (III) has a structure of Formula (IIIa):

or a structure of Formula (IIIb):

wherein the variables Z, R₃, R₄, R₅, R₆, and R₇ are as defined for the compound of Formula (III).

In some embodiments, R₃ is selected from chloro, fluoro, C₁-C₆ alkyl (e.g., methyl, ethyl, propyl, isopropyl, allyl, m-butyl, tert-butyl, pentyl, or hexyl), alkylthio, alkylamino, or aryloxy, optionally wherein the aryl group of the aryl oxy is substituted by one or more aryl group substituents (e.g., alkyl). In some embodiments, R₃ is selected from chloro, methyl, —SH—(CH₂)₃CH₃; —NH(CH₂)₃CH₃; and —O—(C₆H₄)CH₃.

In some embodiments, R₄ is chloro or fluoro.

In some embodiments, Z is selected from the group comprising C₂-C₁₂ acyl (e.g., acetyl, n-hexanoyl, or n-dodecanoyl); cycloalkyl (e.g., cyclohexyl), —S(═O)₂—R₅, —S(═O)₂—N(R₆)₂, —S(═O)₂—O—R₇, and

In some embodiments, Z is —S(═O)₂—R₅, wherein R₅ is heterocyclyl (e.g., morpholine) or substituted phenyl. In some embodiments, the substituted phenyl is an alkoxy- or halo-substituted phenyl (e.g., 4-methoxyphenyl or 4-fluorophenyl).

In some embodiments, Z is —S(═O)₂—N(R₆)₂, wherein each R₆ is selected from alkyl and aralkyl. In some embodiments, at least one R₆ is alkyl, e.g., methyl, ethyl, propyl, butyl, pentyl or hexyl. In some embodiments, both R₆ are alkyl. In some embodiments, both R₆ are ethyl. In some embodiments, one R₆ is aralkyl, e.g., benzyl.

In some embodiments, Z is —S(═O)₂—O—R₇, wherein R₇ is alkyl or aralkyl. In some embodiments, R₇ is alkyl, e.g., methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some embodiments, R₇ is methyl. In some embodiments, R₇ is benzyl.

In some embodiments, Z is selected from the group comprising:

In some embodiments, the compound of Formula (III) is selected from the group comprising: 4-((2,6-dichloro-7H-purin-7-yl)sulfonyl)morpholine (Pi-7), 4-((2,6-dichloro-9H-purin-9-yl)sulfonyl)morpholine (Pi-8), 2,6-dichloro-7-((4-fluorophenyl)sulfonyl)-7H-purine (Pi-13), 2,6-dichloro-9-((4-fluorophenyl)sulfonyl)-9H-purine (Pi-14), 2,6-dichloro-7-((4-methoxyphenyl)sulfonyl)-7H-purine (Pi-15), 2,6-dichloro-9-((4-methoxyphenyl)sulfonyl)-9H-purine (Pi-16), 2,6-dichloro-7-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl)-7H-purine (Pi-11), 2,6-dichloro-9-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl)-9H-purine (Pi-12), 1-(2,6-dichloro-9H-purin-9-yl)dodecan-1-one, 1-(2,6-dichloro-7H-purin-7-yl)hexan-1-one, 1-(2,6-dichloro-9H-purin-9-yl)hexan-1-one, 1-(6-chloro-2-fluoro-7H-purin-7-yl)hexan-1-one, 1-(6-chloro-2-fluoro-9H-purin-9-yl)hexan-1-one, 1-(2-chloro-6-methyl-9H-purin-9-yl)hexan-1-one, 2-chloro-9-cyclohexyl-6-methyl-9H-purine, 9-cyclohexyl-2-fluoro-6-methyl-9H-purine, 1-(6-(butylthio)-2-fluoro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylthio)-2-chloro-9H-purin-9-yl)ethan-1-one (AHL20-001), 1-(6-(butylthio)-2-chloro-7H-purin-7-yl)ethan-1-one, 1-(6-(butylthio)-2-fluoro-7H-purin-7-yl)ethan-1-one, 1-(2-chloro-6-(p-tolyloxy)-9H-purin-9-yl)ethan-1-one, 1-(2-fluoro-6-(p-tolyloxy)-9H-purin-9-yl)ethan-1-one, 1-(2-chloro-6-(p-tolyloxy)-7H-purin-7-yl)ethan-1-one, 1-(2-fluoro-6-(p-tolyloxy)-7H-purin-7-yl)ethan-1-one, 1-(6-(butylamino)-2-chloro-7H-purin-7-yl)ethan-1-one, 1-(6-(butylamino)-2-fluoro-7H-purin-7-yl)ethan-1-one, 1-(6-(butylamino)-2-chloro-9H-purin-9-yl)ethan-1-one, and 1-(6-(butylamino)-2-fluoro-9H-purin-9-yl)ethan-1-one.

In some embodiments, the compound is selected from the group comprising 2,6-dichloro-N,N-diethyl-7H-purine-7-sulfonamide, 2,6-dichloro-N,N-diethyl-9H-purine-9-sulfonamide, N-benzyl-2,6-dichloro-N-methyl-9H-purine-9-sulfonamide, N-benzyl-2,6-dichloro-N-methyl-7H-purine-7-sulfonamide, benzyl 2,6-dichloro-7H-purine-7-sulfonate, benzyl 2,6-dichloro-9H-purine-9-sulfonate, methyl 2,6-dichloro-9H-dichloro-9-purine-9-sulfonate, and methyl 2,6-dichloro-7H-purine-7-sulfonate.

In some embodiments, the compound is 2,6-dichloro-7-(4-nitrobenzyl)-7H-purine.

In some embodiments, the compound is a N7-substituted regioisomer and has a purity of at least about 90% or more (e.g., about 90, 91, 92, 93, 94, 95, 96, 97, 98 or about 99% or more).

The presently disclosed subject matter encompasses the preparation and use of pharmaceutical compositions comprising a ligand compound as described herein useful for treatment of diseases and disorders as would be apparent upon review of the instant disclosure as an active ingredient. Such a pharmaceutical composition can comprise, consist essentially of, or consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition can comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient can be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

As used herein, the term “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.

The compositions of the presently disclosed subject matter can comprise at least one active ingredient, one or more acceptable carriers, and optionally other active ingredients or therapeutic agents.

Pharmaceutically acceptable carriers include physiologically tolerable or acceptable diluents, excipients, solvents, or adjuvants. The compositions are in some embodiments sterile and nonpyrogenic. Examples of 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 and sorbitan esters, microcrystalline cellulose, aluminum methahydroxide, bentonite, kaolin, agar-agar and tragacanth, or mixtures of these substances, and the like.

The pharmaceutical compositions can 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). 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 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). If desired, absorption enhancing or delaying agents (such as liposomes, aluminum monostearate, or gelatin) can be used. The compositions can be prepared in conventional forms, either as liquid solutions 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 or pharmaceutical compositions comprising these compositions can be administered so that the compositions may 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 an approach. Particular parenteral administration methods include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature), peri- and intra-target tissue injection, subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps), intramuscular injection, and direct application to the target area, e.g., intratumoral injection, for example by a catheter or other placement device.

Where the administration of the composition is by injection or direct application, the injection or direct application can be in a single dose or in multiple doses. Where the administration of the compound is by infusion, the infusion can be a single sustained dose over a prolonged period of time or multiple infusions.

The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such 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 desired single- or multi-dose unit.

It will be understood by the skilled artisan that 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 and/or socially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs, birds including commercially and/or socially relevant birds such as chickens, ducks, geese, parrots, and turkeys.

A pharmaceutical composition of the presently disclosed subject matter can be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount 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 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. By way of example, the composition can comprise between 0.1% and 100% (w/w) active ingredient.

In addition to the active ingredient, a pharmaceutical composition of the presently disclosed subject matter can further comprise one or more additional pharmaceutically active agents.

Controlled- or sustained-release formulations of a pharmaceutical composition of the presently disclosed subject matter can be made using conventional technology.

As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring 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 hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the presently disclosed subject matter are known in the art and described, for example in Gennaro (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Pub. Co., Easton, Pa., United States of America and/or Gennaro (ed.) (2003) Remington: The Science and Practice of Pharmacy, 20th edition Lippincott, Williams & Wilkins, Philadelphia, Pa., United States of America, each of which is incorporated herein by reference.

The compositions 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, 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.

Other approaches include but are not limited to nanosizing the composition comprising a ligand compound as described herein to be delivered as a nanoparticle intravenously, intraperitoneal injection, or implanted beads with time release of a ligand compound as described herein.

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 compositions encapsulated in liposomes. The active 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. In addition, if desired, the 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 cell or a tissue of a subject. In some embodiments, 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 and/or a device suitable for administering the composition such as a syringe, injector, or the like or other device as would be apparent to one of ordinary skill in the art upon a review of the instant disclosure.

As used herein, 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 the composition of the presently disclosed subject matter in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, 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 disclosed subject matter can, 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. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the composition be used cooperatively by the recipient.

V. Synthesis

The probes and ligands of the presently disclosed subject matter can be prepared using organic group transformations known in the art of organic synthesis and as further described in the Examples below.

In some embodiments, the presently disclosed purine-based probes and ligands can be prepared by contacting a halo- or di-halo purine with a reagent that can react with one of the amines of the imidazole ring. For example, the halo- or di-halo-substituted purine-based probe or ligand can be prepared by contacting a halo- or di-halo-substituted purine with a halide (e.g., propargyl bromide or another alkyl halide, a benzyl bromide or another aralkyl halide, etc) in the presence of a base (e.g., potassium carbonate or sodium carbonate). See FIG. 2A. The reactions can be performed in a suitable solvent, e.g., an aprotic organic solvent, such as dimethylformamide (DMF) or tetrahydrofuran (THF). These reactions can result in two different regioisomers, the N9 isomer and the more sterically hindered N7 isomer. Typically, the N9 isomer is the major product. See FIG. 2B. However, reports in the literature have described that N-alkylation can occur at the more sterically hindered nitrogen atom of various 1,3-azoles if an organomagnesium reagent is used as a base.¹⁷ Thus, if desired, the N7 isomer of the presently disclosed probes and ligands can be made as the major isomer by addition of, for example, three equivalents of methyl magnesium chloride or another organomagnesium reagent.

Adducts of the halo or dihalo purine probes or ligands, e.g., where the 6-halo substituent is replaced by an alkoxy, aryloxy, alkylthio, arylthiol, alkylamino or arylamino group can be prepared by reacting the halo- or di-halo purines with a thiol, amine, alcohol or phenol in the presence of a hindered/non-nucleophilic base, such as Hunig's base (i.e., N,N-diisopropylethylamine) or triethylamine.

Acylated purine ligands can be prepared by contacting a halo-substituted purine or an alkoxy, alkylthio, alkylamino, aryloxy, arylthiol, or arylamino adduct thereof with an anhydride or acid chloride. Sulfonated purine ligands can be prepared by contacting a halo-substituted purine or an alkoxy, alkylthio, alkylamino, aryloxy, arylthiol, or arylamino adduct thereof with a suitable activated sulfonyl compound, such as a sulfonyl chloride.

Scheme 2, below, shows the compounds prepared according to the methods described above using the following commercially reagents used without further purification: benzyl bromide, allyl bromide, 4-nitrophenyl bromide, 6-(Bromomethyl)-1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene, morpholine-4-sulfonyl chloride, 4-methoxyphenylsulfonyl chloride, 4-fluorophenylsulfonyl chloride 1-butanethiol and acetic anhydride.

Scheme 3, below, shows a synthetic route to sulfonamide- and sulfonate-substituted purine-based ligands of the presently disclosed subject matter. Exemplary sulfonamide- and sulfonate-substituted purine-based ligands can be prepared from a halo-substituted purine using commercially available sulfamoyl halides and esters of halosulfuric acids (e.g., esters of chlorosulfuric acid or sulfurochloridates), such as sulfamoyl chloride, dimethylsulfamoyl chloride, diethyl sulfamoyl chloride, ethyl(phenyl)sulfamoyl chloride, methyl(phenyl)sulfamoyl chloride, diphenylsulfamoyl chloride, benzyl(methyl)sulfamoyl chloride, phenyl sulfurochloridate, isopentyl sulfurochloridate, methyl sulfurochloridate, and 4-methyoxybenzyl sulfurochloridate.

VI. Modified Proteins

In some embodiments, the presently disclosed subject matter provides a modified cysteine-containing protein. The modified protein can be a protein comprising the adduct formed between a cysteine thiol side chain group and a probe or ligand of the presently disclosed subject matter. The modified protein can have a different biological activity than the unmodified protein.

In some embodiments, the presently disclosed subject matter provides a modified cysteine-containing protein comprising a modified cysteine residue wherein the modified cysteine residue is formed by the reaction of a cysteine residue of a non-naturally occurring purine-based compound (e.g., a halo-substituted purine). In some embodiments, the non-naturally occurring purine-based compound is a compound having a structure of Formula (I):

or a compound having a structure of Formula (III′):

wherein X is a monovalent moiety comprising an alkyne moiety, a fluorophore moiety, a detectable labeling group, or a combination thereof; Z′ is selected from the group comprising alkyl (e.g., C₁-C₆ alkyl), substituted alkyl, cycloalkyl (e.g., C₃-C₆ cycloalkyl), heterocycloalkyl, acyl (e.g., C₂-C₂₄ acyl or C₂-C₁₂ acyl), substituted acyl, aralkyl (e.g., benzyl, ethylbenzyl, methylnaphthyl), substituted aralkyl (e.g., substituted benzyl), —S(═O)₂—R₅′, —S(═O)₂—N(R₆)₂, and —S(═O)₂—O—R₇; R₁ and R₂ are independently selected from the group comprising H, halo, hydroxyl, thiol, amino, alkyl (e.g., C₁-C₆ alkyl), alkoxy (e.g., C₁-C₆ alkoxy), alkylamino (e.g., C₁-C₆ alkylamino), alkylthio (e.g., C₁-C₆ alkylthio), aryloxy, arylamino, and arylthio, subject to the proviso that at least one of R₁ and R₂ is halo; R₃′ and R₄′ are independently selected from H, halo, alkyl (e.g., C₁-C₆ alkyl), alkylamino (e.g., C₁-C₆ alkylamino), alkylthio (e.g., C₁-C₆ alkylthio), alkoxy (e.g., C₁-C₆ alkoxy), aryloxy, arylamino, and arylthio, subject to the proviso that at least one of R₃′ and R₄′ is halo; R₅′ is heterocyclyl, substituted heterocyclyl, aryl or substituted aryl; each R₆ is selected from H, alkyl (e.g., C₁-C₆ alkyl), substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl, or wherein the two R₆ together form an alkylene group; and R₇ is selected from alkyl (e.g., C₁-C₆ alkyl), substituted alkyl, aralkyl, substituted aralkyl, aryl and substituted aryl.

In some embodiments, the modified cysteine-containing protein comprises at least one modified cysteine residue comprising a structure of Formula (II-i):

a structure of Formula (II-ii):

a structure of Formula (IV′-i):

or a structure of Formula (IV′-ii):

wherein X, Z′, R₅′, R₆, and R₇ are as defined for the compounds of Formula (I) or Formula (III′) and wherein R₁ is selected from the group consisting of H, halo, hydroxyl, thiol, amino, alkyl, alkoxy, alkylamino, alkylthio, aryloxy, arylamino, and arylthio; R₂ is selected from the group consisting of H, halo, hydroxyl, thiol, amino, alkyl, alkoxy, alkylamino, alkylthio, aryloxy, arylamino, and arylthio; R₃′ selected is from H, halo, alkyl, alkylamino, alkylthio, alkoxy, aryloxy, arylamino, and arylthiol; and R₄′ is selected from H, halo, alkyl, alkylamino, alkylthio, alkoxy, aryloxy, arylamino, and arylthiol. In some embodiments, X or Z′ is attached to the N7 nitrogen atom. In some embodiments, X or Z′ is attached to the N9 nitrogen atom.

In some embodiments, X comprises a fluorophore or a detectable labeling group, such as a fluorophore or detectable labeling group as defined hereinabove. In some embodiments, X is a monovalent moiety comprising an alkyne group. For example, in some embodiments, X comprises or consists of —C≡CH, -alkylene-C≡CH, —C(═O)-alkylene-C≡CH, or —C(═O)—NH-alkylene-C≡CH (e.g., C(═O)—NH—CH₂—C≡CH). In some embodiments, the alkylene group is a C₁-C₅ alkylene group. In some embodiments, the alkylene group is methylene. In some embodiments, X is a propargyl group, i.e., —CH₂—C≡CH.

In some embodiments, Z′ is selected from C₁-C₆ alkyl (e.g., allyl), a sugar residue, benzyl or substituted benzyl (e.g., 4-nitrobenzyl). In some embodiments, Z′ is selected from the group comprising acyl, cycloalkyl (e.g., cyclohexyl), —S(═O)₂—R₅′, —S(═O)₂—N(R₆)₂, —S(═O)₂—O—R₇ and

In some embodiments, Z′ is —S(═O)₂—R₅′, wherein R₅′ is heterocyclyl (e.g., morpholine) or substituted phenyl. In some embodiments, the substituted phenyl is an alkoxy- or halo-substituted phenyl (e.g., 4-methoxyphenyl or 4-fluorophenyl). In some embodiments, Z′ is —S(═O)₂—N(R₆)₂, wherein each R₆ is selected from alkyl and aralkyl. In some embodiments, at least one R₆ is alkyl, e.g., methyl, ethyl, propyl, butyl, pentyl or hexyl. In some embodiments, both R₆ are alkyl. In some embodiments, both R₆ are ethyl. In some embodiments, one R₆ is aralkyl, e.g., benzyl. In some embodiments, Z′ is —S(═O)₂—O—R₇, wherein R₇ is alkyl or aralkyl. In some embodiments, R₇ is alkyl, e.g., methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some embodiments, R₇ is methyl. In some embodiments, R₇ is benzyl.

In some embodiments, Z′ is selected from the group comprising:

In some embodiments, R₁, R₂, R₃′ or R₄′ is selected from chloro, fluoro, C₁-C₆ alkyl (e.g., methyl, ethyl, propyl, isopropyl, allyl, m-butyl, tert-butyl, pentyl, or hexyl), alkylthio, alkylamino, or aryloxy, optionally wherein the aryl group of the aryloxy is substituted by one or more aryl group substituents. In some embodiments, the R₁, R₂, R₃′ or R₄′ is selected from chloro, methyl, —SH—(CH₂)₃CH₃; —NH(CH₂)₃CH₃; and —O—(C₆H₄)CH₃.

In some embodiments, the modified cysteine-containing protein is a cysteine-containing protein listed in Table 3 or Table 4, below, e.g., modified at one of the cysteine residues noted in the tables. In some embodiments, the modified cysteine-containing protein is modified in a domain selected from the group comprising ADF-H domain, calponin-homology (CH) domain, WWE domain, translation-type guanine nucleotide binding (G) domain, elongation factor 1 (EF-1) gamma C-terminal domain, protein kinase domain, Bin3-type S-adenosyl-L-methionine domain, CXC domain, PITH domain, WHEP-TRS domain, mRNA (guanine-N(7)-methyl transferase domain, CoA carboxytransferase domain, and thermonuclease domain.

In some embodiments, the modified cysteine-containing protein is selenocysteine elongation factor (eEF-Sec) modified at cysteine 442, macrophage migration inhibitory factor modified at cysteine 81; or serine/threonine protein kinase 38-like modified at cysteine 235.

VII. Methods of Modulating Protein Activity

In some embodiments, presently disclosed subject matter provides a method of modulating the activity of a protein comprising a reactive amino acid residue by contacting the protein with a halo-substituted purine compound, such as a probe or ligand of the presently disclosed subject matter. In some embodiments, the presently disclosed subject matter provides a method of modulating the activity of a protein comprising a reactive cysteine residue. In some embodiments, the protein with the reactive amino acid residue is an enzyme and modulating the activity of the protein comprises inhibiting or activating the enzyme. In some embodiments, modulating the activity of a protein comprises enhancing or reducing the ability of the protein to interact with other compounds, such as other proteins. Thus, in some embodiments, the modulation results in reducing the protein-protein interactions of the protein comprising the reactive amino acid.

In some embodiments, the presently disclosed subject matter provides a method of modulating the activity of a protein comprising a reactive cysteine residue, wherein the method comprising contacting a protein comprising a reactive cysteine residue with a compound having a structure of Formula (III′):

wherein Z′ is selected from the group comprising alkyl (e.g., C₁-C₆ alkyl), substituted alkyl, cycloalkyl (e.g., C₃-C₆ cycloalkyl), heterocycloalkyl, acyl (e.g., C₂-C₂₄ acyl or C₂-C₁₂ acyl), substituted acyl, aralkyl (e.g., benzyl), substituted aralkyl (e.g., substituted benzyl, ethylbenzyl, methylnaphthyl), —S(═O)₂—R₅′, —S(═O)₂—N(R₆)₂, and —S(═O)₂—O—R₇; R₃′ and R₄′ are independently selected from H, halo, alkyl (e.g., C₁-C₆ alkyl), alkylamino (e.g., C₁-C₆ alkylamino), alkylthio (e.g., C₁-C₆ alkylthio), alkoxy (e.g., C₁-C₆ alkoxy), aryloxy, arylamino, and arylthio, subject to the proviso that at least one of R₃′ and R₄′ is halo (e.g., chloro or fluoro); R₅′ is heterocyclyl, substituted heterocyclyl, aryl or substituted aryl; each R₆ is selected from H, alkyl (e.g., C₁-C₆ alkyl), substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl, or wherein the two R₆ together form an alkylene group; and R₇ is selected from alkyl (e.g., C₁-C₆ alkyl), substituted alkyl, aralkyl, substituted aralkyl, aryl and substituted aryl.

In some embodiments, Z′ is substituted on the N7 or N9 atom and the compound having a structure of Formula (III′) is a compound having a structure of Formula (IIIa′):

or a structure of Formula (IIIb′):

wherein Z′, R₃′, and R₄′ are as defined for Formula (III′).

In some embodiments, R₃′ is halo, alkyl, alkyoxy, alkylthio, alkylamino, or aryloxy. In some embodiments, R₃′ is selected from chloro, fluoro, methyl, n-butylthio, n-butylamino, and —O—(C₆H₄)—OMe. In some embodiments, R₄′ is halo. In some embodiments, R₄′ is fluoro or chloro. In some embodiments, both R₃′ and R₄′ are halo. In some embodiments, R₃′ and R₄′ are each independently selected from chloro and fluoro. In some embodiments, R₃′ and R₄′ are both chloro.

In some embodiments, Z′ is selected from C₁-C₆ alkyl (e.g., allyl), a sugar residue, benzyl or substituted benzyl (e.g., 4-nitrobenzyl). In some embodiments, Z′ is selected from the group comprising acyl, cycloalkyl, —S(═O)₂—R₅′, —S(═O)₂—N(R₆)₂, —S(═O)₂—O—R₇ and

In some embodiments, Z′ is selected from —CH₂—CH═CH₂, C₂-C₁₂ acyl (e.g., acetyl, hexanoyl, or dodecanoyl), cyclohexyl, benzyl, —CH₂—(C₆H₄)—NO₂, —S(═O)₂—R₅′, and

wherein R₅′ is selected from heterocyclyl and substituted aryl (e.g., wherein R₅′ is selected from morpholinyl, 4-halophenyl, and 4-alkoxyphenyl).

In some embodiments, Z′ is —S(═O)₂—R₅′, wherein R₅′ is heterocyclyl (e.g., morpholine) or substituted phenyl. In some embodiments, the substituted phenyl is an alkoxy- or halo-substituted phenyl (e.g., 4-methoxyphenyl or 4-fluorophenyl). In some embodiments, R₅′ is selected from morpholine and 4-substituted phenyl. In some embodiments, R₅′ is selected from morpholine, 4-halophenyl, and 4-alkoxyphenyl. In some embodiments, R₅′ is selected from morpholine, 4-fluorophenyl, and 4-methyoxyphenyl. In some embodiments, Z′ is —S(═O)₂—N(R₆)₂, wherein each R₆ is selected from alkyl and aralkyl. In some embodiments, at least one R₆ is alkyl, e.g., methyl, ethyl, propyl, butyl, pentyl or hexyl. In some embodiments, both R₆ are alkyl. In some embodiments, both R₆ are ethyl. In some embodiments, one R₆ is aralkyl, e.g., benzyl. In some embodiments, Z′ is —S(═O)₂—O—R₇, wherein R₇ is alkyl or aralkyl. In some embodiments, R₇ is alkyl, e.g., methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some embodiments, R₇ is methyl. In some embodiments, R₇ is benzyl.

In some embodiments, Z′ is selected from the group comprising:

In some embodiments, the compound of Formula (III′) is selected from the group comprising 4-((2,6-dichloro-7H-purin-7-yl)sulfonyl)morpholine, 4-((2,6-dichloro-9H-purin-9-yl)sulfonyl)morpholine, 2,6-dichloro-7-((4-fluorophenyl)sulfonyl)-7H-purine, 2,6-dichloro-9-((4-fluorophenyl)sulfonyl)-9H-purine, 2,6-dichloro-7-((4-methoxyphenyl)sulfonyl)-7H-purine, 2,6-dichloro-9-((4-methoxyphenyl)sulfonyl)-9H-purine, 2,6-dichloro-7-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl)-7H-purine, 2,6-dichloro-9-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl)-9H-purine, 2,6-dichloro-7-(4-nitrobenzyl)-7H-purine, 1-(2,6-dichloro-9H-purin-9-yl)dodecan-1-one, 1-(2,6-dichloro-7H-purin-7-yl)hexan-1-one, 1-(2,6-dichloro-9H-purin-9-yl)hexan-1-one, 1-(6-chloro-2-fluoro-7H-purin-7-yl)hexan-1-one, 1-(6-chloro-2-fluoro-9H-purin-9-yl)hexan-1-one, 1-(2-chloro-6-methyl-9H-purin-9-yl)hexan-1-one, 2-chloro-9-cyclohexyl-6-methyl-9H-purine, 9-cyclohexyl-2-fluoro-6-methyl-9H-purine, 1-(6-(butylthio)-2-fluoro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylthio)-2-chloro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylthio)-2-chloro-7H-purin-7-yl)ethan-1-one, 1-(6-(butylthio)-2-fluoro-7H-purin-7-yl)ethan-1-one, 1-(2-chloro-6-(p-tolyloxy)-9H-purin-9-yl)ethan-1-one, 1-(2-fluoro-6-(p-tolyloxy)-9H-purin-9-yl)ethan-1-one, 1-(2-chloro-6-(p-tolyloxy)-7H-purin-7-yl)ethan-1-one, 1-(2-fluoro-6-(p-tolyloxy)-7H-purin-7-yl)ethan-1-one, 1-(6-(butylamino)-2-chloro-7H-purin-7-yl)ethan-1-one, 1-(6-(butylamino)-2-fluoro-7H-purin-7-yl)ethan-1-one, 7-allyl-2,6-dichloro-7H-purine, 9-allyl-2,6-dichloro-9H-purine, 2,6-dichloro-7-benzyl-7H-purine, 2,6-dichloro-9-benzyl-9H-purine, 2,6-dichloro-9-(4-nitrobenzyl-9H-purine, 2-(2,6-dichloro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol, 1-(6-(butylamino)-2-chloro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylamino)-2-fluoro-9H-purin-9-yl)ethan-1-one, 2,6-dichloro-N,N-diethyl-7H-purine-7-sulfonamide, 2,6-dichloro-N,N-diethyl-9H-purine-9-sulfonamide, N-benzyl-2,6-dichloro-N-methyl-9H-purine-9-sulfonamide, N-benzyl-2,6-dichloro-N-methyl-7H-purine-7H-sulfonamide, benzyl 2,6-dichloro-7H-purine-7-sulfonate, benzyl 2,6-dichloro-9H-purine-9-sulfonate, methyl 2,6-dichloro-9H-purine-9-sulfonate, and methyl 2,6-dichloro-7H-purine-7-sulfonate.

In some embodiments, the compound of Formula (III′) is not one of the compounds selected from the group comprising 7-allyl-2,6-dichloro-7H-purine, 9-allyl-2,6-dichloro-9H-purine, 2,6-dichloro-7-benzyl-7H-purine, 2,6-dichloro-9-benzyl-9H-purine, 2,6-dichloro-9-(4-nitrobenzyl-9H-purine, and 2-(2,6-dichloro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol.

In some embodiments, the compound of Formula (III′) is a compound of Formula (IIIb′) and has a purity of at least about 90% (e.g., at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% or more), e.g., by HPLC. Thus, in some embodiments, an N7-substituted purine-based compound is provided substantially free of the N9-substituted regioisomer.

In some embodiments, contacting the protein comprising a reactive cysteine residue with the compound of Formula (III′) provides a modified cysteine-containing protein comprising a structure of one of Formulas (IV′-i) and (IV′-ii) described hereinabove.

In some embodiments, modulating an activity of a protein comprising a reactive cysteine residue comprises inhibiting (partially or substantially completely) an activity (e.g., an enzymatic activity) of the protein comprising a reactive cysteine residue. In some embodiments, modulating an activity of a protein comprising a reactive cysteine residue comprises activating an activity (e.g., an enzymatic activity) of the protein comprising a reactive cysteine residue. In some embodiments, modulating the activity of a protein comprising a reactive cysteine residue comprises inhibiting, blocking (partially or substantially completely) or disrupting a protein-protein interaction, a protein-RNA interaction, a protein-DNA interaction, a protein-lipid interaction, and/or a protein-metabolite interaction of the protein comprising a reactive cysteine residue. In some embodiments, modulating an activity of a protein comprising a reactive cysteine residue comprises inhibiting or disrupting subcellular localization of the protein comprising a reactive cysteine residue. In some embodiments, modulating an activity of a protein comprising a reactive cysteine residue comprises triggering recruitment of an E3 ligase for targeted degradation of the protein comprising a reactive cysteine residue.

VIII. Cells, Analytical Techniques and Instrumentation

In some embodiments, one or more of the methods disclosed herein comprise a sample (e.g., a cell sample, cell lysate sample or a biological organism). In some embodiments, the sample for use with the methods described herein is obtained from cells of an animal. In some instances, the animal cell includes a cell from a marine invertebrate, fish, insects, amphibian, reptile, or mammal. In some instances, the mammalian cell is a primate, ape, equine, bovine, porcine, canine, feline, or rodent. In some instances, the mammal is a primate, ape, dog, cat, rabbit, ferret, or the like. In some cases, the rodent is a mouse, rat, hamster, gerbil, hamster, chinchilla, or guinea pig. In some embodiments, the bird cell is from a canary, parakeet or parrots. In some embodiments, the reptile cell is from a turtles, lizard or snake. In some cases, the fish cell is from a tropical fish. In some cases, the fish cell is from a zebrafish (e.g. Danino rerio). In some cases, the worm cell is from a nematode (e.g. C. elegans). In some cases, the amphibian cell is from a frog. In some embodiments, the arthropod cell is from a tarantula or hermit crab.

In some embodiments, the sample for use with the methods described herein is obtained from a mammalian cell. In some instances, 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.

In some embodiments, the sample for use with the methods described herein is obtained from cells of a tumor cell line. In some instances, the sample is obtained from cells of a solid tumor cell line. In some instances, the solid tumor cell line is a sarcoma cell line. In some instances, the solid tumor cell line is a carcinoma cell line. In some embodiments, the sarcoma cell line is obtained from a cell line of alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastoma, angiosarcoma, 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, infantile fibrosarcoma, inflammatory myofibroblastic tumor, Kaposi sarcoma, leiomyosarcoma of bone, liposarcoma, liposarcoma of bone, malignant fibrous histiocytoma (MFH), malignant fibrous histiocytoma (MFH) of bone, malignant mesenchymoma, malignant peripheral nerve sheath tumor, mesenchymal chondrosarcoma, myxofibrosarcoma, myxoid liposarcoma, myxoinflammatory fibroblastic sarcoma, neoplasms with perivascular epitheioid cell differentiation, osteosarcoma, parosteal osteosarcoma, neoplasm with perivascular epitheioid cell differentiation, periosteal osteosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma, PNET/extraskeletal Ewing tumor, rhabdomyosarcoma, round cell liposarcoma, small cell osteosarcoma, solitary fibrous tumor, synovial sarcoma, and telangiectatic osteosarcoma.

In some embodiments, the carcinoma cell line is obtained from a cell line of adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder 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 cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

In some instances, the sample is obtained from cells of a hematologic malignant cell line. In some instances, the hematologic malignant cell line is a T-cell cell line. In some instances, B-cell cell line. In some instances, 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, 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.

In some instances, the hematologic malignant cell line is obtained from a B-cell cell line of: acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CIVIL), 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, 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, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

In some embodiments, the sample for use with the methods described herein is obtained from a tumor cell line. Exemplary 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, 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, HepG2/SF, OCI-Ly1, OCI-Ly2, OCI-Ly3, OCI-Ly4, OCI-Ly6, OCI-Ly7, OCI-Ly10, OCI-Ly18, OCI-Ly19, U2932, DB, HBL-1, RIVA, SUDHL2, TMD8, MEC1, MEC2, 8E5, CCRF-CEM, MOLT-3, TALL-104, AML-193, THP-1, BDCM, HL-60, Jurkat, RPMI 8226, MOLT-4, RS4, K-562, KASUMI-1, Daudi, GA-10, Raji, JeKo-1, NK-92, and Mino.

In some embodiments, 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, 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. In some embodiments, the sample is a tissue sample, such as a sample obtained from a biopsy or a tumor tissue sample. In some embodiments, the sample is a blood serum sample. In some embodiments, the sample is a blood cell sample containing one or more peripheral blood mononuclear cells (PBMCs). In some embodiments, the sample contains one or more circulating tumor cells (CTCs). In some embodiments, the sample contains one or more disseminated tumor cells (DTC, e.g., in a bone marrow aspirate sample).

In some embodiments, 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 samples from an individual are well known. For example, procedures for drawing and processing tissue sample such as from a needle aspiration biopsy is well-known and is employed to obtain a sample for use in the methods provided. Typically, for collection of such a tissue sample, a thin hollow needle is inserted into a mass such as a tumor mass for sampling of cells that, after being stained, will be examined under a microscope. In some embodiments, the sample is a biological organism. In some embodiments, the biological organism is a rodent, e.g., a mouse or a rat. In some embodiments, the biological organism is a primate, e.g., a monkey. In some embodiments, the biological organism is a bacteria or a fungi.

IX. Sample Preparation and Analysis

In some embodiments, the sample (e.g., cell sample, cell lysate sample, or comprising isolated proteins) is a sample solution. In some instances, the sample solution comprises a solution such as a buffer (e.g. phosphate buffered saline) or a media. In some embodiments, the media is an isotopically labeled media. In some instances, the sample solution is a cell solution.

In some embodiments, the sample (e.g., cell sample, cell lysate sample, or comprising isolated proteins) is incubated with one or more compound probes for analysis of protein-probe interactions. In some instances, the sample (e.g., cell sample, cell lysate sample, or comprising isolated proteins) is further incubated in the presence of an additional compound probe prior to addition of the one or more probes. In other instances, the sample (e.g., cell sample, cell lysate sample, or comprising isolated proteins) 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. In such instances, the sample is incubated with a probe and non-probe small molecule ligand for competitive protein profiling analysis.

In some cases, 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.

In some embodiments, one or more methods are utilized for labeling a sample (e.g. cell sample, 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. cell sample, cell lysate sample, or comprising isolated proteins) is labeled with isotope-labeled amino acids, such as ¹³C or ¹⁵N-labeled amino acids. In some cases, the labeled sample is further compared with a non-labeled sample 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. In some instances, the isotope-labeled method is termed SILAC, stable isotope labeling using amino acids in cell culture.

In some embodiments, 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. In such cases, 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. As described above, the linker can be 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). In some cases, the labeling group is a biotin-linker moiety, which is optionally isotopically labeled with ¹³C and ¹⁵N atoms at one or more amino acid residue positions within the linker. In some cases, the biotin-linker moiety is a isotopically-labeled TEV-tag as previously described.¹⁰

In some embodiments, an isotopic reductive dimethylation (ReDi) method is utilized for processing a sample. In some cases, 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. In some cases, the ReDi labeling method comprises methylating peptides from a first 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. Subsequent proteomic analysis (e.g., mass spectrometry analysis) based on a relative peptide abundance between the heavy and light peptide version might be used for analysis of probe-protein interactions.

In some embodiments, isobaric tags for relative and absolute quantitation (iTRAQ) method is utilized for processing a sample. In some cases, the iTRAQ method is based on the covalent labeling of the N-terminus and side chain amines of peptides from a processed sample. In some cases, reagent such as 4-plex or 8-plex is used for labeling the peptides.

In some embodiments, the probe-protein complex is further conjugated to a chromophore, such as a fluorophore. In some instances, the probe-protein complex is separated and visualized utilizing an 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. In some instances, the probe-protein is subjected to a native electrophoresis condition. In some instances, the probe-protein is subjected to a denaturing electrophoresis condition.

In some instances, the probe-protein after harvesting is further fragmentized to generate protein fragments. In some instances, fragmentation is generated through mechanical stress, pressure, or chemical means. In some instances, the protein from the probe-protein complexes is fragmented by a chemical means. In some embodiments, 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, 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; threonine proteases such as ornithine acetyltransferase; cysteine proteases such as TEV protease, amidophosphoribosyltransferase precursor, gamma-glutamyl hydrolase (Rattus norvegicus), hedgehog protein, DmpA aminopeptidase, papain, bromelain, cathepsin K, calpain, caspase-1, separase, adenain, pyroglutamyl-peptidase I, sortase A, hepatitis C virus peptidase 2, sindbis virus-type nsP2 peptidase, dipeptidyl-peptidase VI, or DeSI-1 peptidase; aspartate proteases such as beta-secretase 1 (BACE1), beta-secretase 2 (BACE2), cathepsin D, cathepsin E, chymosin, napsin-A, nepenthesin, pepsin, plasmepsin, presenilin, or renin; glutamic acid proteases such as AfuGprA; and metalloproteases such as peptidase_M48.

In some instances, 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.

In some instances, the protein fragments are further analyzed by a proteomic method such as by liquid chromatography (LC) (e.g. high performance liquid chromatography), liquid chromatography-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).

In some embodiments, 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 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 chromatography that are classified based on the physical states of those phases. In some embodiments, 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 aqueous normal-phase chromatography.

In some embodiments, the LC method is a high performance liquid chromatography (HPLC) method. In some embodiments, 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, chiral chromatography, and aqueous normal-phase chromatography.

In some embodiments, 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).

In some embodiments, the LC is coupled to a mass spectroscopy as a LC-MS method. In some embodiments, 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 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). In some instances, the LC-MS method is LC/LC-MS/MS. In some embodiments, the LC-MS methods of the present disclosure are performed by standard techniques well known in the art.

In some embodiments, the GC is coupled to a mass spectroscopy as a GC-MS method. In some embodiments, 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-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS).

In some embodiments, CE is coupled to a mass spectroscopy as a CE-MS method. In some embodiments, 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-quadrupole time of flight-mass spectrometry (CE-QTOF-MS).

In some embodiments, the nuclear magnetic resonance (NMR) method is any suitable method well known in the art for the detection of one or more cysteine binding proteins or protein fragments disclosed herein. In some embodiments, the NMR method includes one dimensional (1D) NMR methods, two dimensional (2D) NMR methods, solid state NMR methods and NMR chromatography. Exemplary 1D NMR methods include ¹Hydrogen, ¹³Carbon, ¹⁵Nitrogen, ¹⁷Oxygen, ¹⁹Fluorine, ³¹Phosphorus, ³⁹Potassium, ²³Sodium, ³³Sulfur, ⁸⁷Strontium, ²⁷Aluminium, ⁴³Calcium, ³⁵Chlorine, ³⁷Chlorine, ⁶³Copper, ⁶⁵Copper, ⁵⁷Iron, ²⁵Magnesium, ¹⁹⁹Mercury or ⁶⁷Zinc NMR method, distortionless enhancement by polarization transfer (DEPT) method, attached proton test (APT) method and 1D-incredible natural abundance double quantum transition experiment (INADEQUATE) method. 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. Exemplary solid state NMR method include solid state .sup.13Carbon 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.

In some embodiments, the results from the mass spectroscopy method are analyzed by an algorithm for protein identification. In some embodiments, the algorithm combines the results from the mass spectroscopy method with a protein sequence database for protein identification. In some embodiments, the algorithm comprises ProLuCID algorithm, Probity, Scaffold, SEQUEST, or Mascot.

In accordance with the presently disclosed subject matter, as described above or as discussed in the EXAMPLES below, there can be employed conventional chemical, cellular, histochemical, biochemical, molecular biology, microbiology, recombinant DNA, and clinical techniques which are known to those of skill in the art. Such techniques are explained fully in the literature. See for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Publications, Cold Spring Harbor, N.Y., United States of America; Glover (1985) DNA Cloning: A Practical Approach. Oxford Press, Oxford; Gait (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, England; Harlow & Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York; Roe et al. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley, New York, N.Y., United States of America; and Ausubel et al. (1995) Current Protocols in Molecular Biology, Greene Publishing.

X. Therapeutic Uses and Pharmaceutical Compositions

Small molecules, such as the presently disclosed purine-based ligands and probes, present an alternative method to selectively modulate proteins and to serve as leads for the development of novel therapeutics.

Dysregulated expression of a cysteine-containing protein, in many cases, is associated with or modulates a disease, such as an inflammatory related disease, an immune system related disease, a neurodegenerative disease, or cancer. As such, identification of a potential agonist/antagonist to a cysteine-containing protein aids in improving the disease condition in a patient.

Thus, in some embodiments, disclosed herein are cysteine-containing proteins that comprise one or more ligandable cysteines. In some embodiments, the cysteine-containing protein is selected from a protein listed in Table 3 or Table 4, below. In some embodiments, the cysteine-containing protein is selected from the group comprising the selenocysteine elongation factor (eEF-Sec), macrophage migration inhibitory factor or serine/threonine protein kinase 38-like.

Compounds described herein include isotopically-labeled compounds, which are identical to those recited in the various formulae and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the present compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine and chlorine, such as, for example, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F, ³⁶Cl. In one aspect, isotopically-labeled compounds described herein, for example those into which radioactive isotopes such as ³H and ¹⁴C are incorporated, are useful in drug and/or substrate tissue distribution assays. In one aspect, substitution with isotopes such as deuterium affords certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements.

In some embodiments, the presently disclosed subject matter provides pharmaceutical compositions comprising one or more of the presently disclosed ligands or probes. The pharmaceutical compositions comprise at least one disclosed compound, e.g. selected from compounds of Formula (I), (Ia), (Ib), (III), (IIIa), (IIIb), and (III′) and related formulas described herein in combination with a pharmaceutically acceptable carrier, vehicle, or diluent, such as an aqueous buffer at a physiologically acceptable pH (e.g., pH 7 to 8.5), a non-aqueous liquid, a polymer-based nanoparticle vehicle, a liposome, and the like. The pharmaceutical compositions can be delivered in any suitable dosage form, such as a liquid, gel, solid, cream, or paste dosage form. In one embodiment, the compositions can be adapted to give sustained release of the probe.

In some embodiments, the pharmaceutical compositions include, but are not limited to, those forms suitable for oral, rectal, nasal, topical, (including buccal and sublingual), transdermal, vaginal, parenteral (including intramuscular, subcutaneous, and intravenous), spinal (epidural, intrathecal), central (intracerebroventricular) administration, in a form suitable for administration by inhalation or insufflation. The compositions can, where appropriate, be provided in discrete dosage units. The pharmaceutical compositions of the invention can be prepared by any of the methods well known in the pharmaceutical arts. Some preferred modes of administration include intravenous (i.v.), intraperitoneal (i.p.), topical, subcutaneous, and oral.

Pharmaceutical formulations suitable for oral administration include capsules, cachets, or tablets, each containing a predetermined amount of one or more of the ligands, as a powder or granules. In another embodiment, the oral composition is a solution, a suspension, or an emulsion. Alternatively, the ligands can be provided as a bolus, electuary, or paste. Tablets and capsules for oral administration can contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, colorants, flavoring agents, preservatives, or wetting agents. The tablets can be coated according to methods well known in the art, if desired. Oral liquid preparations include, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs. Alternatively, the compositions can be provided as a dry product for constitution with water or another suitable vehicle before use. Such liquid preparations can contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), preservatives, and the like. The additives, excipients, and the like typically will be included in the compositions for oral administration within a range of concentrations suitable for their intended use or function in the composition, and which are well known in the pharmaceutical formulation art. The presently disclosed ligands will be included in the compositions within a therapeutically useful and effective concentration range, as determined by routine methods that are well known in the medical and pharmaceutical arts. For example, a typical composition can include one or more of the ligands at a concentration in the range of at least about 0.01 nanomolar to about 1 molar, preferably at least about 1 nanomolar to about 100 millimolar.

Pharmaceutical compositions for parenteral, spinal, or central administration (e.g. by bolus injection or continuous infusion) or injection into amniotic fluid can be provided in unit dose form in ampoules, pre-filled syringes, small volume infusion, or in multi-dose containers, and preferably include an added preservative. The compositions for parenteral administration can be suspensions, solutions, or emulsions, and can contain excipients such as suspending agents, stabilizing agents, and dispersing agents. Alternatively, the ligands can be provided in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use. The additives, excipients, and the like typically will be included in the compositions for parenteral administration within a range of concentrations suitable for their intended use or function in the composition, and which are well known in the pharmaceutical formulation art. The ligands of the presently disclosed subject matter can be included in the compositions within a therapeutically useful and effective concentration range, as determined by routine methods that are well known in the medical and pharmaceutical arts. For example, a typical composition can include one or more of the ligands at a concentration in the range of at least about 0.01 nanomolar to about 100 millimolar, preferably at least about 1 nanomolar to about 10 millimolar.

Pharmaceutical compositions for topical administration of the ligands to the epidermis (mucosal or cutaneous surfaces) can be formulated as ointments, creams, lotions, gels, or as a transdermal patch. Such transdermal patches can contain penetration enhancers such as linalool, carvacrol, thymol, citral, menthol, t-anethole, and the like. Ointments and creams can, for example, include an aqueous or oily base with the addition of suitable thickening agents, gelling agents, colorants, and the like. Lotions and creams can include an aqueous or oily base and typically also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, coloring agents, and the like. Gels preferably include an aqueous carrier base and include a gelling agent such as cross-linked polyacrylic acid polymer, a derivatized polysaccharide (e.g., carboxymethyl cellulose), and the like. The additives, excipients, and the like typically will be included in the compositions for topical administration to the epidermis within a range of concentrations suitable for their intended use or function in the composition, and which are well known in the pharmaceutical formulation art. The ligands of the presently disclosed subject matter can be included in the compositions within a therapeutically useful and effective concentration range, as determined by routine methods that are well known in the medical and pharmaceutical arts. For example, a typical composition can include one or more of the ligands at a concentration in the range of at least about 0.01 nanomolar to about 1 molar, preferably at least about 1 nanomolar to about 100 millimolar.

Pharmaceutical compositions suitable for topical administration in the mouth (e.g., buccal or sublingual administration) include lozenges comprising the ligand in a flavored base, such as sucrose, acacia, or tragacanth; pastilles comprising the ligand in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier. The pharmaceutical compositions for topical administration in the mouth can include penetration enhancing agents, if desired. The additives, excipients, and the like typically will be included in the compositions of topical oral administration within a range of concentrations suitable for their intended use or function in the composition, and which are well known in the pharmaceutical formulation art. The ligands of the presently disclosed subject matter invention can be included in the compositions within a therapeutically useful and effective concentration range, as determined by routine methods that are well known in the medical and pharmaceutical arts. For example, a typical composition can include one or more of the ligands at a concentration in the range of at least about 0.01 nanomolar to about 1 molar, preferably at least about 1 nanomolar to about 100 millimolar.

A pharmaceutical composition suitable for rectal administration comprises a ligand of the presently disclosed subject matter in combination with a solid or semisolid (e.g., cream or paste) carrier or vehicle. For example, such rectal compositions can be provided as unit dose suppositories. Suitable carriers or vehicles include cocoa butter and other materials commonly used in the art. The additives, excipients, and the like typically will be included in the compositions of rectal administration within a range of concentrations suitable for their intended use or function in the composition, and which are well known in the pharmaceutical formulation art. The ligands of the presently disclosed subject matter can be included in the compositions within a therapeutically useful and effective concentration range, as determined by routine methods that are well known in the medical and pharmaceutical arts. For example, a typical composition can include one or more of the ligands at a concentration in the range of at least about 0.01 nanomolar to about 1 molar, preferably at least about 1 nanomolar to about 100 millimolar.

According to one embodiment, pharmaceutical compositions of the present invention suitable for vaginal administration are provided as pessaries, tampons, creams, gels, pastes, foams, or sprays containing a ligand of the presently disclosed subject matter in combination with a carriers as are known in the art. Alternatively, compositions suitable for vaginal administration can be delivered in a liquid or solid dosage form. The additives, excipients, and the like typically will be included in the compositions of vaginal administration within a range of concentrations suitable for their intended use or function in the composition, and which are well known in the pharmaceutical formulation art. The ligands of the presently disclosed subject matter will be included in the compositions within a therapeutically useful and effective concentration range, as determined by routine methods that are well known in the medical and pharmaceutical arts. For example, a typical composition can include one or more of the presently disclosed ligands at a concentration in the range of at least about 0.01 nanomolar to about 1 molar, preferably at least about 1 nanomolar to about 100 millimolar.

Pharmaceutical compositions suitable for intra-nasal administration are also encompassed by the present invention. Such intra-nasal compositions comprise a ligand of the presently disclosed subject matter in a vehicle and suitable administration device to deliver a liquid spray, dispersible powder, or drops. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents, or suspending agents. Liquid sprays are conveniently delivered from a pressurized pack, an insufflator, a nebulizer, or other convenient means of delivering an aerosol comprising the ligand. Pressurized packs comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas as is well known in the art. Aerosol dosages can be controlled by providing a valve to deliver a metered amount of the ligand. Alternatively, pharmaceutical compositions for administration by inhalation or insufflation can be provided in the form of a dry powder composition, for example, a powder mix of the ligand and a suitable powder base such as lactose or starch. Such powder composition can be provided in unit dosage form, for example, in capsules, cartridges, gelatin packs, or blister packs, from which the powder can be administered with the aid of an inhalator or insufflator. The additives, excipients, and the like typically will be included in the compositions of intra-nasal administration within a range of concentrations suitable for their intended use or function in the composition, and which are well known in the pharmaceutical formulation art. The ligand of the presently disclosed subject matter will be included in the compositions within a therapeutically useful and effective concentration range, as determined by routine methods that are well known in the medical and pharmaceutical arts. For example, a typical composition can include one or more ligand at a concentration in the range of at least about 0.01 nanomolar to about 1 molar, preferably at least about 1 nanomolar to about 100 millimolar.

Optionally, the pharmaceutical compositions of the presently disclosed subject matter can include one or more other therapeutic agent, e.g., as a combination therapy. The additional therapeutic agent will be included in the compositions within a therapeutically useful and effective concentration range, as determined by routine methods that are well known in the medical and pharmaceutical arts. The concentration of any particular additional therapeutic agent may be in the same range as is typical for use of that agent as a monotherapy, or the concentration can be lower than a typical monotherapy concentration if there is a synergy when combined with a ligand of the presently disclosed subject matter.

XI. Kits/Articles of Manufacture

Disclosed herein, in certain embodiments, are kits and articles of manufacture for use with one or more methods described herein. In some embodiments, described herein is a kit for generating a protein comprising a detectable group and/or a fragment of a ligand compound described herein. In some embodiments, 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. In some instances, the kit further comprises samples, such as a cell sample, and suitable solutions such as buffers or media. In some embodiments, the kit further comprises recombinant proteins for use in one or more of the methods described herein. In some embodiments, 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. In one embodiment, the containers are formed from a variety of 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. For example, the container(s) include probes, ligands, control compounds, and one or more reagents for use in a method 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. For example, 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. In some embodiments, a label is on or associated with the container. In some embodiments, 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. In some embodiments, 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 contents, such as in the methods described herein.

EXAMPLES

The following EXAMPLES provide illustrative embodiments. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following EXAMPLES are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative EXAMPLES, make and utilize the compounds of the presently disclosed subject matter and practice the methods of the presently disclosed subject matter. The following EXAMPLES therefore particularly point out embodiments of the presently disclosed subject matter and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1

Synthetic Methods and Compound Characterization

Preparation of Pu-1 (for Synthesis of N7 Tautomer):

Into a 250 mL round bottom flask was placed 2,6-dichloropurine (5.0 g, 26.5 mmol), dry THF (110 mL) and a stir bar. The reaction was placed under nitrogen and treated with methylmagnesium chloride (MeMgCl, 3.0 M in THF, 9.7 mL, 29.1 mmol). After 30 minutes the reaction was treated with propargyl bromide (80 wt % in toluene, 8.84 ml, 79.4 mmol). The reaction was then heated to 70 C in an oil bath. After 17 hours the reaction was cooled and treated with methanol (25 mL) and concentrated on the rotovap. The residue was re-dissolved in DCM (100 mL) and re-concentrated to give a solid. This material was purified on a single cartridge flash purification system sold under the tradename ISOLERA™ One (Biotage, Uppsala, Sweden) using 5% acetone to 20% acetone/chloroform as the mobile phase. This provided 1.3 grams of the desired N-7 substituted product.

2,6-Dichloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-1)

¹H NMR (600 MHz, Chloroform-d) δ 8.48 (s, 1H), 5.27 (d, J=2.6 Hz, 2H), 2.70 (t, J=2.6 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d6) δ 163.24, 151.84, 151.30, 143.40, 121.61, 78.08, 77.55, 36.62. ESI-TOF (HRMS) m/z [M+H]+ calculated for C₈H₅C₁₂N₄⁺ 226.9886, found 226.9885.

General Procedure for Preparing Probe Compounds (N7 and N9 Tautomers):

As shown in Scheme 4, above, into a 250 mL round bottom flask was placed 2,6-dichloropurine (4.10 g, 21.7 mmol), dimethylformamide (DMF, 100 mL), potassium carbonate (K₂CO₃, 3.00 grams, 21.7 mmol) and propargyl bromide (80 wt % in toluene, 2.4 ml, 21.7 mmol). The reaction mixture was stirred under nitrogen at room temperature for 12 hours. The reaction was partitioned between ethyl acetate and water (200 mL each). The layers were separated and the aqueous layer was extracted with ethyl acetate (2×100 ml). The combined organic layer was dried over sodium sulfate and concentrated to a tan solid (5.3 g). This was dissolved in chloroform (100 mL). The solution was concentrated to approximately 20 mL and heated to reflux to dissolve all the solids. Upon cooling a white crystalline solid formed which was isolated by filtration. The solid was rinsed with fresh chloroform (20 mL) and heptane (20 mL) to give 1.15 g of the N-9 substituted product after air drying. The filtrate contained a mixture of the N-7 and N-9 products. These were separated on a single cartridge flash purification system sold under the tradename ISOLERA™ One (Biotage, Uppsala, Sweden) using 5% acetone to 20% acetone/chloroform as the mobile phase to give 550 mg and 1.55 g, respectively.

2,6-Dichloro-9-(prop-2-yn-1-yl)-9H-purine (Pu-2)

¹H NMR (600 MHz, Chloroform-d) δ 8.33 (s, 1H), 5.04 (d, J=2.6 Hz, 2H), 2.61 (t, J=2.6 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 152.91, 151.21, 149.89, 147.71, 130.44, 77.07, 76.92, 33.52. ESI-TOF (HRMS) m/z [M+H]+ calculated for C₈H₅C₁₂N₄⁺ 226.9886, found 226.9887.

Other purine probes were prepared using the same method using different halogentated purines as the starting material in place of 2,6-dichloropurine.

6-Chloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-3)

¹H NMR (600 MHz, Chloroform-d) δ 8.90 (s, 1H), 8.48 (s, 1H), 5.30 (d, J=2.6 Hz, 2H), 2.68 (t, J=2.6 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 161.59, 152.05, 150.34, 142.43, 121.81, 77.93, 77.76, 36.47. ESI-TOF (HRMS) m/z [M+H]+ calculated for C₈H₆ClN₄⁺ 193.0276, found 193.0276.

6-Chloro-9-(prop-2-yn-1-yl)-9H-purine (Pu-4)

¹H NMR (600 MHz, Chloroform-d) δ 8.78 (s, 1H), 8.34 (s, 1H), 5.07 (d, J=2.6 Hz, 2H), 2.59 (t, J=2.6 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 151.82, 151.39, 149.21, 146.80, 130.73, 77.30, 76.71, 33.27. ESI-TOF (HRMS) m/z [M+H]+ calculated for C₈H₆ClN₄⁺ 193.0276, found 193.0275.

2-Chloro-7-(prop-2-yn-1-yl)-7H-purine (Pu-5)

¹H NMR (600 MHz, DMSO-d₆) δ 9.19 (s, 1H), 8.84 (s, 1H), 5.35 (d, J=2.6 Hz, 2H), 3.69-3.67 (m, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 162.28, 153.15, 150.62, 143.42, 124.25, 77.77, 76.88, 35.42. ESI-TOF (HRMS) m/z [M+H]+ calculated for C₈H₆ClN₄⁺ 193.0276, found 193.0276.

2-Chloro-9-(prop-2-yn-1-yl)-9H-purine (Pu-6)

¹H NMR (600 MHz, DMSO-d₆) δ 9.13 (s, 1H), 8.72 (s, 1H), 5.17 (d, J=2.5 Hz, 2H), 3.57 (t, J=2.6 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 152.96, 152.57, 150.16, 147.43, 132.96, 77.24, 76.76, 32.86. ESI-TOF (HRMS): m/z [M+H]+ calculated for C₈H₆ClN₄⁺ 193.0276, found 193.0275.

2-Amino-6-chloro-9-(prop-2-yn-1-yl)-9H-purine (Pu-8)

¹H NMR (600 MHz, DMSO-d₆) δ 8.17 (s, 1H), 7.02 (s, 2H), 4.93 (d, J=2.5 Hz, 2H), 3.48 (t, J=2.5 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 159.92, 153.56, 149.51, 142.34, 123.06, 77.89, 76.08, 32.40. ESI-TOF (HRMS) m/z [M+H]+ calculated for C₈H₇ClN5⁺ 208.0384, found 208.0384.

6-chloro-2-fluoro-9-(prop-2-yn-1-yl)-9H-purine (Pu-10)

¹H NMR (600 MHz, DMSO-d₆) δ 8.75 (s, 1H), 5.16 (d, J=2.6 Hz, 2H), 3.60 (t, J=2.6 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 156.99, 155.57, 153.42, 150.67, 147.66, 129.93, 77.00, 76.88, 33.45. ESI-TOF (HRMS): m/z [M+H]+ calculated for C₈H₅ClFN₄⁺ 211.0181, found 211.0182.

General Procedure for the Preparation of 1-Alkylthiol Adducts:

As shown in Scheme 5, above, into a 50 mL round bottom flask was placed the dichloropurine compound (831 mg, 3.66 mmol), DMF (10 mL), potassium carbonate (powdered, 556 mg, 4.03 mmol) and n-butanethiol (373 mg, 4.14 mmol). The reaction was stirred under nitrogen at ambient temperature for 16 hours. The reaction was partitioned between ethyl acetate and water (25 mL/40 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (2×25 mL). The combined organic layer was washed with water (25 mL) and brine (25 mL). It was then dried over magnesium sulfate and concentrated to give crude product that was purified on a single cartridge flash purification system sold under the tradename ISOLERA™ One (Biotage, Uppsala, Sweden) to give 650 mg of product as an off-white solid.

6-(Butylthio)-2-chloro-7-(prop-2-yn-1-yl)-7H-purine (Pa-1)

¹H NMR (600 MHz, Chloroform-d) δ 8.27 (s, 1H), 5.26-5.21 (m, 2H), 3.47-3.40 (m, 2H), 2.67 (t, J=2.6 Hz, 1H), 1.82-1.72 (m, 2H), 1.51 (dq, J=14.7, 7.4 Hz, 2H), 0.98 (t, J=7.4 Hz, 3H). ESI-TOF (HRMS): m/z [M+H]+ calculated for C₁₂H₁₄ClN₄S⁺ 281.0622, found 281.0621.

The other adducts were prepared via analogous reactions using other probes as the starting materials. For example, Pa-6 was prepared using Pu-10 as the starting material in place of the Pu-1 used in Scheme 5.

6-(butylthio)-2-fluoro-9-(prop-2-yn-1-yl)-9H-purine (Pa-6)

¹H NMR (600 MHz, Chloroform-d) δ 8.11 (s, 1H), 4.94 (d, J=2.6 Hz, 2H), 3.39-3.33 (m, 2H), 2.54 (t, J=2.6 Hz, 1H), 1.80-1.73 (m, 2H), 1.50 (dq, J=14.8, 7.4 Hz, 2H), 0.96 (t, J=7.4 Hz, 3H). ESI-TOF (HRMS): m/z [M+H]+ calculated for C₁₂H₁₄FN₄S⁺ 265.0918, found 265.0918.

X-Ray crystal structures were obtained for Pu-1, Pu-3, Pu-8, and Pu-10 and were in agreement with the assigned N7- or N9-substituted regioisomer structure.

General Procedure for the Preparation of Purine Inhibitors

As shown in Scheme 6, above, into a 250 mL round bottom flask was placed 2,6-dichloropurine (1.16 g, 6.13 mmol), DMF (50 mL), potassium carbonate powder (848 mg, 6.13 mmol) and benzyl bromide (1.05 g, 6.13 mmol). The reaction was stirred under nitrogen at room temperature. After 16 hours the reaction was partitioned between ethyl acetate and water (100 mL each). The layers were separated and the aqueous layer was extracted with ethyl acetate (2×100 mL). The combined organic layer was dried over MgSO₄ and concentrated on the rotovap (crude weight: 4.10 g). The crude compounds were purified on a single cartridge flash purification system sold under the tradename ISOLERA™ One (Biotage, Uppsala, Sweden) using 20% ethyl acetate/hexanes. This provided the N7 and N9 isomers (220 mg and 800 mg, respectively).

7-Benzyl-2,6-dichloro-7H-purine (P1-1)

¹H NMR (600 MHz, DMSO-d₆) δ 9.05 (s, 1H), 7.36 (t, J=7.3 Hz, 2H), 7.31 (t, J=7.3 Hz, 1H), 7.21 (d, J=6.9 Hz, 2H), 5.74 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 163.38, 152.87, 151.09, 143.19, 136.38, 128.82, 127.94, 126.55, 121.89, 49.54. ESI-TOF (HRMS) m/z [M+H]+ calculated for C₁₂H₉C₁₂N₄⁺ 279.0198, found 279.0197.

9-Benzyl-2,6-9H-purine (Pi-2)

¹H NMR (600 MHz, DMSO-d₆) δ 8.85 (s, 1H), 7.39-7.29 (m, 5H), 5.50 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 153.37, 151.11, 149.79, 148.40, 135.61, 130.49, 128.80, 128.09, 127.59, 47.07. ESI-TOF (HRMS) m/z [M+H]+ calculated for C₁₂H₉C₁₂N₄⁺ 279.0198, found 279.0199.

Other purine inhibitor compounds were prepared via analogous reactions using different halides or sulfonyl halides in place of the benzyl bromide used in Scheme 6.

7-Allyl-2,6-dichloro-7H-purine (P1-3)

¹H NMR (600 MHz, DMSO-d₆) δ 8.89 (s, 1H), 6.17-6.09 (m, 1H), 5.24 (d, J=10.5 Hz, 1H), 5.12 (dt, J=5.0, 1.7 Hz, 2H), 4.97 (d, J=17.2 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 163.22, 152.47, 150.94, 143.16, 133.61, 121.89, 117.48, 48.51. ESI-TOF (HRMS) m/z [M+H]+ calculated for C₈H₇C₁₂N₄⁺ 229.0042, found 229.0042.

9-Allyl-2,6-dichloro-9H-purine (Pi-4)

¹H NMR (600 MHz, DMSO-d₆) δ 8.72 (s, 1H), 6.12-6.03 (m, 1H), 5.25 (d, J=10.4 Hz, 1H), 5.12 (d, J=17.2 Hz, 1H), 4.91 (dt, J=5.5, 1.6 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 153.35, 150.98, 149.64, 148.37, 132.11, 130.43, 118.30, 45.90. ESI-TOF (HRMS) m/z [M+H]+ calculated for C₈H₇C₁₂N₄⁺ 229.0042, found 229.0043.

2,6-Dichloro-7-(4-nitrobenzyl)-7H-purine (Pi-5)

¹H NMR (600 MHz, DMSO-d₆) δ 9.07 (s, 1H), 8.19 (d, J=8.8 Hz, 2H), 7.46 (d, J=9.0 Hz, 2H), 5.90 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 163.48, 152.98, 151.23, 147.08, 144.08, 143.15, 127.73, 123.86, 121.99, 49.06. ESI-TOF (HRMS) m/z [M+H]+ calculated for C₁₂H₈Cl₂N₅O₂⁺ 324.0049, found 324.0051.

2,6-Dichloro-9-(4-nitrobenzyl)-9H-purine (Pi-6)

¹H NMR (600 MHz, DMSO-d₆) δ 8.87 (s, 1H), 8.21 (d, J=8.9 Hz, 2H), 7.57 (d, J=8.9 Hz, 2H), 5.68 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 153.49, 151.19, 149.87, 148.45, 147.20, 143.01, 130.60, 128.70, 123.85, 46.41. ESI-TOF (HRMS) m/z [M−H]− calculated for C₁₂H₆C₁₂N₅O₂⁻ 321.9904, found 321.9902.

4-((2,6-Dichloro-9H-purin-9-yl)sulfonyl)morpholine (Pi-8)

¹H NMR (600 MHz, DMSO-d₆) δ 8.93 (s, 1H), 3.69-3.66 (m, 4H), 3.44-3.40 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 152.36, 152.15, 150.84, 145.87, 131.35, 65.12, 46.13. ESI-TOF (HRMS) m/z [M−H]− calculated for C₉H₈C₁₂N₅O₃S⁻ 335.9730, found 335.9728.

2-(2,6-Dichloro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Pi-10)

¹H NMR (600 MHz, DMSO-d₆) δ 8.98 (s, 1H), 5.97 (d, J=4.9 Hz, 1H), 5.60 (d, J=5.7 Hz, 1H), 5.26 (d, J=5.4 Hz, 1H), 5.08 (t, J=5.4 Hz, 1H), 4.51 (q, J=5.1 Hz, 1H), 4.20-4.15 (m, 1H), 3.99 (q, J=4.0 Hz, 1H), 3.71 (ddd, J=12.0, 5.3, 3.9 Hz, 1H), 3.59 (ddd, J=12.0, 5.5, 3.9 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 153.06, 151.12, 149.85, 146.39, 131.01, 88.25, 85.67, 74.04, 69.83, 60.74.

2,6-Dichloro-7-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl)-7H-purine (Pi-11)

¹H NMR (600 MHz, DMSO-d₆) δ 9.07 (s, 1H), 7.31 (s, 1H), 7.27 (d, J=8.3 Hz, 1H), 6.94-6.90 (m, 1H), 5.65 (s, 2H), 1.61 (s, 4H), 1.19 (d, J=4.5 Hz, 12H). ¹³C NMR (151 MHz, DMSO-d₆) δ 163.32, 152.75, 151.04, 144.77, 144.22, 143.14, 133.14, 126.94, 125.30, 123.99, 121.80, 49.47, 34.39, 33.90, 33.73, 31.48, 31.44. ESI-TOF (HRMS) m/z [M+H]+ calculated for C₂₀H₂₃C₁₂N₄⁺ 389.1294, found 389.1296.

2,6-Dichloro-9-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl)-9H-purine (Pi-12)

¹H NMR (600 MHz, DMSO-d₆) δ 8.86 (s, 1H), 7.46 (d, J=2.0 Hz, 1H), 7.28 (d, J=8.2 Hz, 1H), 7.03 (dd, J=8.1, 2.0 Hz, 1H), 5.41 (s, 2H), 1.60 (s, 4H), 1.20 (d, J=19.9 Hz, 12H). ¹³C NMR (151 MHz, DMSO-d₆) δ 153.29, 151.04, 149.78, 148.31, 144.78, 144.34, 132.57, 130.50, 126.88, 126.28, 124.92, 47.17, 34.40, 34.38, 33.87, 33.72, 31.50, 31.44. ESI-TOF (HRMS) m/z [M+H]+ calculated for C₂₀H₂₃C₁₂N₄⁺ 389.1294, found 389.1298.

2,6-Dichloro-7-((4-methoxyphenyl)sulfonyl)-7H-purine (Pi-15)

¹H NMR (600 MHz, DMSO-d₆) δ 8.74 (s, 1H), 7.52 (d, J=8.8 Hz, 2H), 6.85 (d, J=8.8 Hz, 2H), 3.75 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 165.21, 152.60, 151.34, 151.10, 144.99, 131.50, 131.15, 126.31, 115.39, 56.19.

2,6-Dichloro-7-((4-methoxyphenyl)sulfonyl)-7H-purine (Pi-16)

¹H NMR (600 MHz, DMSO-d₆) δ 9.14 (s, 1H), 8.16 (d, J=9.1 Hz, 2H), 7.25 (d, J=9.1 Hz, 2H), 3.87 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 165.20, 152.60, 151.34, 151.10, 144.98, 131.49, 131.14, 126.31, 115.38, 56.19.

General Procedure for the Preparation of Acyl-Modified Purine Inhibitors:

As shown in Scheme 7, above, into a 250 mL round bottom flask was placed the dichloropurine (7.57 g. 40.0 mmol DMF (100 mL), Hunig's base (8.4 mL, 48.1 mmol) and 1-butanethiol (4.72 mL, 44.1 mmol). The reaction mixture was stirred under nitrogen at ambient temperature. After 16 hours the reaction was diluted with water (150 mL) and extracted with ether (3×100 mL). The combined organic layer was washed with brine (50 mL) and dried over magnesium sulfate. This provided 1.678 grams of an off-white solid after concentration. The proton NMR showed the presence of 1-butanethiol. The solid was triturated with 30 mL of ether for 1 hours. The remaining solid was isolated by filtration to give 1.15 g of a white solid. The proton NMR indicated no remaining 1-butanethiol. NMR data was consistent with data reported in the literature.

This compound was then acylated by placing the monochloro-compound (104.5 mg, 0.431 mmol) into a 1 dram vial along with anhydrous acetonitrile (2 mL) and acetic anhydride (81.4 uL, 0.861 mmol). The reaction mixture was heated to 75° C. under nitrogen for 16 hours. The reaction was concentrated and the residue was purified on a single cartridge flash purification system sold under the tradename ISOLERA™ One (Biotage, Uppsala, Sweden; 4 g silica column, 10% EtOAc/Hexanes to 65% EtOAc/Hexanes). This provided 104.3 mg of product as a white crystalline solid (85% yield).

1-(6-(Butylthio)-2-chloro-9H-purin-9-yl)ethan-1-one (AHL20-001)

¹H NMR (600 MHz, DMSO-d₆): δ 8.89 (s, 1H), 3.32 (t, J=7.3 Hz, 2H), 2.81 (s, 3H), 1.68 (m, 2H), 1.41 (m, 2H), 0.90 (t, J=7.33 Hz, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 167.9, 163.8, 153.7, 149.3, 143.5, 131.5, 31.2, 28.6, 25.3, 21.7, 13.9. ESI-QTOF (HRMS) m/z [M+H]+ calculated for C₁₁H₁₄ClN₄OS⁺ 285.0571, found 285.0576

ESI-QTOF Method (high-resolution mass spectrometry, HRMS): Compounds were dissolved in either methanol or acetone (Pi-6 and Pi-8) (500 ng/mL) and filtered through 0.2 um teflon syringe filters. Compounds were analyzed using a 1260 Infinity II LC with an Agilent 6545 Q-TOF MS (Agilent Technologies, Santa Clara, Calif., United States of America). An atmospheric pressure chemical ionization (APCI source) was utilized for analysis. Analytes were separated using an Agilent ZORBAX™ RRHD Eclipse Plus C18, 2.1 mm ID×50 mm L, 1.8 um particle diameter, 95 angstrom pore size column with 99.9% MeOH+0.1% formic acid (0.4 mL/min flow rate, 40 C column compartment). Data acquisition occurred for 1 minute. MS acquisition used the following parameters: Gas temperature: 325° C.; Vaporizer temperature: 350° C.; Dry gas: 10 L/min; Nebulizer: 60PSI; Corona Voltage: 4 uA; Vcap: 3500V; Fragmentor: 180V; Skimmer: 45V; October 1 RFVpp: 750V; Acquisition: 50-1700 m/z; Rate: 3 spectra/sec; Time: 333.3 ms/spectra; Transients/spectrum: 2665. Positive Mode Deviations: A) 5 μL injection B) Lock masses: 121.050873 & 922.009798; Negative Mode Deviation: A) 10 μL injection B) Lock masses 119.03632 & 966.000725.

HPLC assay for profiling solution purity of purine probes, ligands and fragments: The following reagents were prepared and stored on ice prior to use. 0.1 M solution of caffeine in acetonitrile, 1 M HOAc in ACN and 10 mM solution of purine compound in ACN mixture. 500 μL of purine solution were transferred to a dram vial on ice. 50 μL aliquots were removed quenched with 10 μL of a 1:1 mixture of caffeine and HOAc. Samples were injected (1 μL) and analyzed by reverse-phase HPLC on a Shimadzu 1100 Series spectrometer with UV detection at 254 nm. Chromatographic separation was performed using a Phenomenex Kinetex C18 column (2.6 μm, 50×4.6 mm). Mobile phases A and B were composed of H₂O+0.1% HOAc and ACN+0.1% HOAc, respectively. Samples were analyzed using the following analytical conditions: using a flow rate of 0.8 mL min⁻¹, the gradient was as follows: 0-0.5 min, 15% B; 0.5-6.5 min 85% B; 6.5-7 min 100% B; 7-8.5 min 100% B; 8.5-9 min 15% B; 9-9.8 min 15% B. Note: Probes (i.e Pu-compounds) were not spiked with caffeine but were rather diluted with 10 uL of ACN.

The purities of the compounds as determined by HPLC were as follows:

For the probes (Pu compounds): Pu-1, Pu-2, Pu-3, Pu-4, Pu-5, Pu-7, Pu-8 were all greater than 99% pure; Pu-6 was greater than 95% pure; Pu-9 was greater than 97% pure; and Pu-10 was greater than 98% pure.

For the ligands (Pi compounds): Pi-1 and Pi-12 were each greater than 98% pure; Pi-2, Pi-4, Pi-5, Pi-6 and Pi-10 were each greater than 99% pure; Pi-3 was greater than 95% pure; and Pi-13 was greater than 97% pure.

For the fragments (Pa compounds): Pa-1 was greater than 98% pure and Pa-3 was greater than 95% pure. For AHL20-001, purity was determined to be greater than 96%.

Example 2

Probe Reactivity and Stability

HPLC assay for profiling solution reactivity and stability of purine fragments: The following reagents were prepared and stored on ice prior to use. 0.1 molar (M) solution of caffeine in acetonitrile, 1.0 M solution of amino acid mimetic (butanethiol—cysteine mimetic; n-butylamine—lysine mimetic; p-cresol—tyrosine mimetic; propionamide-asparagine/glutamine mimetic; butyric acid—aspartic/glutamic acid mimetic), tetramethylguanidine (TMG), 1 M acetic acid (HOAc) in acetonitrile (ACN) and 10 mM solution of purine fragment in ACN mixture. 500 μL of fragment solution were transferred to a dram vial on ice. To the mixture, 5.5 μL of TMG and 5.5 μL of respective amino acid mimetic were added and solutions were stirred on ice for 6 h. To monitor reactivity, 50 μL aliquots were removed at indicated time points and quenched with 10 μL of a 1:1 mixture of caffeine and HOAc. Samples were injected (1 μL) and analyzed by reverse-phase HPLC on a Shimadzu 1100 Series spectrometer (Shimadzu Corporation, Kyoto, Japan) with UV detection at 254 nm. Reaction progress was evaluated by monitoring consumption of starting material (purine fragment) normalized to caffeine standard. Chromatographic separation was performed using a Phenomenex Kinetex C18 column (2.6 μm, 50×4.6 mm; Phenomenex, Torrance, Calif., United States of America). Mobile phases A and B were composed of H₂O+0.1% HOAc and ACN+0.1% HOAc, respectively. Samples were analyzed using the following analytical conditions: using a flow rate of 0.8 mL min⁻¹, the gradient was as follows: 0-0.5 min, 15% B; 0.5-6.5 min 85% B; 6.5-7 min 100% B; 7-8.5 min 100% B; 8.5-9 min 15% B; 9-9.8 min 15% B. The amount of purine fragment consumed was calculated using the area under the curve (AUC) for the fragment peak at time (t)=experimental/t=0. All purine fragment peak AUCs used for calculations were normalized to caffeine standard AUCs at respective time points to account for run-to-run variations by HPLC. The amount of purine fragment consumed (% starting material) was plotted as a function of time.

Discussion: An HPLC assay for determining the reactivity of the purine-based probes with mimetics of amino acid residues was performed. FIG. 3A shows the assay using butanethiol as a small molecule mimetic for cysteine. Reaction progress was monitored by monitoring the disappearance of starting material and appearance of product via HPLC.

Results of the HPLC analysis of the solution-based reactions of the purine-based probes Pu-1-Pu-10 with amino acid mimetics are shown in FIGS. 3B and 3C. As shown in FIG. 3B, Pu-1 modifies cysteine (Cys) and tyrosine (Tyr) amino acid mimetics in solution-based studies, while Pu-2 also exhibits mild lysine (Lys) reactivity. These probes are unreactive with Asp/Glu amino acid mimetic butyric acid and Gln/Asn amino acid mimetic propionamide. In both cases, very little starting material mimetic is consumed in 6 hours. The observed reaction kinetics support Cys chemoselectivity for halogenated purines. The N7 tautomer (Pu-1) is more reactive towards nucleophilic attack compared with the N9 counterpart (Pu-2).

As shown in FIG. 3C, Pu-1, Pu-2, Pu-3, Pu-9 and Pu-10 all react with butanethiol, while other probes do not. N7 tautomers (Pu-1, Pu-3, Pu-9) of purine molecules display accelerated reaction kinetics compared to N9 counterparts (Pu-2, Pu-4, Pu-10). Based on the HPLC traces, it appears that Pu-9 and Pu-10 likely form many different adducts (i.e. addition at the 2 position, the 6 position, and dual modification at 2 and 6). The position of the halogen leaving group affects reactivity: the 6 position enhances reactivity compared to the leaving group on the 2 position (Pu-3 versus Pu-5 and Pu-4 versus Pu-6). The addition of an amine group at the 2 position dramatically reduces the electrophilic character of N7 (Pu-7) and N9 (Pu-8) halogenated purines.

Example 3

Inhibitor Reactivity and Stability

HPLC assay for profiling solution reactivity and stability of purine inhibitors—The following reagents were prepared and stored on ice prior to use. 0.1 M solution of caffeine in acetonitrile, 1.0 M solution of amino acid mimetic (butanethiol), tetramethylguanidine (TMG), 1 M HOAc in ACN and 10 mM solution of purine fragment in ACN mixture. 500 μL of fragment solution were transferred to a dram vial on ice. To the mixture, 5.5 μL of TMG and 5.5 μL of respective amino acid mimetic were added and solutions were stirred on ice for 6 h. To monitor reactivity, 50 μL aliquots were removed at indicated time points and quenched with 10 μL of a 1:1 mixture of caffeine and HOAc. Samples were injected (1 μL) and analyzed by reverse-phase HPLC on a Shimadzu 1100 Series spectrometer (Shimadzu Corporation, Kyoto, Japan) with UV detection at 254 nm. Reaction progress was evaluated by monitoring consumption of starting material (purine fragment) normalized to caffeine standard. Chromatographic separation was performed using a Phenomenex Kinetex C18 column (2.6 μm, 50×4.6 mm; Phenomenex, Torrance, Calif., United States of America). Mobile phases A and B were composed of H₂O+0.1% HOAc and ACN+0.1% HOAc, respectively. Samples were analyzed using the following analytical conditions: using a flow rate of 0.8 mL min⁻¹, the gradient was as follows: 0-0.5 min, 15% B; 0.5-6.5 min 85% B; 6.5-7 min 100% B; 7-8.5 min 100% B; 8.5-9 min 15% B; 9-9.8 min 15% B. The amount of purine fragment consumed was calculated using the area under the curve (AUC) for the fragment peak at time (t)=experimental/t=0. All purine fragment peak AUCs used for calculations were normalized to caffeine standard AUCs at respective time points to account for run-to-run variations by HPLC. The amount of purine fragment consumed (% starting material) was plotted as a function of time.

Discussion: An HPLC assay analogous to that shown in FIG. 3A was used to determine the reactivity of the purine ligands for butanethiol, a mimetic of cysteine solution. In general, the N7 tautomers of purine ligands were more reactive with butanethiol nucleophile. FIGS. 3D and 3E show the comparative reactivity of Pi-1 and Pi-2 and Pi-3 and Pi-4 The N9 tautomer counterparts, while overall less reactive, permit tuning capabilities. There were some exceptions to the higher reactivity of the N7 tautomers. Pi-13 and Pi-14 had essentially equivalent reactivity for butanethiol.

Example 4

Solution Reactivity and Stability of Purine Adduct Fragments

HPLC assay for profiling solution reactivity and stability of purine adduct fragments: The following reagents were prepared and stored on ice prior to use. 0.1 M solution of caffeine in acetonitrile, 1.0 M solution of amino acid mimetic (butanethiol, n-butylamine, p-cresol, propionamide and butyric acid), tetramethylguanidine (TMG), 1 M HOAc in ACN and 10 mM solution of purine fragment (e.g., one of the purine adducts shown in Scheme 8, above) in ACN mixture. 500 μL of fragment solution (i.e. purified Pa-1 through Pa-6) were transferred to a dram vial on ice. To the mixture, 5.5 μL of TMG and 5.5 μL of respective amino acid mimetic were added and solutions were stirred on ice for 6 h. To monitor reactivity, 50 μL aliquots were removed at indicated time points and quenched with 10 μL of a 1:1 mixture of caffeine and HOAc. Samples were injected (1 μL) and analyzed by reverse-phase HPLC on a Shimadzu 1100 Series spectrometer with UV detection at 254 nm. Reaction progress was evaluated by monitoring consumption of starting material (purine fragment) normalized to caffeine standard. Chromatographic separation was performed using a Phenomenex Kinetex C18 column (2.6 μm, 50×4.6 mm). Mobile phases A and B were composed of H₂O+0.1% HOAc and ACN+0.1% HOAc, respectively. Samples were analyzed using the following analytical conditions: using a flow rate of 0.8 mL min′, the gradient was as follows: 0-0.5 min, 15% B; 0.5-6.5 min 85% B; 6.5-7 min 100% B; 7-8.5 min 100% B; 8.5-9 min 15% B; 9-9.8 min 15% B. The amount of purine adduct fragment consumed was calculated using the area under the curve (AUC) for the fragment peak at time (t)=experimental/t=0. All purine fragment peak AUCs used for calculations were normalized to caffeine standard AUCs at respective time points to account for run-to-run variations by HPLC. The amount of purine fragment consumed (% starting material) was plotted as a function of time.

Results: The structures of exemplary purine adduct probe compounds are shown in Scheme 8, above. Purine adduct probes (Pa series) Pa-3, Pa-4, and Pa-6 showed mild reactivity with nucleophiles. Pa-1 showed little reactivity with either butanethiol or p-cresol. Pa-3 shows a preference for butanethiol while the other adducts show little preference. The N9 tautomer of Pa compounds (Pa-4) appears to be more reactive against nucleophiles. See FIG. 3F. The presence of the halogen group at the 2-position of 6-substituted purine compounds (Pa-4) results in higher reactivity compared with 6-halogen, 2-substituted counterparts (Pa-3). See FIG. 3G. A fluoro group at the 2-position of 6-substituted purines (Pa-6) results in equivalent reactivity against butanethiol and p-cresol.

Example 5

Activity of Purine Probes in Live Cells and Cell Lysates

Live Cell Activity Method: DM93 cells were grown at 37° C. in 5% CO2 until 90% confluent. Once confluent cells were washed with serum free media and treated with halogenated purine probes at a final concentration of 25 μM (unless stated) for 4 hours (unless stated). Cells were then scraped and washed 3× with cold PBS and lysed in PBS+protease inhibitor. Lysates were spun at 100,000×g for 45 minutes. Halogen probe-modified proteins in soluble fractions were visualized by conjugating rhodamine-azide using copper-catalyzed azide-alkyne cycloaddition (CuAAC; 1 hour, room temperature), subjected to SDS-PAGE, and detected by in-gel fluorescence scanning. SDS-PAGE gels were also stained with Coomassie brilliant blue to determine protein load. All samples were loaded with equivalent amounts of protein. Thus, changes in purine probe labeling is not due to loading of different proteome amounts.

Discussion: A scheme for determining the activity of purine probes in live cells or cell lysates using gel-based analysis is shown in FIG. 4A. As shown in the left panel of FIG. 4B, AHL125 (Pu-1) AHL128 (Pu-2), 6-chloro-2-fluoro-(prop-2-yn-1-yl)-7H-purine (Pu-9) and 6-chloro-2-fluoro-(prop-2-yn-1-yl)-9H-purine (Pu-10) display robust labeling profiles in live DM93 cells. Pu-9 and Pu-10 are likely generating complex adducts (i.e. reaction with cysteine and other nucleophilic amino acids) based on HPLC reactivity data. Enhanced probe labeling with 6-chloro-(prop-2-yn-1-yl)-7H-purine (Pu-3) and 6-chloro-(prop-2-yn-1-yl)-9H-purine (Pu-4) compared to 2-chloro-(prop-2-yn-1-yl)-7H-purine (Pu-5) and 2-chloro-(prop-2-yn-1-yl)-9H-purine (Pu-6) suggest that the 6-chloro position is more electrophilic and reactive towards nucleophilic attack. Comparison between the N7 (Pu-1, Pu-3 and Pu-9) and N9 tautomers (Pu-2, Pu-4, and Pu-10) demonstrate the N7 analogs are more reactive. AHL-Pu-1 and AHL-Pu-1 show concentration dependent labeling in live cells. See FIG. 4B, middle panel. AHL-Pu-1 and AHL-Pu-2 react in a time dependent manner, which supports a covalent reaction mechanism. See FIG. 4B, right panel.

Lysate Activity Method: DM93 cells were grown at 37° C. in 5% CO2 until 90% confluent. Once confluent, cells were scraped and washed 3× times with cold PBS and lysed in PBS+protease inhibitor. Lysates were spun at 100,000×g for 45 minutes. Soluble fractions were treated with 25 μM of halogenated purine probes (unless stated) for 2 hrs (unless stated) at 37° C. Purine probe modified proteins were conjugated to rhodamine-azide by CuAAC, subjected to SDS-PAGE analysis, and detected by in-gel fluorescence scanning. SDS-PAGE gels were stained with Coomassie brilliant blue to determine protein load. All samples were loaded with equivalent amounts of protein to demonstrate changes in purine probe labeling is not due to loading of different proteome amounts.

Discussion: Similar to the live cell treatments, AHL-Pu-1, AHL-Pu-2, AHL-Pu-9 and AHL-Pu-10 show the highest protein labeling activity. See FIG. 4C, left panel. Increased labeling using AHL-Pu-3 and AHL-Pu-4 compared to AHL-Pu-5 and AHL-Pu-6 lane suggest that the 6-Chloro position is more electrophilic towards nucleophilic attack. Comparison between the N7 and N9 tautomers demonstrate the N7 analogs (AHL-Pu-1, AHL-Pu-3 and AHL-Pu-9) are more reactive. AHL-Pu-1 displays concentration dependent labeling of proteomes. See FIG. 4C, middle panel. AHL-Pu-1 and AHL-Pu-2 show time dependence in protein labeling and supports a covalent reaction mechanism with proteins in proteomes. See FIG. 4C, right panel.

Example 6

Activity of Purine Probes in In Vivo

Mouse Treatment and Tissue Preparation Methods: 8-12 week old male and female C57Bl/6 mice were treated intraperitoneally (IP) or by oral gavage (OG) with either AHL125 (Pu-1 or AHL-Pu-1), AHL128 (Pu-2 or AHL-Pu-2), or vehicle (18:1:2, PBS:PEG40:DMSO) at 20 mg/kg unless stated otherwise. Treatment time is indicated in the experiment below. Mice were then euthanized and perfused using PBS. Tissues were then harvested, rinsed and flash frozen using liquid nitrogen and stored at −80° C. until further use. Tissues were lysed using dounce homgenization in the presence of PBS+protease inhibitor. Initial suspensions were centrifuged at 3000×g for 5 minutes to remove insoluble material. The lysate was collected and centrifuged again at 100,000×g for 45 minutes to obtain the soluble fraction. Purine probe modified proteins from treated mice were conjugated to rhodamine-azide by CuAAC, subjected to SDS-PAGE analysis, and detected by in-gel fluorescence scanning. SDS-PAGE gels were stained with Coomassie brilliant blue to determine protein load. All samples were loaded with equivalent amounts of protein. Thus, changes in purine probe labeling is not due to loading of different proteome amounts.

Mice were also treated with 80 mg/kg oral gavage (OG) to determine whether purine probes are orally bioavailable. Mice were treated for four hours.

Discussion: Purine probes show concentration dependent protein labeling activity in animals. As shown in FIGS. 5A-5G, AHL125 (Pu-1) displays the most robust labeling profile in vivo across several tissues (lung, liver, spleen, heart, brain, kidney and white adipose tissue (WAT)) with highest activity in lung. These data support the hypothesis that the N7 tautomer is more electrophilic towards nucleophilic attack. Data supporting oral bioavailability of purine probes was observed in several tissues including lung and spleen. The brain shows some labeling activity, suggesting that purine probes penetrate the blood brain barrier. Pu-5 was used as a negative control.

Time dependent labeling in vivo was studied using 20 mg/kg probe. In general, AHL125 (Pu-1; see FIG. 6, left panel) displayed peak labeling at 2 hours while AHL128 (Pu-2; FIG. 6, right panel) shows the most robust labeling signal at 4 hours. Tissue comparison data suggests robust labeling in the lung with some liver labeling as well. The white adipose tissues also show a very robust labeling profile. Without being bound to any one theory, these results are believed to be related to 1) site of injection (increased time and concentration) and 2) good bioavailiability due to the large amount of lipid.

Example 7

Purine Ligands Targeting Human Selenocysteine-Specific Elongation Factor (eEF-SEC)

Methods: HEK293T cells were grown at 37° C. in 5% CO₂ until 40% confluent. Recombinant human eEF-Sec (Uniprot ID P57772) was expressed by transient transfection for 48 hours. Afterwards, cells were scraped and washed 3× times with cold PBS and lysed in PBS+protease inhibitor. Lysates were spun at 100,000×g for 45 minutes. Soluble or membrane fractions were treated with purine probes for 2 hrs or for a predetermined period of time at 37° C. Purine probe modified proteins were conjugated to rhodamine-azide by CuAAC, subjected to SDS-PAGE analysis, and detected by in-gel fluorescence scanning. Comparison of non-transfected (Mock) and transfected proteomes was used to identify the recombinant eEF-Sec purine probe-labeled band. Purine ligand (Pi compounds) activity was evaluated by pretreating lysates with Pi ligands for a predetermined time followed by labeling with purine probe. Reduction in fluorescent signals from purine probe labeling was indicative of Pi ligand inhibitory activity. Mut represents eEF-Sec mutant where cysteine residue 442 is mutated to alanine (C₄₄₂A).

SDS-PAGE gels were transferred to nitrocellulose membrane. Nitrocellulose blots were blocked with 5% BSA. Blots were washed five times with TBS-T. Recombinant protein expression was detected using an anti-FLAG primary antibody (1:1,000) followed by fluorescent secondary antibody (1:10,000) to determine if there was equivalent protein expression across different treatment conditions.

Discussion: The selenocysteine elongation factor (eEF-Sec) was identified as a target from initial LC-MS/MS experiment (see FIG. 7) using AHL125 (Pu-1). eEF-Sec is a translation factor that assists in the production of Sec-proteins (of which the human proteome contains 25). eEF-Sec is a 4 domain GTP binding protein that acts as the translation factor responsible for inserting selenocysteine (Sec) into proteins. Dysregulation of selenoproteins have been identified in a variety of disease pathologies, including cardiac, muscular, nervous system, endocrine system, immune system, and reproductive system disorders and diseases. Currently, there are no available inhibitors to study this protein. Based on the initial LC-MS/MS experiment, it appears that Pu-1 (AHL125) binds at C422 of eEF-Sec. See FIG. 10A.

A recombinant protein band at ˜65 kDa in the gels of transfected but not mock samples supported expression of eEF-Sec in HEK293T cells. Equivalent expression across different treatment conditions support changes in purine probe labeling is not due to differences in recombinant protein expression.

Recombinant eEF-SEC was labeled in a time dependent manner by purine probes (50 μM AHL-Pu-1 or AHL-Pu-2) in live cells. See FIG. 10B. The absence of probe labeling in the C442A EEF SEC mutant supports purine probe labeling activity at cysteine 442 site of this protein. The lack of purine probe labeling in eEF-SEC C442A mutant (CS) compared with wild-type (WT) protein at different purine probe concentrations from in vitro labeling experiments (1 hr at 37° C.) is shown in FIG. 10C. Using a competition assay (see FIG. 8), purine ligands Pi-5 and Pi-8 (10 μM, see FIG. 9) can block AHL-Pu-1 probe labeling (25 μM) in a time dependent and concentration dependent manner in live cells. See FIGS. 10D and 10E. Pi-5 shows greater than 50% inhibition at 10 μM, while its regioisomer Pi-6 loses all apparent activity against the target at the same 10 μM.

Example 8

LCMS Detection of Purine Protein Adducts

Methods: SILAC DM93 cells were cultured at 37° C. with 5% CO2 in either “light” or “heavy” media supplemented with 10% dialyzed fetal bovine serum (Omega Scientific), 1% L-glutamine (Fisher Scientific), and isotopically labeled amino acids. Light media was supplemented with 100 μg mL⁻¹ L-arginine and 100 μg mL⁻¹ L-lysine. Heavy media was supplemented with 100 μg mL⁻¹ [¹³C₆¹⁵N₄] L-arginine and 100 μg mL⁻¹ [¹³C₆¹⁵N₂] L-lysine. Labelled amino acids were incorporated for at least five passages before utilizing SILAC cells for experiments. Cells grown to ˜90% confluency in 10 cm plates were treated with DMSO vehicle or purine compound in serum-free media at a final concentration of 25 μM for 4 hours at 37° C. with 5% CO₂. After treatment, cells were washed with cold PBS twice before collection and preparation for chemical proteomic evaluation. Protein concentrations were normalized to 2.3 mg mL⁻¹ and 432 μL (for 1 mg final protein amount) were used for sample preparation. Probe-modified proteomes were conjugated to desthiobiotin-PEG3-azide followed by enrichment of probe-modified peptides for nano-electrospray ionization-LC-MS/MS analyses as previously described¹⁵. Identification of peptides and proteins from tandem mass spectrometry analyses was accomplished using bioinformatics software and quality control criteria as previously described¹⁵.

Discussion: Proteomes were prepared according to the methods described above. Macrophage migration inhibitor factor (MIF, Uniprot ID P14174) was selected because it passed all quality control parameters (Byonic score>300, ratio dot product [RDOTP] and isotope dot product [IDOTP]>0.8). Additionally, this protein contained a single modified Cys residue (C81) and was only observed with Pu-1 treatments. Covalent reaction with Pu-1 adds +604.2631 Da to the modified amino acid C81 from MIF and supports the proposed purine reaction mechanism whereby the halogen (Cl) serves as the leaving group during modification with nucleophilic residues on proteins.

As an additional example of a purine probe protein adduct, the protein serine/threonine-protein kinase 38-like (STK38L, Uniprot ID Q9Y2H1) was selected because it passed all quality control parameters (Byonic score>300, ratio dot product [RDOTP] and isotope dot product [IDOTP]>0.8). Additionally, this protein contained a single modified Cys residue (C235) and was only observed with Pu-1 treatment. Covalent reaction with Pu-1 adds +604.2631 Da to the modified amino acid C235 from STK38L and supports the proposed purine reaction mechanism.

Example 9

Quantitative Chemical Proteomics and Bioinformatics Analysis of Pu-1 and Pu-2 Modified Proteins from Lysate and Live Cell Treatments

Cells were grown at 37° C. in 5% CO₂ until 90% confluent. Once confluent cells were washed with serum free media and treated with purine probes (Pu-1, Pu-2) at a final concentration of 25 μM for 4 hours. Cells were then scraped and washed 3× with cold PBS and lysed in PBS+protease inhibitor. Lysates were spun at 100,000×g for 45 minutes. Purine probe-modified proteins in soluble fractions were coupled to desthiobiotin-azide using copper-catalyzed azide-alkyne cycloaddition (CuAAC; 1 hour, room temperature), probe-modified proteins digested into peptides using trypsin protease, probe-modified peptides enriched by avidin affinity chromatography, and subjected to LC-MS quantitative chemical proteomics as previously described.¹⁵ The following cell lines were used for analysis: DM93, Hela, A549, HEK293T, and Jurkat. All data shown are for proteins with a cysteine site modified by purine probes.

Functional protein domains that are statistically significantly enriched by Pu-1 and Pu-2 purine probes were determined by Q<0.05 after Benjamini-Hochberg correction of a two-sided binomial test following previously described methods.′⁵ Evaluation of probe-enriched domains (cysteine site on target protein) revealed enriched functions that include proteins involved in nucleotide recognition, protein ubiquitination, ADP ribosylation, and protein kinases. See FIG. 11.

Pu-1- and Pu-2-modified proteins (cysteine site) were compared with DrugBank proteins (DBP proteins). Only twenty-six percent of the purine-modified proteins (137/525) were DPB proteins. The DBP proteins were subdivided into proteins with associated compounds that are FDA-approved drugs. Twenty percent of the purine-modified proteins were proteins with associated FDA-approved drugs. Non-DBP proteins are proteins that did not match a DrugBank entry. A large fraction of purine-modified proteins (74%, 388/525) were non-DBP proteins, and thus lack pharmacological probes and/or drugs.

Subcellular location analysis of Pu-1- and Pu-2-modified proteins from live cell studies was performed. Proteins with a modified cysteine site were grouped based on subcellular location using a published subcellular location analysis (SLA) algorithm.′ The analysis is summarized in the graph shown in FIG. 12, where the number of modified proteins compared with the number of proteins from the SwissProt database for each subcellular compartment (x-axis) using SLA analyses are shown (Proteins in Database, y-axis). The shading in the bars depicts the percentage of modified proteins from each subcellular compartment compared with all modified proteins quantified in datasets.

Tables 1 and 2, below, show the distribution of Pu-1- and Pu-2-modified sites (high confidence sites; Byonic score>300) among the nucleophilic amino acid residues detected in proteomes. Purine probes were chemoselective for cysteine residues on target proteins (˜80% of all purine probe-modified peptides).

TABLE 1
Amino Acid Selectivity of AHL125 (Pu-1).
Modified Amino	Number of Unique	Percentage of
Acid Residue	Modified Residues	Modified Residues
Cysteine (C)	196	82
Aspartic Acid (D)	9	3.8
Glutamic Acid (E)	12	5.0
Histidine (H)	1	0.42
Lysine (K)	4	1.7
Methionine (M)	2	0.84
Asparagine (N)	0	0
Glutamine (Q)	3	1.3
Arginine (R)	2	0.84
Serine (S)	1	0.42
Threonine (T)	2	0.84
Tryptophan (W)	1	0.42
Tyrosine (Y)	6	2.5

TABLE 2
Amino Acid Selectivity of AHL128 (Pu-2).
Modified Amino	Number of Unique	Percentage of
Acid Residue	Modified Residues	Modified Residues
Cysteine (C)	952	80
Aspartic Acid (D)	44	3.7
Glutamic Acid (E)	68	5.7
Histidine (H)	12	1.0
Lysine (K)	24	2.0
Methionine (M)	7	0.59
Asparagine (N)	0	0
Glutamine (Q)	39	3.3
Arginine (R)	19	1.6
Serine (S)	5	0.42
Threonine (T)	6	0.50
Tryptophan (W)	3	0.25
Tyrosine (Y)	17	21.4

Example 10

Proteins Modified by Purine-Based Probes

Chemical proteomics performed as described in Example 9 determined several protein modification sites targeted by Pu-1 and/or Pu-2. Tables 3 and 4, below, lists sites of modification targeted by Pu-1 and Pu-2, respectively. The format of the tables is as follows: protein species, protein Uniprot accession number, cysteine sites (amino acid positions) modified (where if multiple sites are modified in the same protein, the sites are separated by vertical lines).

TABLE 3
Protein Modification Sites Targeted by Pu-1.
Gene name	UniProt	Pu-1-modified Site
TACC3_HUMAN	Q9Y6A5	242|
TNPO3_HUMAN	Q9Y5L0	511|
CD2AP_HUMAN	Q9Y5K6	540|
PPME1_HUMAN	Q9Y570	381|
PRC2C_HUMAN	Q9Y520	895|
FARP1_HUMAN	Q9Y4F1	522|
WAC2C_HUMAN	Q9Y4E1	828|
KDM3A_HUMAN	Q9Y4C1	1140|
TLN1_HUMAN	Q9Y490	1939|
RBGP1_HUMAN	Q9Y3P9	476|
STRAP_HUMAN	Q9Y3F4	340|305|
UFC1_HUMAN	Q9Y3C8	116|
SF3B6_HUMAN	Q9Y3B4	74|
LC7L2_HUMAN	Q9Y383	348|
AR2BP_HUMAN	Q9Y2Y0	149|
2ABG_HUMAN	Q9Y2T4	330|
GUAD_HUMAN	Q9Y2T3	24|
PDIP2_HUMAN	Q9Y2S7	143|
RRP44_HUMAN	Q9Y2L1	533|
ST38L_HUMAN	Q9Y2H1	235|
AKAP2_HUMAN	Q9Y2D5	296|
LZTS1_HUMAN	Q9Y250	385|261|
SRRM2_HUMAN	Q9UQ35	1029|
UBP24_HUMAN	Q9UPU5	1556|
PZRN3_HUMAN	Q9UPQ7	1002|
LIMC1_HUMAN	Q9UPQ0	577|333|182|
TRI33_HUMAN	Q9UPN9	943|786
TTF2_HUMAN	Q9UNY4	204|
TIM_HUMAN	Q9UNS1	1126|
PACN2_HUMAN	Q9UNF0	163|
SOX13_HUMAN	Q9UN79	33|
NOL7_HUMAN	Q9UMY1	149|
TPX2_HUMAN	Q9ULW0	536|301|
COR1C_HUMAN	Q9ULV4	456|
MRTFB_HUMAN	Q9ULH7	282|
NUP50_HUMAN	Q9UKX7	333|
ACINU_HUMAN	Q9UKV3	546|
TF3C4_HUMAN	Q9UKN8	116|
BAZ1B_HUMAN	Q9UIG0	338|
BI2L1_HUMAN	Q9UHR4	182|
NARF_HUMAN	Q9UHQ1	99|
CHRD1_HUMAN	Q9UHD1	86|
ARP21_HUMAN	Q9UBL0	321|
ORC3_HUMAN	Q9UBD5	483|
NCDN_HUMAN	Q9UBB6	98|
RBM27_HUMAN	Q9P2N5	840|
SYLC_HUMAN	Q9P2J5	554|
RRBP1_HUMAN	Q9P2E9	892|1216|
BCCIP_HUMAN	Q9P287	213|
UBP36_HUMAN	Q9P275	638|
RCC2_HUMAN	Q9P258	280|
K1522_HUMAN	Q9P206	491|
F120A_HUMAN	Q9NZB2	531|
TRM1_HUMAN	Q9NXH9	620|
NHP2_HUMAN	Q9NX24	18|
WDR70_HUMAN	Q9NW82	227|
PNPO_HUMAN	Q9NVS9	156|
TYDP1_HUMAN	Q9NUW8	135|
KIF15_HUMAN	Q9NS87	576|
STRN4_HUMAN	Q9NRL3	337|
EI2BG_HUMAN	Q9NR50	281|
SIAS_HUMAN	Q9NR45	283|
DDX21_HUMAN	Q9NR30	378|
ANLN_HUMAN	Q9NQW6	819|512|1117|
AVEN_HUMAN	Q9NQS1	169|
RRAGD_HUMAN	Q9NQL2	359|
GPCP1_HUMAN	Q9NPB8	205|
SYSM_HUMAN	Q9NP81	64|
NCK5L_HUMAN	Q9HCH0	783|
MCCB_HUMAN	Q9HCC0	267|
SPC25_HUMAN	Q9HBM1	27|
RRAGC_HUMAN	Q9HB90	377|358|
XPO5_HUMAN	Q9HAV4	941|1157|
PKHA5_HUMAN	Q9HAU0	891|
CSN7B_HUMAN	Q9H9Q2	240|
CNO10_HUMAN	Q9H9A5	504|
DCTP1_HUMAN	Q9H773	162|
RANB3_HUMAN	Q9H6Z4	249|228|
CCD86_HUMAN	Q9H6F5	116|
RABE2_HUMAN	Q9H5N1	482|115|
WNK1_HUMAN	Q9H4A3	2292|
NELFA_HUMAN	Q9H3P2	44|
SLK_HUMAN	Q9H2G2	1153|
TDIF1_HUMAN	Q9H147	156|
CSTFT_HUMAN	Q9H0L4	222|
XRN2_HUMAN	Q9H0D6	21|
KLC2_HUMAN	Q9H0B6	474|
PIP30_HUMAN	Q9GZU8	187|
DDX24_HUMAN	Q9GZR7	832|
PITH1_HUMAN	Q9GZP4	187|
MTMRC_HUMAN	Q9C0I1	67|
CEP44_HUMAN	Q9C0F1	28|
UBE2O_HUMAN	Q9C0C9	341|
TB182_HUMAN	Q9C0C2	794|716|
NIBA1_HUMAN	Q9BZQ8	516|
TBD2A_HUMAN	Q9BYX2	686|
FACD2_HUMAN	Q9BXW9	893|
NAA15_HUMAN	Q9BXJ9	721|214|
SSBP3_HUMAN	Q9BWW4	80|
NADAP_HUMAN	Q9BWU0	723|
SSBP4_HUMAN	Q9BWG4	81|
NUDT9_HUMAN	Q9BW91	347|
GNL3_HUMAN	Q9BVP2	15|
NUP58_HUMAN	Q9BVL2	252|
MELPH_HUMAN	Q9BV36	277|
DIDO1_HUMAN	Q9BTC0	350|
NTPCR_HUMAN	Q9BSD7	101|
CPPED_HUMAN	Q9BRF8	54|
TBA1C_HUMAN	Q9BQE3	347|
CND3_HUMAN	Q9BPX3	667|
SH3G2_HUMAN	Q99962	108|
SCAFB_HUMAN	Q99590	566|1020|
NUP88_HUMAN	Q99567	608|
DNJC2_HUMAN	Q99543	240|
VAT1_HUMAN	Q99536	86|
PARK7_HUMAN	Q99497	46|
PSMD1_HUMAN	Q99460	112|
CNN2_HUMAN	Q99439	240|
NIBA2_HUMAN	Q96TA1	466|
RANB9_HUMAN	Q96S59	610|594|
NUDC1_HUMAN	Q96RS6	111|
TGS1_HUMAN	Q96RS0	357|
UIMC1_HUMAN	Q96RL1	257|220|
NACC1_HUMAN	Q96RE7	178|
TRNT1_HUMAN	Q96Q11	373|
DOCK7_HUMAN	Q96N67	2125|
TRI47_HUMAN	Q96LD4	454|
SNX27_HUMAN	Q96L92	30|
SCYL1_HUMAN	Q96KG9	241|
TOPK_HUMAN	Q96KB5	70|22|
UBP47_HUMAN	Q96K76	856|
LRCH3_HUMAN	Q96II8	531|
PDLI5_HUMAN	Q96HC4	213|
ZCCHL_HUMAN	Q96H79	121|
DTBP1_HUMAN	Q96EV8	302|
DAZP1_HUMAN	Q96EP5	85|
TCAL4_HUMAN	Q96EI5	34|
ELP4_HUMAN	Q96EB1	218|
NMD3_HUMAN	Q96D46	214|
OPTN_HUMAN	Q96CV9	472|
FWCH2_HUMAN	Q96CP2	132|
EFHD2_HUMAN	Q96C19	53|
NTAN1_HUMAN	Q96AB6	118|
EXOC4_HUMAN	Q96A65	957|
SYAP1_HUMAN	Q96A49	283|
LPP_HUMAN	Q93052	364|262|
DDX17_HUMAN	Q92841	584|
EZH1_HUMAN	Q92800	504|
HS105_HUMAN	Q92598	845|
PHF3_HUMAN	Q92576	402|
TOPB1_HUMAN	Q92547	1166|
DDB2_HUMAN	Q92466	364|322|
PALLD_HUMAN	Q8WX93	964|429|
LMO7_HUMAN	Q8WWI1	228|
DNJA4_HUMAN	Q8WW22	368|
LEO1_HUMAN	Q8WVC0	530|
PDC6I_HUMAN	Q8WUM4	512|40|
GEMI5_HUMAN	Q8TEQ6	806|
TBCK_HUMAN	Q8TEA7	737|
DDX54_HUMAN	Q8TDD1	73|
NEK9_HUMAN	Q8TD19	11|
CIP2A_HUMAN	Q8TCG1	615|
UBA3_HUMAN	Q8TBC4	28|
THOC2_HUMAN	Q8NI27	1518|
TTL_HUMAN	Q8NG68	238|
BD1L1_HUMAN	Q8NFC6	686|607|2438|1947|
LS14A_HUMAN	Q8ND56	375|
TXND5_HUMAN	Q8NBS9	350|
UBR7_HUMAN	Q8N806	374|
EH1L1_HUMAN	Q8N3D4	221|1364
CMTR1_HUMAN	Q8N1G2	9|
NUP93_HUMAN	Q8N1F7	522|422|
CCAR2_HUMAN	Q8N163	644|
SPART_HUMAN	Q8N0X7	405|
PALM2_HUMAN	Q8IXS6	238|
DHX40_HUMAN	Q8IX18	33|
CHERP_HUMAN	Q8IWX8	69|
GCC2_HUMAN	Q8IWJ2	416|
MA7D3_HUMAN	Q8IWC1	494|
AHNK2_HUMAN	Q8IVF2	5415|225|1872|
ANKY2_HUMAN	Q8IV38	277|
I2BP1_HUMAN	Q8IU81	363|
DAAF5_HUMAN	Q86Y56	365|
GA2L3_HUMAN	Q86XJ1	427|
PRSR2_HUMAN	Q86WR7	367|
NIPA_HUMAN	Q86WB0	208|
MET16_HUMAN	Q86W50	448|432|
NOP9_HUMAN	Q86U38	242|
HUWE1_HUMAN	Q7Z6Z7	790|3658|2721|1628|1421|1401|
RHG30_HUMAN	Q7Z6I6	654|
I2BP2_HUMAN	Q7Z5L9	65|
WAPL_HUMAN	Q7Z5K2	160|
RAI1_HUMAN	Q7Z5J4	594|
HDGR2_HUMAN	Q7Z4V5	631|
HAUS6_HUMAN	Q7Z4H7	743|
NUFP2_HUMAN	Q7Z417	234|
MYH14_HUMAN	Q7Z406	954|
ZFY16_HUMAN	Q7Z3T8	863|304|
SETX_HUMAN	Q7Z333	637|
ZCCHV_HUMAN	Q7Z2W4	645|272|
TRM1L_HUMAN	Q7Z2T5	239|
RSRC2_HUMAN	Q7L4I2	382|
MEPCE_HUMAN	Q7L2J0	153|
BZW1_HUMAN	Q7L1Q6	35|
SND1_HUMAN	Q7KZF4	152|
TBA1A_HUMAN	Q71U36	347|
DUSTY_HUMAN	Q6XUX3	57|
CPLX2_HUMAN	Q6PUV4	90|
LARP1_HUMAN	Q6PKG0	238|
PRP8_HUMAN	Q6P2Q9	435|
CDC73_HUMAN	Q6P1J9	145|
ZCHC8_HUMAN	Q6NZY4	607|
PPR18_HUMAN	Q6NYC8	276|
TTI2_HUMAN	Q6NXR4	36|
IF5AL_HUMAN	Q6IS14	73|
TWF2_HUMAN	Q6IBS0	141|
CPIN1_HUMAN	Q6FI81	274|237|
TENS3_HUMAN	Q68CZ2	1241|
KANK2_HUMAN	Q63ZY3	389|
RPRD2_HUMAN	Q5VT52	903|
LYRM7_HUMAN	Q5U5X0	97|
UBAP2_HUMAN	Q5T6F2	208|
UBR4_HUMAN	Q5T4S7	3430|
RRP12_HUMAN	Q5JTH9	31|
PRC2B_HUMAN	Q5JSZ5	1184|1121|
PP6R3_HUMAN	Q5H9R7	844|830|
SGO1_HUMAN	Q5FBB7	503|
RIPL1_HUMAN	Q5EBL4	47|
RHG15_HUMAN	Q53QZ3	140|
LRRF1_HUMAN	Q32MZ4	726|644|14|
TSR1_HUMAN	Q2NL82	126|
ERC6L_HUMAN	Q2NKX8	825|
PDS5A_HUMAN	Q29RF7	532|
INF2_HUMAN	Q27J81	971|1029|
UPP1_HUMAN	Q16831	89|225|17|
KYNU_HUMAN	Q16719	45|
MAPK3_HUMAN	Q16644	379|
DREB_HUMAN	Q16643	613|
RBBP7_HUMAN	Q16576	97|
DPYL2_HUMAN	Q16555	504|
SNPC1_HUMAN	Q16533	256|
ADRM1_HUMAN	Q16186	121|
EZH2_HUMAN	Q15910	503|
NAB2_HUMAN	Q15742	499|
PCH2_HUMAN	Q15645	14|
TSN_HUMAN	Q15631	225|
SURF2_HUMAN	Q15527	127|
SKIV2_HUMAN	Q15477	913|
KS6A1_HUMAN	Q15418	432|223|
CNN3_HUMAN	Q15417	173|
PCBP2_HUMAN	Q15366	217|
PCBP1_HUMAN	Q15365	54|355|158|
TEBP_HUMAN	Q15185	58|
IPYR_HUMAN	Q15181	270|
PLEC_HUMAN	Q15149	4494|
KIF14_HUMAN	Q15058	1640|
FL2D_HUMAN	Q15007	270|
CND2_HUMAN	Q15003	418|255|114|
GAPD1_HUMAN	Q14C86	568|
NUMA1_HUMAN	Q14980	961|1937|1907|
CHD4_HUMAN	Q14839	1594|
GOGB1_HUMAN	Q14789	681|
UBP10_HUMAN	Q14694	254|
LAGE3_HUMAN	Q14657	23|
ZN460_HUMAN	Q14592	181|
PDE3A_HUMAN	Q14432	526|407|
SRC8_HUMAN	Q14247	246|112|
ELOA1_HUMAN	Q14241	339|
NPAT_HUMAN	Q14207	1172|
DYHC1_HUMAN	Q14204	633|4121|
DCTN1_HUMAN	Q14203	1252|
DPOA2_HUMAN	Q14181	82|
MORC3_HUMAN	Q14149	694|15|
DSG2_HUMAN	Q14126	871|
CKAP5_HUMAN	Q14008	1795|1113|
CUL4B_HUMAN	Q13620	787|
CUL4A_HUMAN	Q13619	633|241|
CUL3_HUMAN	Q13618	636|
HDAC1_HUMAN	Q13547	408|
SQSTM_HUMAN	Q13501	331|113|
GOGA4_HUMAN	Q13439	853|1729|
SNTB2_HUMAN	Q13425	391|
ATM_HUMAN	Q13315	564|
STK3_HUMAN	Q13188	410|
PRDX4_HUMAN	Q13162	51|
AIMP2_HUMAN	Q13155	306|
AAPK1_HUMAN	Q13131	425|
TRAF2_HUMAN	Q12933	124|
TRAP1_HUMAN	Q12931	573|
TP53B_HUMAN	Q12888	896|319|1703|1280|
CHD3_HUMAN	Q12873	1838|
AKP13_HUMAN	Q12802	1666|
NU160_HUMAN	Q12769	1166|
AHNK_HUMAN	Q09666	5502|2806|1967|1900|
NCBP1_HUMAN	Q09161	503|
NSUN2_HUMAN	Q08J23	758|599|502|
PRDX1_HUMAN	Q06830	71|173|
PSME1_HUMAN	Q06323	101|
GFPT1_HUMAN	Q06210	264|
CALD1_HUMAN	Q05682	636|
LGUL_HUMAN	Q04760	139|
K1C17_HUMAN	Q04695	40|358|
IF4G1_HUMAN	Q04637	662|
GLGB_HUMAN	Q04446	81|221|
DYST_HUMAN	Q03001	3168|
AKA12_HUMAN	Q02952	578|1521|1314|
KIF23_HUMAN	Q02241	629|
AMPD2_HUMAN	Q01433	123|
HNRPU_HUMAN	Q00839	594|
CDK17_HUMAN	Q00537	233|
CDK16_HUMAN	Q00536	206|
CDK6_HUMAN	Q00534	15|
VIGLN_HUMAN	Q00341	940|
2ABB_HUMAN	Q00005	330|
H33_HUMAN	P84243	111|
SRSF3_HUMAN	P84103	6|
COG7_HUMAN	P83436	505|
PRKDC_HUMAN	P78527	3837|
GSTO1_HUMAN	P78417	192|
TCPB_HUMAN	P78371	535|
GTF2I_HUMAN	P78347	215|
RPP30_HUMAN	P78346	257|
TBB4B_HUMAN	P68371	303|12|
TBA4A_HUMAN	P68366	54|
TBA1B_HUMAN	P68363	347|315|295|
PP2AA_HUMAN	P67775	266|251|
ACTG_HUMAN	P63261	285|257|
IF5A1_HUMAN	P63241	73|
SKP1_HUMAN	P63208	160|
2ABA_HUMAN	P63151	334|
1433Z_HUMAN	P63104	25|
RAC1_HUMAN	P63000	178|105|
GRB2_HUMAN	P62993	32|
HNRPK_HUMAN	P61978	184|145|132|
PSME3_HUMAN	P61289	92|
DEST_HUMAN	P60981	39|23|135|
RAC3_HUMAN	P60763	178|
GSDMD_HUMAN	P57764	268|
NU107_HUMAN	P57740	78|
WDR4_HUMAN	P57081	412|
SOX10_HUMAN	P56693	22|
HNRH2_HUMAN	P55795	267|
PSA_HUMAN	P55786	265|
DSRAD_HUMAN	P55265	773|
CASP6_HUMAN	P55212	163|
NP1L1_HUMAN	P55209	132|
ELL_HUMAN	P55199	411|
PMS2_HUMAN	P54278	591|
SYRC_HUMAN	P54136	32|
ICLN_HUMAN	P54105	73|
COPB_HUMAN	P53618	684|623|
ACLY_HUMAN	P53396	20|
PLK1_HUMAN	P53350	372|
BIEA_HUMAN	P53004	204|
AK1C2_HUMAN	P52895	7|206|188|
THOP1_HUMAN	P52888	350|
MSH6_HUMAN	P52701	615|
HNRPF_HUMAN	P52597	267|
6PGD_HUMAN	P52209	402|170|
KS6A3_HUMAN	P51812	229|
MECP2_HUMAN	P51608	429|
LRBA_HUMAN	P50851	1228|
DYN2_HUMAN	P50570	86|
VASP_HUMAN	P50552	334|
ERF_HUMAN	P50548	477|
SPB9_HUMAN	P50453	259|
GUAA_HUMAN	P49915	523|449|
RBP2_HUMAN	P49792	3071|2696|220|
NU153_HUMAN	P49790	593|21|1129|
SYAC_HUMAN	P49588	773|
SRP09_HUMAN	P49458	39|
FAS_HUMAN	P49327	779|2202|1548|
NASP_HUMAN	P49321	254|
NEST_HUMAN	P48681	575|
TCPE_HUMAN	P48643	407|
PPCE_HUMAN	P48147	255|
CAPZB_HUMAN	P47756	206|
UTRO_HUMAN	P46939	277|
MAP1B_HUMAN	P46821	2065|2041|1228|
ATRX_HUMAN	P46100	2404|
RECQ1_HUMAN	P46063	606|49|
KI67_HUMAN	P46013	226|1479|
CBX5_HUMAN	P45973	133|
RANG_HUMAN	P43487	158|
MAGA3_HUMAN	P43357	10|
MSH2_HUMAN	P43246	873|
CASP2_HUMAN	P42575	343|
MTOR_HUMAN	P42345	300|
STAT1_HUMAN	P42224	492|
LAP2A_HUMAN	P42166	684|287|
GARS_HUMAN	P41250	616|466|
CSK_HUMAN	P41240	31|
NAA10_HUMAN	P41227	194|
IF2G_HUMAN	P41091	105|
UBP8_HUMAN	P40818	809|
STAT3_HUMAN	P40763	687|
TAGL2_HUMAN	P37802	124|
MYH9_HUMAN	P35579	917|172|1379|
SPB6_HUMAN	P35237	350|
CTNA1_HUMAN	P35221	116|
HSP74_HUMAN	P34932	270|
MCM5_HUMAN	P33992	197|
CSTF2_HUMAN	P33240	150|
PRDX2_HUMAN	P32119	172|
1433S_HUMAN	P31947	38|
HNRH1_HUMAN	P31943	267|
CPSM_HUMAN	P31327	816|
PRDX6_HUMAN	P30041	91|
PML_HUMAN	P29590	389|
TKT_HUMAN	P29401	41|133|
TPP2_HUMAN	P29144	28|
ERCC5_HUMAN	P28715	550|
MAP4_HUMAN	P27816	1098|
PYR1_HUMAN	P27708	736|1889|
DCK_HUMAN	P27707	59|45|
ARNT_HUMAN	P27540	62|
EF1G_HUMAN	P26641	266|210|
DNMT1_HUMAN	P26358	62|41|
DDX6_HUMAN	P26196	102|
RS12_HUMAN	P25398	130|
KTHY_HUMAN	P23919	31|
IF4B_HUMAN	P23588	457|
COF1_HUMAN	P23528	147|139|
SYWC_HUMAN	P23381	62|
PUR6_HUMAN	P22234	63|
OSBP1_HUMAN	P22059	343|
TGM2_HUMAN	P21980	545|230|
FLNA_HUMAN	P21333	733|717|623|53|478|2582|2543|205|1353|1157|
CD11B_HUMAN	P21127	440|
ICAL_HUMAN	P20810	661|328|241|
ANXA7_HUMAN	P20073	363|
NFKB1_HUMAN	P19838	61|
CSK22_HUMAN	P19784	336|
NUCL_HUMAN	P19338	543|
PYRG1_HUMAN	P17812	491|362|
CAN2_HUMAN	P17655	82|374|
ZNF24_HUMAN	P17028	189|
RS2_HUMAN	P15880	182|
ERF3A_HUMAN	P15170	327|
PLAK_HUMAN	P14923	49|
HNRPL_HUMAN	P14866	472|
MIF_HUMAN	P14174	81|
PLST_HUMAN	P13797	104|
PLSL_HUMAN	P13796	101|
TCTP_HUMAN	P13693	172|
EF2_HUMAN	P13639	67|651|41|369|
ACTN1_HUMAN	P12814	370|154|
IMDH2_HUMAN	P12268	173|
PABP1_HUMAN	P11940	132|
G6PD_HUMAN	P11413	446|385|158|13|
PYGB_HUMAN	P11216	437|319|
KAP0_HUMAN	P10644	18|
THIO_HUMAN	P10599	73|32|
ARAF_HUMAN	P10398	192|
CTF8_HUMAN	P0CG13	83|
UCHL1_HUMAN	P09936	90|
ROA1_HUMAN	P09651	175|
HMGB1_HUMAN	P09429	23|106|
HS90B_HUMAN	P08238	589|564|
SYEP_HUMAN	P07814	856|1480|
TBB5_HUMAN	P07437	303|12|
LDHB_HUMAN	P07195	36|
GELS_HUMAN	P06396	331|
P53_HUMAN	P04637	141|
KITH_HUMAN	P04183	206|
GCR_HUMAN	P04150	287|
ALDOA_HUMAN	P04075	178|
PNPH_HUMAN	P00491	31|
AL1A1_HUMAN	P00352	50|370|186|133|126|
LDHA_HUMAN	P00338	163|
NSD2_HUMAN	O96028	406|
BAG3_HUMAN	O95817	179|151|
DDX58_HUMAN	O95786	490|
HS74L_HUMAN	O95757	540|417|
IPO7_HUMAN	O95373	90|757|
TRI16_HUMAN	O95361	178|
KIF4A_HUMAN	O95239	1224|1171|
ELP1_HUMAN	O95163	453|
UBXN7_HUMAN	O94888	160|
STK10_HUMAN	O94804	947|888|
WDHD1_HUMAN	O75717	773|
NU155_HUMAN	O75694	1344|
SURF6_HUMAN	O75683	189|
TIPRL_HUMAN	O75663	87|
CSDE1_HUMAN	O75534	680|
NCOR1_HUMAN	O75376	2322|
FLNB_HUMAN	O75369	1868|1326|
VATG1_HUMAN	O75348	69|
BRE1B_HUMAN	O75150	890|
ROCK2_HUMAN	O75116	649|1257|
IF2P_HUMAN	O60841	1158|
DIAP1_HUMAN	O60610	1227|
TBCD4_HUMAN	O60343	74|
GANP_HUMAN	O60318	1864|
MYO1B_HUMAN	O43795	613|
SGTA_HUMAN	O43765	148|
HTSF1_HUMAN	O43719	512|480|462|
ACTN4_HUMAN	O43707	173|
XPOT_HUMAN	O43592	650|
WIPF1_HUMAN	O43516	446|
IF4G3_HUMAN	O43432	1411|
HNRPR_HUMAN	O43390	226|
SNUT1_HUMAN	O43290	645|
SERA_HUMAN	O43175	281|18|
MCES_HUMAN	O43148	95|
EIF3D_HUMAN	O15371	19|
PPM1G_HUMAN	O15355	241|
IKKA_HUMAN	O15111	406|
PUR4_HUMAN	O15067	606|
ARHGA_HUMAN	O15013	401|
XPO1_HUMAN	O14980	34|1070|
CSKP_HUMAN	O14936	418|
HAT1_HUMAN	O14929	101|
GEMI2_HUMAN	O14893	63|
IRS4_HUMAN	O14654	522|
PDXK_HUMAN	O00764	273|
EXOC5_HUMAN	O00471	194|
DNM1L_HUMAN	O00429	470|
IPO5_HUMAN	O00410	915|
CLIC1_HUMAN	O00299	59|
SPT5H_HUMAN	O00267	740|
PSMD9_HUMAN	O00233	81|59|
SBNO1_HUMAN	A3KN83	445|
SPD2B_HUMAN	A1X283	598|

TABLE 4
Protein Modification Sites Targeted by Pu-2.
Gene name	UniProt	Pu-2-modified Site
UBA6_HUMAN	A0AVT1	347|
MED19_HUMAN	A0JLT2	62|
SPD2B_HUMAN	A1X283	783|598|
SBNO1_HUMAN	A3KN83	445|699|
WDR91_HUMAN	A4D1P6	351|366|
SMHD1_HUMAN	A6NHR9	1656|897|
PSD11_HUMAN	O00231	289|
DFFA_HUMAN	O00273	165|38|
WASL_HUMAN	O00401	431|
ARI1A_HUMAN	O14497	1874|
UB2L6_HUMAN	O14933	86|
AURKA_HUMAN	O14965	49|
BTAF1_HUMAN	O14981	109|
SPTN2_HUMAN	O15020	231|
PUR4_HUMAN	O15067	1285|606|66|1044|
INP4_BHUMAN	O15327	534|
PPM1G_HUMAN	O15355	241|164|
R113A_HUMAN	O15541	15|
MCES_HUMAN	O43148	73|95|
SERA_HUMAN	O43175	369|18|
DC1L2_HUMAN	O43237	191|
ERI3_HUMAN	O43414	285|
IF4G3_HUMAN	O43432	1411|
XPOT_HUMAN	O43592	650|
HTSF1_HUMAN	O43719	512|462|
STRN_HUMAN	O43815	765|
TBCD4_HUMAN	O60343	1286|316|45|1277|
GSDME_HUMAN	O60443	180|
OGA_HUMAN	O60502	596|
HNRPQ_HUMAN	O60506	96|
USO1_HUMAN	O60763	678|
KIN17_HUMAN	O60870	212|
NBN_HUMAN	O60934	487|
SRGP2_HUMAN	O75044	820|357|
N4BP1_HUMAN	O75113	483|
PP6R2_HUMAN	O75170	880|761|870|
MPPB_HUMAN	O75439	265|
KS6A5_HUMAN	O75582	214|
TIPRL_HUMAN	O75663	14|87|
GLRX3_HUMAN	O76003	146|
CIAO1_HUMAN	O76071	234|
STK10_HUMAN	O94804	888|
PLPHP_HUMAN	O94903	261|
HEXI1_HUMAN	O94992	79|
UBR5_HUMAN	O95071	2267|
UBE4B_HUMAN	O95155	113|
ATE1_HUMAN	O95260	138|
6PGL_HUMAN	O95336	32|
SMC2_HUMAN	O95347	1174|
SVIL_HUMAN	O95425	26|671|
BAG3_HUMAN	O95817	179|151|
LMNA_HUMAN	P02545	588|
ALDOA_HUMAN	P04075	290|
KITH_HUMAN	P04183	206|
P53_HUMAN	P04637	135|182|141|
TBB5_HUMAN	P07437	12|127|303|
SYEP_HUMAN	P07814	744|856|1377|
ARAF_HUMAN	P10398	597|192|
THIO_HUMAN	P10599	32|73|
G6PD_HUMAN	P11413	13|
ACTN1_HUMAN	P12814	370|
PEPD_HUMAN	P12955	467|478|
RINI_HUMAN	P13489	409|
EF2_HUMAN	P13639	41|
FAAA_HUMAN	P16930	105|
ZNF24_HUMAN	P17028	233|
SON_HUMAN	P18583	2070|
NFKB1_HUMAN	P19838	61|
VATC1_HUMAN	P21283	15|
FLNA_HUMAN	P21333	1157|1260|2543|574|717|
NF1_HUMAN	P21359	1032|454|
OSBP1_HUMAN	P22059	343|
PUR2_HUMAN	P22102	41|
SP100_HUMAN	P23497	238|
COF1_HUMAN	P23528	147|139|
IF4B_HUMAN	P23588	457|
CDK2_HUMAN	P24941	177|
SYVC_HUMAN	P26640	41|
MAP4_HUMAN	P27816	1008|635|535|1098|
IP3KB_HUMAN	P27987	379|
RXRB_HUMAN	P28702	191|
PML_HUMAN	P29590	338|389|
TF2H1_HUMAN	P32780	181|
CGL_HUMAN	P32929	229|
HSP74_HUMAN	P34932	34|417|
CAH8_HUMAN	P35219	200|266|
MYH9_HUMAN	P35579	988|1437|569|1379|
MYH10_HUMAN	P35580	1238|
ADDA_HUMAN	P35611	430|525|
NU214_HUMAN	P35658	1003|
TAGL2_HUMAN	P37802	124|
NAA10_HUMAN	P41227	194|
GARS_HUMAN	P41250	466|
LAP2A_HUMAN	P42166	287|280|658|330|
EPS15_HUMAN	P42566	657|
RANG_HUMAN	P43487	132|99|
CBX5_HUMAN	P45973	133|
KI67_HUMAN	P46013	1843|226|
CRKL_HUMAN	P46109	249|
MP2K3_HUMAN	P46734	227|
MAP1B_HUMAN	P46821	2041|2065|905|1814|2083|1228|
UTRO_HUMAN	P46939	277|
CAPZB_HUMAN	P47756	206|147|
NEST_HUMAN	P48681	157|575|161|
NASP_HUMAN	P49321	708|254|
FAS_HUMAN	P49327	1448|2202|
NU153_HUMAN	P49790	1065|404|585|1129|
RBP2_HUMAN	P49792	3032|
GUAA_HUMAN	P49915	631|
PAPOA_HUMAN	P51003	36|
HCFC1_HUMAN	P51610	1886|227|
DYLT3_HUMAN	P51808	8|
HDGF_HUMAN	P51858	12|108|
HNRPM_HUMAN	P52272	653|
HNRPF_HUMAN	P52597	267|
STAT2_HUMAN	P52630	174|
NUBP1_HUMAN	P53384	31|
SC24C_HUMAN	P53992	78|
ICLN_HUMAN	P54105	73|
PMS2_HUMAN	P54278	216|591|
HSP72_HUMAN	P54652	191|
XPO2_HUMAN	P55060	939|
SYMC_HUMAN	P56192	441|
GSDMD_HUMAN	P57764	191|
DEST_HUMAN	P60981	135|147|23|
HNRPK_HUMAN	P61978	184|
PPIA_HUMAN	P62937	62|
RAC1_HUMAN	P63000	178|
DYL1_HUMAN	P63167	24|
IF5A1_HUMAN	P63241	73|
TBA1B_HUMAN	P68363	315|347|
TBB4B_HUMAN	P68371	12|303|
HSF1_HUMAN	Q00613	153|
HNRPU_HUMAN	Q00839	594|
CAP1_HUMAN	Q01518	93|
RL18A_HUMAN	Q02543	22|
TF65_HUMAN	Q04206	109|105|
GLGB_HUMAN	Q04446	221|81|
IF4G1_HUMAN	Q04637	662|
PTN12_HUMAN	Q05209	495|
PTN11_HUMAN	Q06124	259|
PSME1_HUMAN	Q06323	101|22|
PRDX1_HUMAN	Q06830	173|
KLC1_HUMAN	Q07866	114|
GOGA3_HUMAN	Q08378	45|455|
CAMP2_HUMAN	Q08AD1	675|
SLFN5_HUMAN	Q08AF3	362|
VAC14_HUMAN	Q08AM6	516|
NCBP1_HUMAN	Q09161	36|44|
AHNK_HUMAN	Q09666	1900|2806|1833|
GRSF1_HUMAN	Q12849	476|
TP53B_HUMAN	Q12888	1038|1159|319|101|772|1375|
TRAF2_HUMAN	Q12933	287|
CDC16_HUMAN	Q13042	544|
ATM_HUMAN	Q13315	219|2323|2607|2991|564|384|1396|
ILK_HUMAN	Q13418	346|422|
GOGA4_HUMAN	Q13439	1729|1340|1771|
MYO9B_HUMAN	Q13459	1169|
SQSTM_HUMAN	Q13501	331|289|113|
ITPK1_HUMAN	Q13572	391|
RIN1_HUMAN	Q13671	733|
SHRM2_HUMAN	Q13796	1231|
MORC3_HUMAN	Q14149	619|671|694|
SAFB2_HUMAN	Q14151	361|
SRC8_HUMAN	Q14247	112|246|
HLTF_HUMAN	Q14527	442|461|
LAGE3_HUMAN	Q14657	23|
SMC1A_HUMAN	Q14683	987|
UBP10_HUMAN	Q14694	94|254|
GOGB1_HUMAN	Q14789	1462|3070|3144|1811|
MEF2D_HUMAN	Q14814	217|
NUMA1_HUMAN	Q14980	1136|1729|1907|2009|1937|961|
GAPD1_HUMAN	Q14C86	293|568|741|
SPCS2_HUMAN	Q15005	17|
SART3_HUMAN	Q15020	537|670|
IPYR_HUMAN	Q15181	270|
TEBP_HUMAN	Q15185	40|75|58|
PCBP1_HUMAN	Q15365	109|54|163|
PCBP2_HUMAN	Q15366	109|
CNN3_HUMAN	Q15417	273|173|
SAFB1_HUMAN	Q15424	362|
SKIV2_HUMAN	Q15477	247|
SURF2_HUMAN	Q15527	116|127|
TSN_HUMAN	Q15631	225|
TRIPB_HUMAN	Q15643	212|
CDC37_HUMAN	Q16543	64|
SMN_HUMAN	Q16637	60|
DREB_HUMAN	Q16643	96|613|
MAR1_HUMAN	Q16655	68|
IF16_HUMAN	Q16666	191|637|
UPP1_HUMAN	Q16831	17|71|162|
PDS5A_HUMAN	Q29RF7	532|1093|
FA98B_HUMAN	Q52LJ0	216|
RIPL1_HUMAN	Q5EBL4	47|
ATPF1_HUMAN	Q5TC12	321|
NT5D1_HUMAN	Q5TFE4	119|
PR38B_HUMAN	Q5VTL8	113|
MYOME_HUMAN	Q5VU43	603|
LYPL1_HUMAN	Q5VWZ2	12|
DEN4C_HUMAN	Q5VZ89	1113|
RN213_HUMAN	Q63HN8	310|
TENS3_HUMAN	Q68CZ2	928|615|
FMN1_HUMAN	Q68DA7	228|660|
ZFY26_HUMAN	Q68DK2	715|
CPIN1_HUMAN	Q6FI81	237|249|
OTU7B_HUMAN	Q6GQQ9	708|
TWF2_HUMAN	Q6IBS0	141|
IF5AL_HUMAN	Q6IS14	73|
PPR18_HUMAN	Q6NYC8	611|
EDC4_HUMAN	Q6P2E9	54|838|
ANM9_HUMAN	Q6P2P2	290|
ZC3HE_HUMAN	Q6PJT7	261|
LARP1_HUMAN	Q6PKG0	238|
LRSM1_HUMAN	Q6UWE0	23|
DNMBP_HUMAN	Q6XZF7	691|
YJ005_HUMAN	Q6ZSR9	251|
RAPH1_HUMAN	Q70E73	847|
EIF3M_HUMAN	Q7L2H7	175|
MEPCE_HUMAN	Q7L2J0	429|54|
CYFP1_HUMAN	Q7L576	98|
ZFY16_HUMAN	Q7Z3T8	1002|122|269|304|313|377|41|62|829|863|174|
HDGR2_HUMAN	Q7Z4V5	631|
WAPL_HUMAN	Q7Z5K2	160|365|906|
PXK_HUMAN	Q7Z7A4	196|570|
CK096_HUMAN	Q7Z7L8	402|
PRUN1_HUMAN	Q86TP1	437|
MTA70_HUMAN	Q86U44	336|
UBP48_HUMAN	Q86UV5	409|986|850|
MET16_HUMAN	Q86W50	448|480|432|
CACL1_HUMAN	Q86Y37	94|
RB6I2_HUMAN	Q8IUD2	258|
RHG12_HUMAN	Q8IWW6	199|
ANKH1_HUMAN	Q8IWZ3	615|873|
SUGP1_HUMAN	Q8IWZ8	78|
DHX40_HUMAN	Q8IX18	33|
SPART_HUMAN	Q8N0X7	499|
CMTR1_HUMAN	Q8N1G2	9|
VPS8_HUMAN	Q8N3P4	974|
OTU6B_HUMAN	Q8N6M0	172|
CB069_HUMAN	Q8N8R5	230|
NHLC2_HUMAN	Q8NBF2	716|
MTMRE_HUMAN	Q8NCE2	429|
LS14A_HUMAN	Q8ND56	375|
DP13B_HUMAN	Q8NEU8	412|
BD1L1_HUMAN	Q8NFC6	1742|2386|
NEUA_HUMAN	Q8NFW8	432|
NEDD1_HUMAN	Q8NHV4	314|
TM10A_HUMAN	Q8TBZ6	285|
NEK9_HUMAN	Q8TD19	878|
PKHO2_HUMAN	Q8TD55	295|
MICA1_HUMAN	Q8TDZ2	837|
IPO4_HUMAN	Q8TEX9	708|
PRUN2_HUMAN	Q8WUY3	2536|2598|653|1377|357|
LEO1_HUMAN	Q8WVC0	530|
TPC12_HUMAN	Q8WVT3	160|
HNRLL_HUMAN	Q8WVV9	505|
NELFB_HUMAN	Q8WX92	115|
PPM1E_HUMAN	Q8WY54	525|
IRGQ_HUMAN	Q8WZA9	340|
GBF1_HUMAN	Q92538	661|
HS105_HUMAN	Q92598	376|845|
TBCD5_HUMAN	Q92609	676|
ANS1A_HUMAN	Q92625	651|849|
AKAP1_HUMAN	Q92667	147|165|376|438|
ARC1A_HUMAN	Q92747	279|
DDX17_HUMAN	Q92841	584|
ARHG1_HUMAN	Q92888	911|
FUBP2_HUMAN	Q92945	176|436|
ARHG2_HUMAN	Q92974	306|478|715|
UBP7_HUMAN	Q93009	315|
TSR2_HUMAN	Q969E8	114|
NTAN1_HUMAN	Q96AB6	118|
OTUL_HUMAN	Q96BN8	17|47|
EFHD2_HUMAN	Q96C19	53|
FWCH2_HUMAN	Q96CP2	132|64|
F122A_HUMAN	Q96E09	193|
SIR1_HUMAN	Q96EB6	574|
RBM33_HUMAN	Q96EV2	726|
DTBP1_HUMAN	Q96EV8	302|
CYFP2_HUMAN	Q96F07	98|
CCD97_HUMAN	Q96F63	78|
MTNB_HUMAN	Q96GX9	16|
ZC21A_HUMAN	Q96GY0	276|
SAHH3_HUMAN	Q96HN2	353|
MET2A_HUMAN	Q96IZ6	171|
ZFP91_HUMAN	Q96JP5	182|520|
TOPK_HUMAN	Q96KB5	112|70|22|
TRI47_HUMAN	Q96LD4	454|
DOCK7_HUMAN	Q96N67	193|1944|2125|1902|
TRNT1_HUMAN	Q96Q11	373|
XPO6_HUMAN	Q96QU8	299|
UIMC1_HUMAN	Q96RL1	601|
RANB9_HUMAN	Q96S59	513|527|
CK5P2_HUMAN	Q96SN8	249|
CNN22_HUMAN	Q99439	164|215|175|
PSMD1_HUMAN	Q99460	806|
ANM1_HUMAN	Q99873	119|
AKAP9_HUMAN	Q99996	1367|3521|
F118B_HUMAN	Q9BPY3	319|
TRIR_HUMAN	Q9BQ61	165|
MACD1_HUMAN	Q9BQ69	186|
MBB1A_HUMAN	Q9BQG0	942|
WAC_HUMAN	Q9BTA9	15|553|
DIDO1_HUMAN	Q9BTC0	498|350|
MCMBP_HUMAN	Q9BTE3	325|200|
KIFC1_HUMAN	Q9BW19	95|
NADAP_HUMAN	Q9BWU0	730|
NAA15_HUMAN	Q9BXJ9	238|721|
SRRT_HUMAN	Q9BXP5	412|
FACD2_HUMAN	Q9BXW9	893|310|
ITPA_HUMAN	Q9BY32	146|
TB182_HUMAN	Q9C0C2	1296|1443|794|1114|136|1208|1373|1175|749|
UBE2O_HUMAN	Q9C0C9	375|400|
TANC1_HUMAN	Q9C0D5	1594|1785|
PITH1_HUMAN	Q9GZP4	14|
EGLN1_HUMAN	Q9GZT9	127|
ILKAP_HUMAN	Q9H0C8	301|
DHX36_HUMAN	Q9H2U1	135|
NELFA_HUMAN	Q9H3P2	471|
SFR19_HUMAN	Q9H7N4	950|
PHAX_HUMAN	Q9H814	51|
CNO10_HUMAN	Q9H9A5	504|
KT3K_HUMAN	Q9HA64	24|
PKHA5_HUMAN	Q9HAU0	359|
XPO5_HUMAN	Q9HAV4	1131|
ARFG3_HUMAN	Q9NP61	241|
ANLN_HUMAN	Q9NQW6	309|469|512|353|260|
GEPH_HUMAN	Q9NQX3	284|293|419|
DIAP3_HUMAN	Q9NSV4	1125|
TXLNG_HUMAN	Q9NUQ3	101|
EXOC1_HUMAN	Q9NV70	566|
TBC13_HUMAN	Q9NVG8	387|282|
INT10_HUMAN	Q9NVR2	387|
RPC5_HUMAN	Q9NVU0	456|
NRK1_HUMAN	Q9NWW6	72|
HPF1_HUMAN	Q9NWY4	29|
CARF_HUMAN	Q9NXV6	178|
TLS1_HUMAN	Q9NZ63	145|
F120A_HUMAN	Q9NZB2	531|919|
SMAL1_HUMAN	Q9NZC9	108|
CNOT2_HUMAN	Q9NZN8	175|
OGFR_HUMAN	Q9NZT2	443|417|330|
CHMP5_HUMAN	Q9NZZ3	20|
VPS18_HUMAN	Q9P253	22|
RCC2_HUMAN	Q9P258	428|
ORC3_HUMAN	Q9UBD5	483|
MALT1_HUMAN	Q9UDY8	71|
CGBP1_HUMAN	Q9UFW8	92|
LIMD1_HUMAN	Q9UGP4	148|
CHRD1_HUMAN	Q9UHD1	86|
EI2BD_HUMAN	Q9UI10	69|
TR112_HUMAN	Q9UI30	33|
GGA2_HUMAN	Q9UJY4	429|
TF3C4_HUMAN	Q9UKN8	129|
NUP50_HUMAN	Q9UKX7	151|
PSME2_HUMAN	Q9UL46	91|
DNPEP_HUMAN	Q9ULA0	144|327|
ASAP1_HUMAN	Q9ULH1	740|
COR1C_HUMAN	Q9ULV4	420|456|
AKP8L_HUMAN	Q9ULX6	211|128|
POLI_HUMAN	Q9UNA4	560|
CHIP_HUMAN	Q9UNE7	199|
PP6R1_HUMAN	Q9UPN7	326|
DICER_HUMAN	Q9UPY3	1641|1621|
MARE3_HUMAN	Q9UPY8	182|
MTMR6_HUMAN	Q9Y217	569|
LZTS1_HUMAN	Q9Y250	167|385|
VDAC3_HUMAN	Q9Y277	36|
SAC2_HUMAN	Q9Y2H2	268|
RRP44_HUMAN	Q9Y2L1	213|799|
TPPC8_HUMAN	Q9Y2L5	265|
CRYL1_HUMAN	Q9Y2S2	125|
AP3M1_HUMAN	Q9Y2T2	288|
GIT1_HUMAN	Q9Y2X7	576|
SNX24_HUMAN	Q9Y343	114|
LC7L2_HUMAN	Q9Y383	348|
PHOCN_HUMAN	Q9Y3A3	134|
STRAP_HUMAN	Q9Y3F4	305|
RBGP1_HUMAN	Q9Y3P9	476|
WAC2C_HUMAN	Q9Y4E1	828|
IRS2_HUMAN	Q9Y4H2	514|1231|
ATG4B_HUMAN	Q9Y4P1	189|
PRC2C_HUMAN	Q9Y520	177|953|
PPME1_HUMAN	Q9Y570	238|
TNPO3_HUMAN	Q9Y5L0	511|
GMPPB_HUMAN	Q9Y5P6	245|
TSSC4_HUMAN	Q9Y5U2	99|
NS1BP_HUMAN	Q9Y6Y0	143|274|
S23IP_HUMAN	Q9Y6Y8	604|
SHOT1_HUMAN	A0MZ66	550|
LRC47_HUMAN	Q8N1G4	249|
CASP2_HUMAN	P42575	343|
LIN37_HUMAN	Q96GY3	28|
CE022_HUMAN	Q49AR2	165|195|
STIP1_HUMAN	P31948	461|26|
RBBP7_HUMAN	Q16576	116|97|
SEP10_HUMAN	Q9P0V9	100|
GEMI7_HUMAN	Q9H840	44|
RECQ1_HUMAN	P46063	606|
FTO_HUMAN	Q9C0B1	171|
AGAP3_HUMAN	Q96P47	263|
ERI1_HUMAN	Q8IV48	75|
PI3R4_HUMAN	Q99570	899|
RIPK1_HUMAN	Q13546	325|
GCN1_HUMAN	Q92616	648|
PRDX6_HUMAN	P30041	47|91|
SRP09_HUMAN	P49458	48|
SYAC_HUMAN	P49588	671|773|
PLIN3_HUMAN	O60664	60|39|
LPP_HUMAN	Q93052	364|
ZCCHL_HUMAN	Q96H79	121|
PNPO_HUMAN	Q9NVS9	156|
DUS3L_HUMAN	Q96G46	282|54|
DCTN4_HUMAN	Q9UJW0	258|
TBC9B_HUMAN	Q66K14	444|
CBX8_HUMAN	Q9HC52	261|
SYTC_HUMAN	P26639	656|
XPO1_HUMAN	O14980	119|
KTNB1_HUMAN	Q9BVA0	344|
ACLY_HUMAN	P53396	20|845|
IRAK4_HUMAN	Q9NWZ3	13|128|
TMF1_HUMAN	P82094	431|
DIAP1_HUMAN	O60610	1227|
NSUN2_HUMAN	Q08J23	502|93|
PTN23_HUMAN	Q9H3S7	628|
ZCHC8_HUMAN	Q6NZY4	607|393|
L2GL1_HUMAN	Q15334	1059|
PLST_HUMAN	P13797	104|33|
CKAP5_HUMAN	Q14008	1113|
UBP8_HUMAN	P40818	809|
ICAL_HUMAN	P20810	661|381|413|328|241|
SRSF3_HUMAN	P84103	6|
KNL1_HUMAN	Q8NG31	562|
INT12_HUMAN	Q96CB8	379|
PARN_HUMAN	O95453	169|543|
NTM1A_HUMAN	Q9BV86	195|
TBC24_HUMAN	Q9ULP9	29|
F263_HUMAN	Q16875	493|
ZBT21_HUMAN	Q9ULJ3	152|378|
DYHC1_HUMAN	Q14204	4121|1977|
CND1_HUMAN	Q15021	286|596|
TCAL4_HUMAN	Q96EI5	34|
CLAP2_HUMAN	O75122	976|
ARFG1_HUMAN	Q8N6T3	96|
OTUB1_HUMAN	Q96FW1	23|
MSH6_HUMAN	P52701	88|
CE170_HUMAN	Q5SW79	967|
SEPT9_HUMAN	Q9UHD8	248|
ELF2_HUMAN	Q15723	470|
ACINU_HUMAN	Q9UKV3	546|
AKA12_HUMAN	Q02952	470|1314|1139|
SHIP2_HUMAN	O15357	926|
CPPED_HUMAN	Q9BRF8	54|
RANB3_HUMAN	Q9H6Z4	249|
I2BP2_HUMAN	Q7Z5L9	65|
DAXX_HUMAN	Q9UER7	427|699|
U520_HUMAN	O75643	238|
ARPIN_HUMAN	Q7Z6K5	183|
SYRC_HUMAN	P54136	32|
AKAP2_HUMAN	Q9Y2D5	205|
NR2C2_HUMAN	P49116	204|
SRRM2_HUMAN	Q9UQ35	872|1029|
RN214_HUMAN	Q8ND24	149|
PKHA4_HUMAN	Q9H4M7	438|
PYGB_HUMAN	P11216	437|
AEDO_HUMAN	Q96SZ5	239|
ASPC1_HUMAN	Q9BZE9	48|109|224|
STA5B_HUMAN	P51692	688|
LANC2_HUMAN	Q9NS86	187|
G3P_HUMAN	P04406	152|
LRRF1_HUMAN	Q32MZ4	750|644|
SYMPK_HUMAN	Q92797	472|
YAP1_HUMAN	P46937	343|
ACSL3_HUMAN	O95573	649|
CLU_HUMAN	O75153	1196|
CASP8_HUMAN	Q14790	360|
PSME3_HUMAN	P61289	92|
TRI33_HUMAN	Q9UPN9	582|786|
PDCD4_HUMAN	Q53EL6	275|
UBA3_HUMAN	Q8TBC4	28|
UBR7_HUMAN	Q8N806	260|
MAVS_HUMAN	Q7Z434	283|
SOX10_HUMAN	P56693	190|
VATG1_HUMAN	O75348	69|
NIBA1_HUMAN	Q9BZQ8	891|
SPF30_HUMAN	O75940	214|
NF2IP_HUMAN	Q8NCF5	232|
PDE12_HUMAN	Q6L8Q7	108|
MACF1_HUMAN	Q9UPN3	5131|7245|
KTN1_HUMAN	Q86UP2	736|
RB_HUMAN	P06400	853|
RICTR_HUMAN	Q6R327	1576|
CAND1_HUMAN	Q86VP6	413|
RAN_HUMAN	P62826	112|
PSMD2_HUMAN	Q13200	459|
SOX6_HUMAN	P35712	594|
LIPB2_HUMAN	Q8ND30	446|
SYFB_HUMAN	Q9NSD9	195|
BZW2_HUMAN	Q9Y6E2	270|
COG7_HUMAN	P83436	505|
EF1G_HUMAN	P26641	194|266|339|
COPB_HUMAN	P53618	684|623|
2A5D_HUMAN	Q14738	17|
VAPA_HUMAN	Q9P0L0	60|
EH1L1_HUMAN	Q8N3D4	346|
PP6R3_HUMAN	Q5H9R7	844|830|
HNRH1_HUMAN	P31943	267|
PR40A_HUMAN	O75400	39|
PSMD9_HUMAN	O00233	59|
WAC2A_HUMAN	Q641Q2	594|
GTF2I_HUMAN	P78347	215|
CHD4_HUMAN	Q14839	1594|1827|
HEM6_HUMAN	P36551	192|
PP4R2_HUMAN	Q9NY27	22|
NSL1_HUMAN	Q96IY1	224|
KIF11_HUMAN	P52732	723|
DCAF1_HUMAN	Q9Y4B6	784|
HUWE1_HUMAN	Q7Z6Z7	2721|3361|3259|
CD2AP_HUMAN	Q9Y5K6	540|
MCM3_HUMAN	P25205	148|
UBP24_HUMAN	Q9UPU5	1362|
PK3CD_HUMAN	O00329	132|
UACA_HUMAN	Q9BZF9	408|
RS28_HUMAN	P62857	27|
CSDE1_HUMAN	O75534	680|
GPTC8_HUMAN	Q9UKJ3	508|
VIME_HUMAN	P08670	328|
TIGAR_HUMAN	Q9NQ88	114|161|
IMDH2_HUMAN	P12268	331|
DNJA4_HUMAN	Q8WW22	368|
I2BP1_HUMAN	Q8IU81	363|
KAP2_HUMAN	P13861	101|
DDX6_HUMAN	P26196	102|
LIMA1_HUMAN	Q9UHB6	316|
OGT1_HUMAN	O15294	323|
NU160_HUMAN	Q12769	1166|
TDRD7_HUMAN	Q8NHU6	333|
INT9_HUMAN	Q9NV88	578|
DCPS_HUMAN	Q96C86	37|
VASP_HUMAN	P50552	334|
HNRL2_HUMAN	Q1KMD3	308|
FAD1_HUMAN	Q8NFF5	236|
TBC15_HUMAN	Q8TC07	197|
EFL1_HUMAN	Q7Z2Z2	953|
ELP4_HUMAN	Q96EB1	218|
DNMT1_HUMAN	P26358	1125|
HDAC1_HUMAN	Q13547	408|
CSK22_HUMAN	P19784	336|
TCAL3_HUMAN	Q969E4	44|
RO60_HUMAN	P10155	71|
STRN4_HUMAN	Q9NRL3	337|
STK24_HUMAN	Q9Y6E0	394|
CLIC1_HUMAN	O00299	59|
PABP1_HUMAN	P11940	339|132|
PABP4_HUMAN	Q13310	339|132|
HMGCL_HUMAN	P35914	323|
UBP15_HUMAN	Q9Y4E8	264|
IMPCT_HUMAN	Q9P2X3	226|
MSH2_HUMAN	P43246	527|
MCTS1_HUMAN	Q9ULC4	14|
EEA1_HUMAN	Q15075	1134|
PPIL4_HUMAN	Q8WUA2	426|
MCAF1_HUMAN	Q6VMQ6	955|
NCDN_HUMAN	Q9UBB6	98|
SURF6_HUMAN	O75683	19|
1433T_HUMAN	P27348	134|
RL10_HUMAN	P27635	105|
NOP58_HUMAN	Q9Y2X3	439|139|
CPSF3_HUMAN	Q9UKF6	498|
REN3B_HUMAN	Q9BZI7	319|
JUPI2_HUMAN	Q9H910	118|
PRKDC_HUMAN	P78527	2342|25|
ACTN4_HUMAN	O43707	499|
IKKA_HUMAN	O15111	406|
LRBA_HUMAN	P50851	1228|950|
UBR1_HUMAN	Q8IWV7	993|
NOSIP_HUMAN	Q9Y314	8|
UBE3A_HUMAN	Q05086	198|108|
STAT3_HUMAN	P40763	712|
I2BPL_HUMAN	Q9H1B7	63|
GNL3_HUMAN	Q9BVP2	113|
CLMN_HUMAN	Q96JQ2	353|
AAPK2_HUMAN	P54646	174|
AAPK1_HUMAN	Q13131	185|
COMT_HUMAN	P21964	223|
NCK5L_HUMAN	Q9HCH0	783|
PYRG1_HUMAN	P17812	491|
MAAI_HUMAN	O43708	205|
ATG7_HUMAN	O95352	524|
DSN1_HUMAN	Q9H410	65|
ESS2_HUMAN	Q96DF8	263|
PURA_HUMAN	Q00577	272|
RAI14_HUMAN	Q9P0K7	584|
OSBL9_HUMAN	Q96SU4	720|
DDX20_HUMAN	Q9UHI6	524|
TRIP6_HUMAN	Q15654	47|
CCD50_HUMAN	Q8IVM0	85|
DPYL2_HUMAN	Q16555	504|
RS16_HUMAN	P62249	25|
NFKB2_HUMAN	Q00653	57|432|
SYCC_HUMAN	P49589	27|
TOE1_HUMAN	Q96GM8	371|
DIP2B_HUMAN	Q9P265	1019|
POP1_HUMAN	Q99575	530|358|
FOXK1_HUMAN	P85037	404|
R3HD4_HUMAN	Q96D70	29|
NAA30_HUMAN	Q147X3	74|
SPAG7_HUMAN	O75391	191|
BORG4_HUMAN	Q9H3Q1	313|
PIMT_HUMAN	P22061	102|
LRCH3_HUMAN	Q96II8	531|
CD11B_HUMAN	P21127	440|
TUT7_HUMAN	Q5VYS8	911|
GPX4_HUMAN	P36969	93|134|
MAGD2_HUMAN	Q9UNF1	516|
KAD2_HUMAN	P54819	40|
KLC4_HUMAN	Q9NSK0	113|
TES_HUMAN	Q9UGI8	196|
TADBP_HUMAN	Q13148	50|
CUL4A_HUMAN	Q13619	633|
CUL4B_HUMAN	Q13620	787|
PLEC_HUMAN	Q15149	530|
TBCK_HUMAN	Q8TEA7	386|
NUCL_HUMAN	P19338	543|
GABPA_HUMAN	Q06546	421|
TMED8_HUMAN	Q6PL24	161|
NUDC1_HUMAN	Q96RS6	376|
CAN7_HUMAN	Q9Y6W3	197|
TBA1A_HUMAN	Q71U36	347|
TBA1C_HUMAN	Q9BQE3	347|
2ABA_HUMAN	P63151	334|
2ABB_HUMAN	Q00005	330|
SIN3A_HUMAN	Q96ST3	1167|
HNRPL_HUMAN	P14866	472|
HS74L_HUMAN	O95757	540|
ROCK2_HUMAN	O75116	649|
APC7_HUMAN	Q9UJX3	131|
ACACA_HUMAN	Q13085	1297|
BCCIP_HUMAN	Q9P287	213|
TPX2_HUMAN	Q9ULW0	536|
PTBP1_HUMAN	P26599	23|
CND2_HUMAN	Q15003	418|
HERC4_HUMAN	Q5GLZ8	175|
MRGBP_HUMAN	Q9NV56	170|
CSTFT_HUMAN	Q9H0L4	222|
RRAGC_HUMAN	Q9HB90	377|
HPBP1_HUMAN	Q9NZL4	22|
GCR_HUMAN	P04150	302|
EI2BG_HUMAN	Q9NR50	281|
CUL3_HUMAN	Q13618	636|
TRM2A_HUMAN	Q8IZ69	463|
MED15_HUMAN	Q96RN5	660|
BRX1_HUMAN	Q8TDN6	52|
UBR4_HUMAN	Q5T4S7	2554|
KAZRN_HUMAN	Q674X7	312|
DCAF8_HUMAN	Q5TAQ9	272|
RUFY1_HUMAN	Q96T51	320|
PRC2A_HUMAN	P48634	437|
SCLY_HUMAN	Q96I15	22|
SASH1_HUMAN	O94885	727|
TPR_HUMAN	P12270	1149|
CYBP_HUMAN	Q9HB71	173|
DHX57_HUMAN	Q6P158	453|
1433E_HUMAN	P62258	97|
DLGP5_HUMAN	Q15398	592|
FUBP3_HUMAN	Q96I24	460|
DAZP1_HUMAN	Q96EP5	85|
ABRAL_HUMAN	Q9P1F3	39|
LAD1_HUMAN	O00515	428|
WIPI2_HUMAN	Q9Y4P8	393|
SIN1_HUMAN	Q9BPZ7	149|
PRDX3_HUMAN	P30048	229|
ECHM_HUMAN	P30084	62|
HS90B_HUMAN	P08238	564|
FN3K_HUMAN	Q9H479	24|
PPCE_HUMAN	P48147	57|
TTC1_HUMAN	Q99614	8|
KS6A3_HUMAN	P51812	229|
KS6A1_HUMAN	Q15418	223|
SH3G1_HUMAN	Q99961	277|
PPR37_HUMAN	O75864	660|
AR6P4_HUMAN	Q66PJ3	220|
TLN1_HUMAN	Q9Y490	1353|
SMRC1_HUMAN	Q92922	761|
SNX8_HUMAN	Q9Y5X2	192|
CBL_HUMAN	P22681	508|
PDLI5_HUMAN	Q96HC4	213|
GSK3B_HUMAN	P49841	14|
DPH2_HUMAN	Q9BQC3	250|
GSTO1_HUMAN	P78417	192|
ZCCHV_HUMAN	Q7Z2W4	645|
TACC1_HUMAN	O75410	219|
DNM1L_HUMAN	O00429	505|
IRF4_HUMAN	Q15306	194|
SREK1_HUMAN	Q8WXA9	494|
EIF3D_HUMAN	O15371	19|
ABCF3_HUMAN	Q9NUQ8	102|
LN28B_HUMAN	Q6ZN17	187|
HPRT_HUMAN	P00492	106|
YJU2_HUMAN	Q9BW85	275|
RPP25_HUMAN	Q9BUL9	16|
MITF_HUMAN	O75030	209|
KIF1B_HUMAN	O60333	1635|

REFERENCES

All references listed in the instant disclosure, including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries (including but not limited to UniProt, EMBL, and GENBANK® biosequence database entries and including all annotations available therein) are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, and/or teach methodology, techniques, and/or compositions employed herein. The discussion of the references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of any cited reference.

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• 16. Brulet, et al. Liganding Functional Tyrosine Sites on Proteins Using Sulfur-Triazole Exchange Chemistry. J Am Chem Soc 2020, 142, 8270-8280.
• 17. Chen et al. Direct, Regioselective N-Alkylation of 1,3-Azoles, Org. Lett. 2016, 18 (1), 16-19.

It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

1. A method for identifying a reactive amino acid residue of a protein, the method comprising: (a) providing a protein sample comprising isolated proteins, living cells, a cell lysate, or a biological organism;(b) contacting the protein sample with a probe compound of Formula (I) for a period of time sufficient for the probe compound to react with at least one reactive amino acid in a protein in the protein sample, thereby forming at least one modified amino acid residue; and(c) analyzing proteins in the protein sample or removed from the protein sample to identify at least one modified 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):

2. The method of claim 1, where the probe compound of Formula (I) has a structure of Formula (Ia):

3. The method of claim 1, wherein the reactive amino acid residue is a cysteine residue.

4. The method of claim 1, wherein the modified amino acid residue has a structure of Formula (IIa-i):

5. The method of claim 1, wherein R1 and R2 are selected from H, halo, and amino or wherein R1 and R2 are selected from H and halo.

6. The method of claim 1, wherein R1 is chloro or fluoro.

7. The method of claim 1, wherein R2 is chloro or fluoro.

8. The method of claim 1, wherein X is —CH2—C≡CH.

9. The method of claim 1, wherein the probe compound is selected from the group consisting of 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine, 2,6-dichloro-9-(prop-2-yn-1-yl)-9H-purine, 6-chloro-7-(prop-2-yn-1-yl)-7H-purine, 6-chloro-9-(prop-2-yn-1-yl)-9H-purine, 2-chloro-7-(prop-2-yn-1-yl)-7H-purine, 2-chloro-9-(prop-2-yn-1-yl)-9H-purine, 2,6-difluoro-7-(prop-2-yn-1-yl)-7H-purine, 2,6,-difluoro-9-(prop-2-yn-1-yl)-9H-purine, 6-chloro-2-fluoro-7-(prop-2-yn-1-yl)-7H-purine, 6-chloro-2-fluoro-9-(prop-2-yn-1-yl)-9H-purine, 6-chloro-2-amino-7-(prop-2-yn-1-yl)-7H-purine, and 6-chloro-2-amino-9-(prop-2-yn-1-yl)-9H-purine.

10. The method of claim 1, wherein the probe compound has a structure of Formula (Ib).

11. The method of claim 1, wherein the probe compound is 2,6-dichloro-7-(prop-2-yn-1-yl)-7H-purine.

12. The method of claim 1, wherein the analyzing of step (c) further comprises tagging the at least one modified reactive amino acid residue with a compound comprising a detectable labeling group, thereby forming at least one tagged reactive amino acid residue comprising said detectable labeling group.

13. The method of claim 12, wherein the detectable labeling group comprises biotin or a biotin derivative, optionally wherein the biotin derivative is desthiobiotin.

14. The method of claim 12, wherein the tagging comprises reacting an alkyne group in the X moiety of the at least one modified reactive amino acid residue with a compound comprising (i) an azide moiety and (ii) the detectable labeling group, optionally via a copper-catalyzed azide-alkyne cycloaddition (CuAAC) coupling reaction.

15. The method of claim 12, wherein the analyzing further comprises digesting proteins with trypsin to provide a digested protein sample comprising a protein fragment comprising the at least one tagged reactive amino acid moiety comprising the detectable labeling group.

16. The method of claim 15, wherein the analyzing further comprises enriching the digested protein sample for the detectable labeling group, optionally wherein the enriching comprises contacting the digested protein sample with a solid support comprising a binding partner of the detectable labeling group.

17. The method of claim 16, wherein the analyzing further comprises analyzing the enriched digested protein sample via liquid chromatography-mass spectrometry (LC-MS).

18. The method of claim 1, wherein the protein sample is a biological organism, optionally a mammal; wherein contacting the protein sample with the probe compound of Formula (I) comprises administering the probe compound of Formula (I) to the biological organism, optionally via oral administration or injection; and wherein prior to analyzing the proteins, tissues are removed from the biological organism and homogenized.

19. The method of claim 1, wherein: providing the protein sample further comprises separating the protein sample into a first protein sample and a second protein sample;contacting the protein sample with a probe compound of Formula (I) comprises contacting the first protein sample with a first probe compound of Formula (I) at a first probe concentration for a first period of time and contacting the second protein sample with one of the group consisting of: (b1) a second probe compound of Formula (I) at the first probe concentration for the first period of time, (b2) the first probe compound of Formula (I) at a second probe concentration for the first period of time, and (b3) the first probe compound of Formula (I) at the first probe concentration for a second period of time; thereby forming at least one modified reactive amino acid residue in said first and/or said second protein sample; andanalyzing 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.

20. The method of claim 1, wherein the protein sample comprises living cells and wherein 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), optionally wherein the first cell culture medium comprises 13C- and/or 15N-labeled amino acids, further optionally wherein the first cell culture medium comprises 13C-,15N-labeled lysine and arginine; and culturing the second protein sample in a second cell culture medium, wherein said second cell culture medium comprises a naturally occurring isotope distribution, prior to the contacting of step (b).

21. The method of claim 20, wherein one of the first and the second protein sample is cultured in the presence of an inhibitor of an enzyme known or suspected of being present in said first or second protein sample.

22. The method of claim 1, wherein the probe compound of Formula (I) comprises a detectable labeling group comprising a heavy isotope or wherein the analyzing of step (c) further comprises tagging the at least one modified amino acid residue with a compound comprising a detectable labeling group comprising a heavy isotope, optionally wherein the heavy isotope is carbon-13.

23. A probe compound for detecting a reactive amino acid residue, optionally a reactive cysteine residue, in a protein, wherein the probe compound is selected from the group consisting of 2,6-difluoro-7-(prop-2-yn-1-yl)-7H-purine, 2,6-difluoro-9-(prop-2-yn-1-yl)-9H-purine, and 6-chloro-2-fluoro-7-(prop-2-yn-1-yl)-7H-purine.

24. A compound having the structure of Formula (III):

25. The compound of claim 24, wherein the compound of Formula (III) has a structure of Formula (IIIa):

26. The compound of claim 24, wherein R3 is selected from chloro, methyl, —SH—(CH2)3CH3; —NH(CH2)3CH3; and —O—(C6H4)CH3.

27. The compound of claim 24, wherein R4 is chloro or fluoro.

28. The compound of claim 24, wherein Z is acetyl, n-hexanoyl, n-dodecanoyl; cyclohexyl, —S(═O)2—R5, —S(═O)2—N(R6)2, —S(═O)2—O—R7, and

29. The compound of claim 28, wherein Z is selected from

30. The compound of claim 24, wherein the compound is selected from the group consisting of 4-((2,6-dichloro-7H-purin-7-yl)sulfonyl)morpholine, 4-((2,6-dichloro-9H-purin-9-yl)sulfonyl)morpholine, 2,6-dichloro-7-((4-fluorophenyl)sulfonyl)-7H-purine, 2,6-dichloro-9-((fluorophenyl)sulfonyl)-9H-purine, 2,6-dichloro-7-((4-methoxyphenyl)sulfonyl)-7H-purine, 2,6-dichloro-9-((4-methoxyphenyl)sulfonyl)-9H-purine, 2,6-dichloro-7-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl)-7H-purine, 2,6-dichloro-9-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl)-9H-purine, 1-(2,6-dichloro-9H-purin-9-yl)dodecan-1-one, 1-(2,6-dichloro-7H-purin-7-yl)hexan-1-one, 1-(2,6-dichloro-9H-purin-9-yl)hexan-1-one, 1-(6-chloro-2-fluoro-7H-purin-7-yl)hexan-1-one, 1-(6-chloro-2-fluoro-9H-purin-9-yl)hexan-1-one, 1-(2-chloro-6-methyl-9H-purin-9-yl)hexan-1-one, 2-chloro-9-cyclohexyl-6-methyl-9H-purine, 9-cyclohexyl-2-fluoro-6-methyl-9H-purine, 1-(6-(butylthio)-2-fluoro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylthio)-2-chloro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylthio)-2-chloro-7H-purin-7-yl)ethan-1-one, 1-(6-(butylthio)-2-fluoro-7H-purin-7-yl)ethan-1-one, 1-(2-chloro-6-(p-tolyloxy)-9H-purin-9-yl)ethan-1-one, 1-(2-fluoro-6-(p-tolyloxy)-9H-purin-9-yl)ethan-1-one, 1-(2-chloro-6-(p-tolyloxy)-7H-purin-7-yl)ethan-1-one, 1-(2-fluoro-6-(p-tolyloxy)-7H-purin-7-yl)ethan-1-one, 1-(6-(butylamino)-2-chloro-7H-purin-7-yl)ethan-1-one, 1-(6-(butylamino)-2-fluoro-7H-purin-7-yl)ethan-1-one, 1-(6-(butylamino)-2-chloro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylamino)-2-fluoro-9H-purin-9-yl)ethan-1-one, 2,6-dichloro-N, N-diethyl-7H-purine-7-sulfonamide, 2,6-dichloro-N, N-diethyl-9H-purine-9-sulfonamide, N-benzyl-2,6-dichloro-N-methyl-9H-purine-9-sulfonamide, N-benzyl-2,6-dichloro-N-methyl-7H-purine-7H-sulfonamide, benzyl 2,6-dichloro-7H-purine-7-sulfonate, benzyl 2,6-dichloro-9H-purine-9-sulfonate, methyl 2,6-dichloro-9H-purine-9-sulfonate, and methyl 2,6-dichloro-7H-purine-7-sulfonate.

31. A compound, wherein said compound is 2,6-dichloro-7-(4-nitrobenzyl)-7H-purine.

32. A modified cysteine-containing protein comprising a modified cysteine residue wherein the modified cysteine residue is formed by the reaction of a cysteine residue with a non-naturally occurring purine-based compound wherein said non-naturally occurring purine-based compound is a compound having a structure of Formula (I):

33. The modified cysteine-containing protein of claim 32, wherein said modified cysteine-containing protein comprises at least one modified cysteine residue comprising a structure of Formula (II-i):

34. The modified cysteine containing protein of claim 32, wherein the modified cysteine-containing protein is selenocysteine elongation factor (eEF-Sec) modified at cysteine 442, macrophage migration inhibitory factor modified at cysteine 81; or serine/threonine protein kinase 38-like modified at cysteine 235.

35. A method for modulating the activity of a protein comprising a reactive cysteine residue, wherein the method comprising contacting a protein comprising a reactive cysteine residue with a compound having a structure of Formula (III′):

36. The method of claim 35, wherein the compound having a structure of Formula (III′) is a compound having a structure of Formula (IIIa′):

37. The method of claim 35, wherein R3′ is selected from chloro, fluoro, methyl, n-butylthio, n-butylamino, or —O—(C6H4)—OMe.

38. The method of claim 35, wherein Z′ is selected from —CH2—CH═CH2, C2-C12 acyl, cyclohexyl, benzyl, —CH2—(C6H4)—NO2, —S(═O)2—R5′, and

39. The method of claim 35, wherein both R3′ and R4′ are chloro.

40. The method of claim 35, wherein the compound of Formula (III′) is selected from the group consisting of 4-((2,6-dichloro-7H-purin-7-yl)sulfonyl)morpholine, 4-((2,6-dichloro-9H-purin-9-yl)sulfonyl)morpholine, 2,6-dichloro-7-((4-fluorophenyl)sulfonyl)-7H-purine, 2,6-dichloro-9-((4-fluorophenyl)sulfonyl)-9H-purine, 2,6-dichloro-7-((4-methoxyphenyl)sulfonyl)-7H-purine, 2,6-dichloro-9-((4-methoxyphenyl)sulfonyl)-9H-purine, 2,6-dichloro-7-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl)-7H-purine, 2,6-dichloro-9-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl)-9H-purine, 2,6-dichloro-7-(4-nitrobenzyl)-7H-purine, 1-(2,6-dichloro-9H-purin-9-yl)dodecan-1-one, 1-(2,6-dichloro-7H-purin-7-yl)hexan-1-one, 1-(2,6-dichloro-9H-purin-9-yl)hexan-1-one, 1-(6-chloro-2-fluoro-7H-purin-7-yl)hexan-1-one, 1-(6-chloro-2-fluoro-9H-purin-9-yl)hexan-1-one, 1-(2-chloro-6-methyl-9H-purin-9-yl)hexan-1-one, 2-chloro-9-cyclohexyl-6-methyl-9H-purine, 9-cyclohexyl-2-fluoro-6-methyl-9H-purine, 1-(6-(butylthio)-2-fluoro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylthio)-2-chloro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylthio)-2-chloro-7H-purin-7-yl)ethan-1-one, 1-(6-(butylthio)-2-fluoro-7H-purin-7-yl)ethan-1-one, 1-(2-chloro-6-(p-tolyloxy)-9H-purin-9-yl)ethan-1-one, 1-(2-fluoro-6-(p-tolyloxy)-9H-purin-9-yl)ethan-1-one, 1-(2-chloro-6-(p-tolyloxy)-7H-purin-7-yl)ethan-1-one, 1-(2-fluoro-6-(p-tolyloxy)-7H-purin-7-yl)ethan-1-one, 1-(6-(butylamino)-2-chloro-7H-purin-7-yl)ethan-1-one, 1-(6-(butylamino)-2-fluoro-7H-purin-7-yl)ethan-1-one, 7-allyl-2,6-dichloro-7H-purine, 9-allyl-2,6-dichloro-9H-purine, 2,6-dichloro-7-benzyl-7H-purine, 2,6-dichloro-9-benzyl-9H-purine, 2,6-dichloro-7-(4-nitrobenzyl)-7H-purine, 2,6-dichloro-9-(4-nitrobenzyl-9H-purine, 2-(2,6-dichloro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol, 1-(6-(butylamino)-2-chloro-9H-purin-9-yl)ethan-1-one, 1-(6-(butylamino)-2-fluoro-9H-purin-9-yl)ethan-1-one, 2,6-dichloro-N,N-diethyl-7H-purine-7-sulfonamide, 2,6-dichloro-N, N-diethyl-9H-purine-9-sulfonamide, N-benzyl-2,6-dichloro-N-methyl-9H-purine-9-sulfonamide, N-benzyl-2,6-dichloro-N-methyl-7H-purine-7H-sulfonamide, benzyl 2,6-dichloro-7H-purine-7-sulfonate, benzyl 2,6-dichloro-9H-purine-9-sulfonate, methyl 2,6-dichloro-9H-purine-9-sulfonate, and methyl 2,6-dichloro-7H-purine-7-sulfonate.

41. The method of claim 35, wherein modulating an activity of a protein comprising a reactive cysteine residue comprises inhibiting an activity of the protein comprising a reactive cysteine residue.

42. The method of claim 35, wherein modulating an activity of a protein comprising a reactive cysteine residue comprises activating an activity of the protein comprising a reactive cysteine residue.

43. The method of claim 35, wherein modulating an activity of a protein comprising a reactive cysteine residue comprises blocking a protein-protein interaction of the protein comprising a reactive cysteine residue.

44. The method of claim 35, wherein modulating an activity of a protein comprising a reactive cysteine residue comprises disrupting a protein-RNA interaction of the protein comprising a reactive cysteine residue.

45. The method of claim 35, wherein modulating an activity of a protein comprising a reactive cysteine residue comprises disrupting a protein-DNA interaction of the protein comprising a reactive cysteine residue.

46. The method of claim 35, wherein modulating an activity of a protein comprising a reactive cysteine residue comprises disrupting a protein-lipid interaction of the protein comprising a reactive cysteine residue.

47. The method of claim 35, wherein modulating an activity of a protein comprising a reactive cysteine residue comprises disrupting a protein-metabolite interaction of the protein comprising a reactive cysteine residue.

48. The method of claim 35, wherein modulating an activity of a protein comprising a reactive cysteine residue comprises disrupting subcellular localization of the protein comprising a reactive cysteine residue.

49. The method of claim 35, wherein modulating an activity of a protein comprising a reactive cysteine residue comprises triggering recruitment of an E3 ligase for targeted degradation of the protein comprising a reactive cysteine residue.