G protein coupled receptor signaling modulation

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 G protein coupled receptor (GPCR)-G protein signaling modifying peptide that binds to the G protein binding domain of said GPCR with high-affinity identified by the method which comprises:
 (a) providing a library of candidate compounds to screen for binding to said G protein binding domain of said GPCR;   (b) providing a GPCR G protein binding domain binding peptide, wherein said GPCR G protein binding domain binding peptide binds to the GPCR with higher affinity than the native G protein;   (c) screening said library of candidate compounds for high affinity binding to said G protein binding domain of said GPCR in the presence of said GPCR G protein binding domain binding peptide;   (d) determining whether (i) a member of said library of candidate compounds has binding to said G protein binding domain of said GPCR of equal or higher affinity than that of the peptide of (b) or (ii) a member of said library of candidate compounds results in modified binding affinity of the peptide of (b) when it binds to said G protein binding domain of said GPCR; and   (e) identifying compounds that bind to said G protein binding domain of said GPCR of equal or higher affinity than that of the peptide of (b) or the binding of which to said G protein binding domain of said GPCR result in modified binding affinity of the peptide of (b).   
     
     
         2 . A GPCR-G protein signaling modifying peptide of  claim 1  which 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. 
     
     
         3 . A compound selected from the group consisting of SEQ ID Nos: 2, 13, 15, 17, 21, 25, 26, 27, 30, 34, 38, 40 and 45-85. 
     
     
         4 . A method for identifying a GPCR-G protein signaling modifying compound, which comprises:
 (a) providing a GPCR-G protein signaling modifying peptide that binds to the G protein binding domain of said GPCR with high affinity, wherein said peptide is labeled to provide a detectable signal;   (b) providing a library of candidate GPCR signaling modifying compounds;   (c) contacting said peptide of (a) with said GPCR under conditions such that said peptide binds to said GPCR;   (d) optionally removing unbound peptide from said GPCR;   (e) measuring the signaling activity of said peptide-bound GPCR and measuring said detectable label on said peptide;   (f) contacting the members of said library with said peptide-bound GPCR;   (g) measuring the signaling activity of said peptide-bound GPCR and measuring said detectable label on said peptide;   (h) determining whether said GPCR signaling activity is increased or decreased after contact with said library members and whether GPCR-peptide binding is increased or decreased after contact with said library members; and   (i) identifying library members for which contact with said peptide-bound GPCR results in modified GPCR signaling.   
     
     
         5 . A method of  claim 4 , wherein said measuring the signaling activity is selected from the group consisting of:
 (a) measuring inositol phosphate accumulation;   (b) measuring intracellular Ca 2+  levels;   (c) measuring adenyl cyclase activity;   (d) measuring transendothelial electrical resistance;   (e) measuring stress fiber formation;   (f) measuring ligand binding;   (g) measuring receptor expression;   (h) measuring receptor desensitization;   (i) measuring kinase activity;   (j) measuring phosphatase activity;   (k) measuring nuclear transcription factors;   (l) measuring cell migration (chemotaxis);   (m) measuring superoxide formation;   (n) measuring nitric oxide formation;   (o) measuring cell degranulation;   (p) measuring GIRK activity;   (q) measuring actin polymerization;   (r) measuring vasoconstriction;   (s) measuring cell permeability;   (t) measuring apoptosis;   (u) measuring cell differentiation;   (v) measuring membrane association of a protein that translocates upon GPCR activation;   (w) measuring cytosolic accumulation of a protein that translocates upon GPCR activation; and   (x) measuring nuclear association of a protein that translocates upon GPCR activation.   
     
     
         6 . A method of modifying GPCR signaling in a cell having a GPCR which comprises administering a compound of  claim 1  to said cell in vitro. 
     
     
         7 . A method of modifying GPCR signaling in a cell having a GPCR which comprises administering a compound of  claim 3  to said cell in vitro. 
     
     
         8 . A method of identifying a GPCR signaling enhancer, which comprises:
 (a) providing a peptide library based on a native GPCR binding peptide that binds said GPCR;   (b) screening said peptide library for high affinity binding to said GPCR;   (c) selecting a peptide library member that binds to said GPCR with higher affinity than that of the native peptide of (a);   (d) providing a library of candidate compounds to screen for binding to said GPCR;   (e) screening said library of candidate compounds for high affinity binding to said GPCR in competition with a peptide library member selected in (c); and   (f) identifying a candidate compound library member having binding to said GPCR of equal or higher affinity than that of the peptide library member selected in (c) or identifying a candidate compound library member the binding of which results in increased binding affinity of the peptide library member of step (c).   
     
     
         9 . A method of  claim 8 , wherein said screening of step (b) or step (e) is performed by testing for binding to an intact GPCR. 
     
     
         10 . A method of  claim 8 , wherein said screening of step (b) or step (e) is performed by testing for binding to at least an intracellular fragment of a GPCR. 
     
     
         11 . A method of  claim 8 , wherein said native GPCR binding peptide of (a) is a G protein subunit or fragment thereof. 
     
     
         12 . A method of  claim 11 , wherein said G protein subunit fragment is about 8 to about 50 amino acids long. 
     
     
         13 . A method of  claim 11 , wherein said G protein subunit fragment is about 9 to about 23 amino acids long. 
     
     
         14 . A method of  claim 11 , wherein said G protein subunit fragment is about 11 amino acids long. 
     
     
         15 . A method of  claim 11 , wherein said G protein subunit is a Gα subunit. 
     
     
         16 . A method of  claim 11 , wherein said G protein coupled receptor binding peptide is a Gα subunit carboxyl terminal peptide. 
     
     
         17 . A method of  claim 11 , wherein said G protein subunit is a Gβγ dimer. 
     
     
         18 . A method of  claim 8 , wherein said screening of step (b) comprises a competitive binding assay. 
     
     
         19 . A method of  claim 8 , wherein said peptide library members are capable of providing a detectable signal. 
     
     
         20 . A method of  claim 8 , wherein binding to said G protein coupled receptor is determined by measuring a signal generated from interaction of said signalling enhancer with said G protein coupled receptor. 
     
     
         21 . A method of  claim 8 , wherein said peptide library is a combinatorial peptide library. 
     
     
         22 . A method of  claim 8 , wherein said peptide library is a peptide display library. 
     
     
         23 . A method of  claim 8 , wherein said library of candidate compounds of step (d) is a focused library of candidate compounds based on the structure of a compound selected in step (c). 
     
     
         24 . A method of isolating a GPCR binding partner, which comprises:
 (a) providing a solid support comprising bound compound identified by a method according to  claim 8 ;   (b) providing a library of candidate GPCR binding partner compounds;   (c) contacting said library of candidate compounds with said solid support under conditions such that binding of said candidate compounds to said compound can occur;   (d) eluting unbound and nonspecifically bound candidate compounds from said solid support; and   (e) recovering bound candidate compounds from said solid support.   
     
     
         25 . A nucleic acid which comprises a DNA that encodes a peptide identified by a method according to  claim 8 , wherein said DNA is operably linked to a heterologous transcriptional regulatory sequence. 
     
     
         26 . An expression vector which comprises a nucleic acid of  claim 25 . 
     
     
         27 . A cell transfected with the expression vector of  claim 26 .