Method for identifying inhibitors of G protein coupled receptor signaling

Abstract

This invention relates to methods for identifying peptides and other compounds which block G protein coupled receptor mediated signaling with high affinity and specificity. Assays developed in conjunction with these methods also are disclosed.

1 . A method of identifying a G protein coupled receptor (GPCR) signaling inhibitor, which comprises: 
 (a) providing a first library comprising peptide members, wherein the primary sequences of said peptide members are based on the primary sequence of the native G protein peptide that binds to said GPCR on the G protein binding domain of said GPCR;    (b) screening said peptide first library members for binding to said G protein binding domain of said GPCR in competition with a native peptide that comprises said primary sequence of the native G protein of (a), to identify peptide first library members that bind to said GPCR G protein binding domain with higher affinity than that of said native peptide;    (c) selecting a high-affinity peptide first library member identified in (b);    (d) providing a second library of member compounds;    (e) screening said second library member compounds for binding to said GPCR G protein binding domain, wherein said screening is a binding assay performed in the presence of the peptide selected in (c), to determine whether a second library member compound binds to said GPCR G protein binding domain with equal or higher affinity than that of said second competitive peptide.    
     
     
         2 . The method of  claim 1 , wherein said screening of (b) or (e) is testing for binding to a GPCR molecule that comprises at least the intracellular fragment of said GPCR.  
     
     
         3 . The method of  claim 1 , wherein said native G protein peptide is selected from the group consisting of a Gα subunit or carboxyl terminal fragment thereof.  
     
     
         4 . The method of  claim 3 , wherein said native G protein peptide is a Gα subunit carboxyl terminal fragment from about 7 to about 70 amino acids long.  
     
     
         5 . The method of  claim 3 , wherein said native G protein peptide is a Gα subunit carboxyl terminal fragment from about 9 to about 23 amino acids long.  
     
     
         6 . The method of  claim 3 , wherein said native G protein peptide is a Gα subunit carboxyl terminal fragment from about 11 amino acids long.  
     
     
         7 . The method of  claim 3 , wherein said G protein subunit is a Gβγ dimer.  
     
     
         8 . The method of  claim 1 , wherein said first library peptide members provide signal to detect binding.  
     
     
         9 . The method of  claim 1 , wherein said first library is a combinatorial peptide library.  
     
     
         10 . The method of  claim 9 , wherein said combinatorial peptide library is a protein-peptide fusion protein library.  
     
     
         11 . The method of  claim 10 , wherein said protein-peptide fusion protein library is a maltose binding protein-peptide fusion protein library.  
     
     
         12 . The method of  claim 11 , wherein said peptide library is a peptide display library.  
     
     
         13 . The method of  claim 1 , wherein said second library of member compounds is a small molecule library.  
     
     
         14 . A compound identified by a method of  claim 1 .  
     
     
         15 . A method of selecting a G protein coupled receptor (GPCR) signaling inhibitor peptide, which comprises: 
 (a) providing a library comprising peptide members, wherein the primary sequences of said peptide members are based on the primary sequence of the native G protein peptide that binds to said GPCR on the G protein binding domain of said GPCR;    (b) screening said peptide library members for binding to said G protein binding domain of said GPCR in competition with a native peptide that comprises said primary sequence of the native G protein of (a), to identify peptide first library members that bind to said GPCR G protein binding domain with higher affinity than that of said native peptide;    (c) selecting a high-affinity peptide first library member identified in (b).    
     
     
         16 . The method of  claim 15 , wherein said screening of (b) is testing for binding to a GPCR molecule that comprises at least the intracellular fragment of said GPCR.  
     
     
         17 . The method of  claim 15 , wherein said native G protein peptide is selected from the group consisting of a Gα subunit or carboxyl terminal fragment thereof.  
     
     
         18 . The method of  claim 17 , wherein said native G protein peptide is a Gα subunit carboxyl terminal fragment from about 7 to about 70 amino acids long.  
     
     
         19 . The method of  claim 17 , wherein said native G protein peptide is a Gα subunit carboxyl terminal fragment from about 9 to about 23 amino acids long.  
     
     
         20 . The method of  claim 17 , wherein said native G protein peptide is a Gα subunit carboxyl terminal fragment from about 11 amino acids long.  
     
     
         21 . The method of  claim 17 , wherein said G protein subunit is a Gβγ dimer.  
     
     
         22 . The method of  claim 15 , wherein said first library peptide members provide signal to detect binding.  
     
     
         23 . The method of  claim 15 , wherein said first library is a combinatorial peptide library.  
     
     
         24 . The method of  claim 23 , wherein said combinatorial peptide library is a protein-peptide fusion protein library.  
     
     
         25 . The method of  claim 24 , wherein said protein-peptide fusion protein library is a maltose binding protein-peptide fusion protein library.  
     
     
         26 . The method of  claim 25 , wherein said peptide library is a peptide display library.  
     
     
         27 . The method of  claim 15 , wherein said second library of member compounds is a small molecule library.  
     
     
         28 . A compound identified by a method of  claim 15 .  
     
     
         29 . A method of identifying a G protein coupled receptor (GPCR) signaling inhibitor, which comprises: 
 (a) providing a library of candidate compounds to screen for binding to said G protein coupled receptor;    (b) providing a peptide that binds to said GPCR G protein binding domain with higher affinity than that of its native peptide;    (c) screening said library of candidate compounds for binding to said GPCR G protein binding domain in the presence of said peptide of (b), to determine whether a candidate compound binds to said GPCR G protein binding domain with equal or higher affinity than that of said peptide of (b).    
     
     
         30 . The method of  claim 29 , wherein said screening of (c) is testing for binding to a GPCR molecule that comprises at least the intracellular fragment of said GPCR.  
     
     
         31 . The method of  claim 29 , wherein said library of candidate compounds is a small molecule library.  
     
     
         32 . A compound identified by a method of  claim 29 .  
     
     
         33 . The method of  claim 31 , wherein said library of candidate compounds is a focused library of candidate compounds based on the structure of the peptide of (b).  
     
     
         34 . The method of  claim 31 , wherein said library of candidate compounds of step (a) is a combinatorial library.  
     
     
         35 . The method of  claim 34 , wherein said combinatorial library is a diverse small molecule library.  
     
     
         36 . The method of  claim 35 , wherein said diverse small molecule combinational library comprises drug-like molecules.  
     
     
         37 . The method of  claim 35 , wherein said diverse small molecule combinational library is a focused small molecule library.  
     
     
         38 . The method of inhibiting G protein coupled receptor signaling in a cell having a G protein coupled receptor which comprises administering to said cell in vitro a compound identified according to a method of  claim 1 .  
     
     
         39 . The method of inhibiting G protein coupled receptor signaling in a cell having a G protein coupled receptor which comprises administering to said cell in vitro a compound identified according to a method of  claim 15 .  
     
     
         40 . The method of inhibiting G protein coupled receptor signaling in a cell having a G protein coupled receptor which comprises administering to said cell in vitro a compound identified according to a method of  claim 29 .  
     
     
         41 . A compound identified by the method of  claim 15 , which comprises a peptide selected from the group consisting of SEQ ID NOS: 2, 13, 15, 17, 19, 21, 23, 25, 27, 30, 32, 34, 36, 38, 40, 42 and 45-111.  
     
     
         42 . A compound selected from the group consisting of SEQ ID NOS: 2, 13, 15, 17, 19, 21, 23, 25, 27, 30, 32, 34, 36, 38, 40, 42 and 45-111.