Methods for Identifying Compounds that Modulate Enzymatic Activities by Employing Covalently Bonded Target-Extender Complexes with Ligand Candidates

Abstract

The present invention relates to the use of “tethering” to identify compounds that modulate enzymatic activity.

1 . A method comprising:
 a) providing a target having a reactive thiol located outside of a site of interest;   b) contacting the target with an extender thereby forming a target-extender complex wherein the extender comprises a first functionality that forms a first covalent bond with the thiol and a second functionality that is capable of forming a second covalent bond;   c) contacting the target-extender complex with a candidate ligand that comprises a group that is capable of forming a second covalent bond with the second functionality;   d) forming a second covalent bond between the target-extender complex and the candidate ligand thereby forming a target-extender-ligand conjugate; and, e) identifying the candidate ligand present in the target-extender-ligand conjugate; wherein at least the second covalent bond formed is a disulfide bond.   
     
     
         2 . (canceled) 
     
     
         3 . A method for identifying a candidate ligand comprising:
 a) providing a target having a first reactive nucleophile located inside of a site of interest and a second reactive nucleophile located outside of the site of interest;   b) contacting the target with an extender thereby forming a target-extender complex, the extender comprising a first functionality and a latent second functionality, a cleavable linker and a binding determinant wherein the first functionality forms a first covalent bond with the second reactive nucleophile and the binding determinant binds to the site of interest;   c) cleaving the extender at the cleavable linker thereby forming a modified target-extender complex thereby exposing the second functionality and releasing the binding determinant from the site of interest;   d) contacting the modified target-extender complex with a candidate ligand that comprises a group that is capable of forming a second covalent bond with the second functionality;   e) forming a second covalent bond between the modified target-extender complex and the candidate ligand thereby forming a target-extender-ligand conjugate; and,   f) identifying the candidate ligand present in the target-extender-ligand conjugate.   
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the first covalent bond formed is a thioether bond and the second covalent bond is a disulfide bond, whereby the target-extender-ligand complex formed has a thioether bond between the target and extender and a disulfide bond between the extender and the ligand candidate. 
     
     
         6 . The method of  claim 5 , wherein the second functionality is a disulfide. 
     
     
         7 . The method of  claim 5 , wherein the second functionality is a thioester. 
     
     
         8 . The method of  claim 1 , wherein the first covalent bond formed is a first disulfide bond and the second covalent bond formed is a second disulfide bond, whereby the target-extender-ligand complex formed has a first disulfide bond between the target and the extender and a second disulfide bond between the extender and the ligand candidate. 
     
     
         9 . The method of  claim 8 , wherein the second functionality is a disulfide. 
     
     
         10 . The method of  claim 8 , wherein the second functionality is a thioester. 
     
     
         11 . The method of  claim 1  wherein the extender includes a double or triple bond adjacent to a carbonyl, imide, quinine, CN, NO 2 , —S(═O)— or vinyl sulfone. 
     
     
         12 . The method of  claim 1  wherein the extender is selected from the group consisting of 
       
         
           
           
               
               
           
         
       
       where  is a binding determinant and R′ is is H or —SR 1  wherein R 1  is where R 1  is unsubstituted C 1 -C 10  aliphatic, substituted C 1 -C 10  aliphatic, unsubstituted aromatic or substituted aromatic. 
     
     
         13 . The method of  claim 12  wherein  is selected from the group consisting of unsubstituted C 1 -C 20  aliphatic, substituted C 1 -C 20  aliphatic, unsubstituted aryl and substituted aryl. 
     
     
         14 . The method of  claim 1 , wherein the reactive thiol on the target is a naturally occurring —SH from a cysteine that is part of a naturally occurring protein sequence. 
     
     
         15 . The method of  claim 1  wherein the reactive thiol on the target is from a cysteine where mutagenesis was used to replace a naturally occurring amino acid. 
     
     
         16 . The method of  claim 1  wherein the reactive thiol is a masked a disulfide. 
     
     
         17 . The method of  claim 1  wherein the target-extender complex is contacted with a candidate ligand in the presence of a reducing agent. 
     
     
         18 . The method of  claim 17  wherein the reducing agent is selected from the group consisting of: cysteine, cysteamine, dithiothreitol, dithioerythritol, glutathione, 2-mercaptoethanol, 3-mercaptoproprionic acid, a phosphine and sodium borohydride. 
     
     
         19 . A method comprising:
 a) providing a target having a reactive nucleophile located outside of a site of interest;   b) contacting the target with an extender thereby forming a target-extender complex wherein the extender comprises a first functionality that reacts with the nucleophile in the target to form a covalent bond with the nucleophile and a second functionality that is capable of forming a disulfide bond, wherein the extender is selected from the group consisting of   
       
         
           
           
               
               
           
         
       
       where  is a binding determinant and R′ is is H or —SR 1  wherein R 1  is is selected from the group consisting of unsubstituted C 1 -C 10  aliphatic, substituted C 1 -C 10  aliphatic, unsubstituted aromatic or substituted aromatic;
 c) contacting the target-extender complex with a candidate ligand that comprises a group that is capable of forming a disulfide bond; 
 d) forming a disulfide bond between the target-extender complex and the candidate ligand thereby forming a target-extender-ligand conjugate; and 
 e) identifying the candidate ligand present in the target-extender-ligand conjugate. 
 
     
     
         20 . The method of  claim 19  wherein the candidate ligand present in the target-extender-ligand conjugage is identified using mass spectrometry. 
     
     
         21 . The method of  claim 19  wherein the candidate ligand present in the target-extender-ligand conjugage is identified using a labeled probe. 
     
     
         22 . The method of  claim 19  wherein the candidate ligand present in the target-extender-ligand conjugage is identified using a functional assay. 
     
     
         23 . The method of  claim 19  wherein the candidate ligand present in the target-extender-ligand conjugage is identified using chromatography. 
     
     
         24 . A method comprising:
 a) providing a target having a first reactive thiol located inside of a site of interest and a second reactive thiol located outside of the site of interest;   b) contacting the target with an extender thereby forming a target-extender complex, the extender comprising a first functionality and a latent second functionality, a cleavable linker and a binding determinant wherein the first functionality forms a thioether bond with the second reactive thiol and the binding determinant binds to the site of interest;   c) cleaving the extender at the cleavable linker thereby forming a modified target-extender complex by exposing the second functionality and releasing the binding determinant from the site of interest;   d) contacting the modified target-extender complex with a candidate ligand that comprises a group that is capable of forming a disulfide bond with the second functionality;   e) forming a disulfide bond between the modified target-extender complex and the candidate ligand thereby forming a target-extender-ligand conjugate; and   f) identifying the candidate ligand present in the target-extender-ligand conjugate.   
     
     
         25 . The method of  claim 24  wherein the extender includes a double or triple bond adjacent to a carbonyl, imide, quinine, CN, NO 2 , —S(═O)— or vinyl sulfone. 
     
     
         26 . The method of  claim 24  wherein the extender is selected from the group consisting of 
       
         
           
           
               
               
           
         
       
       where   is the binding determinant and R′ is is H or —SR 1  wherein R 1  is selected from the group consisting of unsubstituted C 1 -C 10  aliphatic, substituted C 1 -C 10  aliphatic, unsubstituted aromatic and substituted aromatic. 
     
     
         27 . The method of  claim 26  wherein   is selected from the group consisting of unsubstituted C 1 -C 20  aliphatic, substituted C 1 -C 20  aliphatic, unsubstituted aryl and substituted aryl.