Methods for ligand discovery

Abstract

The present invention provides novel methods for ligand discovery. The inventive methods rely on a process termed “tethering” where potential ligands are covalently bonded or “tethered” to a target and subsequently identified.

What is claimed is:  
     
         1 . A method comprising 
 a) contacting a target that comprises a chemically reactive group at or near a site of interest with a compound that is capable of forming a covalent bond with the chemically reactive group;    b) forming a covalent bond between the target and the compound thereby forming a target-compound conjugate; and,    c) identifying the target-compound conjugate by subjecting the target-compound conjugate to mass spectrometry.    
     
     
         2 . A mass spectrometer comprising a target-compound conjugate.  
     
     
         3 . A target-compound conjugate selected from the group consisting of  
       
         
           
           
               
               
           
         
       
       wherein  
       
         
           
           
               
               
           
         
       
       is the target, R and R′ are each independently unsubstituted C 1 -C 20  aliphatic, substituted C 1 -C 20  aliphatic, unsubstituted aryl, or substituted aryl; 
 m is 0, 1, or 2; and  
 n is 1 or 2.  
 
     
     
         4 . The target-compound conjugate of  claim 3  wherein the target is a polypeptide.  
     
     
         5 . The target-compound conjugate of  claim 4  wherein the covalent bond between the —S—S— group and the  
       
         
           
           
               
               
           
         
       
       target is reversible.  
     
     
         6 . The target-compound conjugate of  claim 4  wherein the covalent bond between the —S—S— group and the  
       
         
           
           
               
               
           
         
       
       target is irreversible.  
     
     
         7 . The target-compound conjugate of  claim 4  wherein the target is selected from the group consisting of enzymes, receptors, transcription factors, ligands for receptors, growth factors, cytokines, immunoglobulins, nuclear proteins, signal transduction components, and allosteric enzyme regulators.  
     
     
         8 . A method comprising: 
 a) contacting a target protein that is capable of forming a disulfide bond with a ligand candidate that is also capable of forming a disulfide bond;    b) forming a disulfide bond between the target protein and the ligand candidate thereby forming a target-ligand conjugate; and    c) identifying the ligand present in the target protein-ligand conjugate.    
     
     
         9 . The method as in  claim 8  wherein the contacting step occurs in the presence of a reducing agent.  
     
     
         10 . The method as in  claim 8  wherein the identification occurs using mass spectrometry.  
     
     
         11 . The method as in  claim 8  wherein the identification occurs using a labeled probe.  
     
     
         12 . The method as in  claim 8  wherein the identification occurs using a functional assay.  
     
     
         13 . The method as in  claim 8  wherein the identification occurs using chromatography.  
     
     
         14 . The method as in  claim 8  wherein the identification occurs using surface plasmon resonance.  
     
     
         15 . The method as in  claim 8  wherein the ligand candidate is selected from the group comprising  
       
         
           
           
               
               
           
         
       
       wherein R and R′ are each independently unsubstituted C 1 -C 20  aliphatic, substituted C 1 -C 20  aliphatic, unsubstituted aryl, or substituted aryl; 
 m is 0, 1, or 2; and,  
 n is 1 or 2.  
 
     
     
         16 . The method of  claim 8  wherein the target protein comprises an —SH group from a cysteine that is part of the native amino acid sequence of said protein.  
     
     
         17 . The method of  claim 8  wherein the target protein has comprises an engineered —SH group from a cysteine introduced into the amino acid sequence of said protein.  
     
     
         18 . A library of compounds wherein each member comprises a moiety —SSR 1  where R 1  is unsubstituted C 1 -C 10  aliphatic, substituted C 1 -C 10  aliphatic, unsubstituted aryl, and wherein each member has a different mass.  
     
     
         19 . The library of  claim 18  having at least 5 members.  
     
     
         20 . The library of  claim 18  having at least 100 members.  
     
     
         21 . The library of  claim 18  wherein each member library has a mass that differs from another member of the library by at least 5 atomic mass units.  
     
     
         22 . The library of  claim 18  wherein each member has a mass that differs from another member of the library by at least 10 atomic mass units.  
     
     
         23 . A method comprising: 
 a) identifying a first compound of the formula R D SSR 1  that binds to a target protein;    b) identifying a second compound of the formula R E SSR 1  that binds to a target protein; and    c) forming a conjugate compound comprising R D  and R E  wherein    R D  and R E  are each independently C 1 -C 20  unsubstituted aliphatic, C 1 -C 20  substituted aliphatic, unsubstituted aryl, and substituted aryl; and R 1  is unsubstituted C 1 -C 10  aliphatic, substituted C 1 -C 10  aliphatic, unsubstituted aryl.    
     
     
         24 . The method of  claim 23  wherein the identification of the second compound that binds to the target occurs in the presence of the first compound.  
     
     
         25 . The method of  claim 23  wherein R D SSR 1  and R E SSR 1  are each independently selected from the group consisting of  
       
         
           
           
               
               
           
         
       
       wherein R and R′ are each independently unsubstituted C 1 -C 20  aliphatic, substituted C 1 -C 20  aliphatic, unsubstituted aryl, or substituted aryl; 
 m is 0, 1, or 2; and,  
 n is 1 or 2.  
 
     
     
         26 . A method comprising 
 a) providing a target having an anchoring group that is capable of forming a covalent bond or coordinating a metal at or near 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 either a covalent bond or coordinates a metal and a second functionality that is capable of forming a covalent bond;    c) contacting the target-extender complex with a candidate ligand that comprises a group that is capable of forming a covalent bond with the second functionality;    d) forming a covalent bond between the target-extender complex and the candidate ligand; and,    e) identifying the candidate ligand present in the target-extender-ligand conjugate.    
     
     
         27 . The method of  claim 26  wherein the anchoring group is selected from a group consisting of a reactive electrophile, a reactive nucleophile, and a metal coordination site.  
     
     
         28 . A method comprising: 
 a) providing a target having a reactive nucleophile at or near 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 and a second functionality that is capable of forming a disulfide bond;    c) contacting the target-extender complex with a ligand candidate that is capable of forming a disulfide bond;    d) forming a disulfide bond between the target-extender complex and the ligand candidate thereby forming a target-extender-ligand conjugate; and,    e) identifying the ligand candidate present in the target-extender-ligand conjugate.    
     
     
         29 . The method as in  claim 28  wherein the reactive nucleophile on the target is a thiol or a masked thiol.  
     
     
         30 . The method of  claim 28  wherein the extender is of the formula:  
       
         
           
           
               
               
           
         
       
       where R is unsubstituted C 1 -C 20  aliphatic, substituted C 1 -C 20  aliphatic, unsubstituted aryl, and substituted aryl; R′ is H, —SR 1  wherein R 1  is unsubstituted C 1 -C 10  aliphatic, substituted C 1 -C 10  aliphatic, unsubstituted aryl, and substituted aryl; X is a leaving group, and the boxes in each formula represent a binding determinant.  
     
     
         31 . The method of  claim 28  wherein the extender is of the formula:  
       
         
           
           
               
               
           
         
       
       where R′ is H, —SR 1  wherein R 1  is unsubstituted C 1 -C 10  aliphatic, substituted C 1 -C 10  aliphatic, unsubstituted aryl, and substituted aryl, and the boxes represent a binding determinant.