Cysteine reactive probes and uses thereof

Abstract

Disclosed herein are methods, compositions, probes, polypeptides, assays, and kits for identifying a cysteine containing protein as a binding target for a small molecule fragment. Also disclosed herein are methods, compositions, and probes for mapping a biologically active cysteine site on a protein and screening a small molecule fragment for interaction with a cysteine containing protein.

What is claimed is: 
     
         1 . A method of identifying a cysteine containing protein as a binding target for a small molecule fragment, comprising:
 a) obtaining a set of cysteine-reactive probe-protein complexes from a sample comprising a first cell solution treated with a small molecule fragment and a cysteine reactive probe wherein the cysteine-reactive probe comprises a reactive moiety capable of forming a covalent bond with a cysteine residue located on the cysteine containing protein;   b) analyzing the set of cysteine-reactive probe-protein complexes by a proteomic analysis means; and   c) based on step b), identifying a cysteine containing protein as the binding target for the small molecule fragment.   
     
     
         2 . The method of  claim 1 , further comprising determining a value of each of the cysteine containing protein from the set of cysteine-reactive probe-protein complexes for identifying a cysteine containing protein as the binding target for the small molecule fragment, wherein the value is determined based on the proteomic analysis means of step b). 
     
     
         3 . The method of  claim 1 , wherein the sample further comprises a second cell solution. 
     
     
         4 . The method of  claim 1 , further comprising contacting the first cell solution with a small molecule fragment for an extended period of time prior to incubating the first cell solution with a first cysteine-reactive probe to generate a first group of cysteine-reactive probe-protein complexes. 
     
     
         5 . The method of  claim 4 , wherein the extended period of time is about 5, 10, 15, 20, 30, 60, 90, 120 minutes or longer. 
     
     
         6 . The method of  claim 3 , further comprising contacting the second cell solution with a second cysteine-reactive probe to generate a second group of cysteine-reactive probe-protein complexes. 
     
     
         7 . The method of  claim 3 , wherein the first cysteine-reactive probe and the second cysteine-reactive probe are the same. 
     
     
         8 . The method of  claim 3 , wherein the first group and the second group of cysteine-reactive probe-protein complexes comprise the set of cysteine-reactive probe-protein complexes. 
     
     
         9 . The method of  claim 1 , wherein the cysteine containing protein is an enzyme, a transporter, a receptor, a channel protein, an adaptor protein, a chaperone, a signaling protein, a plasma protein, transcription related protein, translation related protein, mitochondrial protein, or cytoskeleton related protein. 
     
     
         10 . The method of  claim 1 , wherein the cysteine containing protein is a protein illustrated in Table 3. 
     
     
         11 . The method of  claim 1 , wherein the cysteine containing protein is a protein illustrated in Table 1, Table 2, Table 8, Table 9, Table 10A, Table 10B, Table 10C, Table 10D or Table 10E. 
     
     
         12 . The method of  claim 1 , wherein the small molecule fragment is a small molecule fragment of Formula (I): 
       
         
           
           
               
               
           
         
         wherein: 
         RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and 
         F is a small molecule fragment moiety. 
       
     
     
         13 . The method of  claim 12 , wherein the Michael acceptor moiety comprises an alkene or an alkyne moiety. 
     
     
         14 . The method of  claim 12 , wherein F is obtained from a compound library. 
     
     
         15 . The method of  claim 14 , wherein 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. 
     
     
         16 . The method of  claim 12 , wherein F is a small molecule fragment moiety illustrated in  FIG. 3 . 
     
     
         17 . The method of  claim 1 , wherein the cysteine-reactive probe is a cysteine-reactive probe of Formula (II): 
       
         
           
           
               
               
           
         
         wherein: 
         RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond to the thiol group of a cysteine residue; and 
         AHM is an affinity handle moiety. 
       
     
     
         18 . The method of  claim 17 , wherein the Michael acceptor moiety comprises an alkene or an alkyne moiety. 
     
     
         19 . The method of  claim 17 , wherein the affinity handle moiety comprises an affinity handle and a binding moiety that facilitates covalent interaction of the cysteine-reactive probe to a cysteine residue of a cysteine-containing protein. 
     
     
         20 . The method of  claim 17 , wherein the affinity handle comprises a carbodiimide, N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymethyl phosphine, maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, alkyne, azide, or isocyanate group.