Method for identifying modulators of G protein coupled receptor signaling

Abstract

This invention relates to methods for identifying peptides and other compounds which block or enhance G protein coupled receptor mediated signaling with high affinity and specificity and/or which stabilize a particular conformer of a G protein coupled receptor. Assays, methods of treatment and other methods developed in conjunction with these methods also are disclosed.

1 . A two-screen method of detecting high-affinity G protein coupled receptor (GPCR) G protein interaction site binding compounds, which comprises: 
 (a) providing a peptide library the members of which are based on the primary sequence of a native G protein Gα subunit carboxyl terminal peptide sequence that binds to said GPCR on a G protein interaction site of said GPCR, wherein said G protein Gα subunit carboxyl terminal peptide sequence is selected from the group consisting of SEQ ID NOs: 2, 13, 15, 17, 21, 25, 26, 27, 30, 34, 38, 40, and 45-85;    (b) screening said peptide library for in vitro high affinity binding to said G protein interaction site of said GPCR to identify library members that bind to said G protein interaction site with higher affinity than that of said native G protein Gα subunit carboxyl terminal peptide sequence;    (c) selecting a member of said peptide library having binding to said GPCR of higher affinity than that of said native G protein Gα subunit carboxyl terminal peptide sequence    (d) providing a library of candidate compounds to screen for binding to said G protein interaction site of said GPCR;    (e) screening said library of candidate compounds in vitro for binding to said GPCR in competition with a member of said peptide library selected in step (c) to detect candidate compounds having high-affinity binding to said G protein interaction site of said GPCR.    
     
     
         2 . A two-screen method of detecting high-affinity GPCR G protein interaction site binding compounds, which comprises: 
 (a) providing a peptide library the members of which are based on the primary sequence of a native G protein Gα subunit carboxyl terminal peptide sequence that binds to said GPCR on a G protein interaction site of said GPCR;    (b) screening said peptide library for in vitro high affinity binding to said G protein interaction site of said GPCR to identify library members that bind to said G protein interaction site with higher affinity than that of said native G protein Gα subunit carboxyl terminal peptide sequence;    (c) selecting a member of said peptide library having binding to said GPCR of higher affinity than that of said native G protein Gα subunit carboxyl terminal peptide sequence;    (d) providing a library of candidate compounds to screen for binding to said G protein interaction site of said GPCR;    (e) screening said library of candidate compounds in vitro for binding to said GPCR in competition with a member of said peptide library selected in step (c) to detect candidate compounds having high-affinity binding to said G protein interaction site of said GPCR.    
     
     
         3 . A method of  claim 1 , wherein said screening of step (b) is performed by testing for binding to an intact G protein coupled receptor.  
     
     
         4 . A method of  claim 1 , wherein said screening of step (b) is performed by testing for binding to an intracellular fragment of a GPCR.  
     
     
         5 . A method of  claim 1 , wherein said screening of step (b) comprises a competitive binding assay.  
     
     
         6 . A method of  claim 5 , wherein said competitive binding assay is characterized by co-incubation of members of said peptide library with said native Gα subunit carboxyl terminal peptide sequence.  
     
     
         7 . A method of  claim 1 , wherein binding to said GPCR is determined by measuring a signal generated from interaction of an activating ligand with said GPCR.  
     
     
         8 . A method of  claim 1 , wherein said library of variant peptides is a combinatorial peptide library.  
     
     
         9 . A method of  claim 8 , wherein said library of variant peptides is a protein-peptide fusion protein library.  
     
     
         10 . A method of  claim 9 , wherein said protein-peptide fusion protein library is a maltose binding protein-peptide fusion protein library.  
     
     
         11 . A method of  claim 1 , wherein said library of variant peptides is a peptide display library.  
     
     
         12 . A method of  claim 2 , wherein said native G protein Gα subunit carboxyl terminal peptide sequence that binds to said GPCR on a G protein interaction site of said GPCR is from about 7 to about 70 amino acids long.  
     
     
         13 . A method of  claim 2 , wherein said native G protein Gα subunit carboxyl terminal peptide sequence that binds to said GPCR on a G protein interaction site of said GPCR is from about 7 to about 55 amino acids long.  
     
     
         14 . A method of  claim 2 , wherein said native G protein Gα subunit carboxyl terminal peptide sequence that binds to said GPCR on a G protein interaction site of said GPCR is from about 8 to about 50 amino acids long.  
     
     
         15 . A method of  claim 2 , wherein said native G protein Gα subunit carboxyl terminal peptide sequence that binds to said GPCR on a G protein interaction site of said GPCR is from about 9 to about 23 amino acids long.  
     
     
         16 . A method of  claim 2 , wherein said native G protein Gα subunit carboxyl terminal peptide sequence that binds to said GPCR on a G protein interaction site of said GPCR is about 11 amino acids long.  
     
     
         17 . A method of  claim 16 , wherein said G protein Gα subunit carboxyl terminal peptide sequence is selected from the group consisting of SEQ ID NOs: 2, 13, 15, 17, 21, 25, 26, 27, 30, 34, 38, 40, and 45-85.