Protein binding domains stabilizing functional conformational states of gpcrs and uses thereof

Abstract

The present invention relates to the field of GPCR structure biology and signaling. In particular, the present invention relates to protein binding domains directed against or capable of specifically binding to a functional conformational state of a G-protein-coupled receptor (GPCR). More specifically, the present invention provides protein binding domains that are capable of increasing the stability of a functional conformational state of a GPCR, in particular, increasing the stability of a GPCR in its active conformational state. The protein binding domains of the present invention can be used as a tool for the structural and functional characterization of G-protein-coupled receptors bound to various natural and synthetic ligands, as well as for screening and drug discovery efforts targeting GPCRs. Moreover, the invention also encompasses the diagnostic, prognostic and therapeutic usefulness of these protein binding domains for GPCR-related diseases.

1 - 42 . (canceled) 
     
     
         43 . A method for identifying a test compound that binds to an active conformational state of a GPCR, the method comprising:
 (i) providing a GPCR and a protein binding domain that binds to an active conformational state of the GPCR;   (ii) providing a test compound; and   (iii) evaluating whether the test compound binds to the active conformational state of the GPCR.   
     
     
         44 . The method of  claim 43 , further comprising:
 (iv) selecting a compound that binds to the active conformational state of the GPCR.   
     
     
         45 . The method of  claim 43 , wherein the test compound is selected from the group consisting of a polypeptide, a peptide, a small molecule, a natural product, a peptidomimetic, a nucleic acid, a lipid, a lipopeptide, a carbohydrate, an antibody or an antigen-binding fragment thereof, a heavy chain antibody (hcAb), a single domain antibody (sdAb), a minibody, a variable domain derived from a camelid heavy chain antibody, a variable domain of an immunoglobulin new antigen receptor (V NAR ), and a protein scaffold. 
     
     
         46 . The method of  claim 45 , wherein the antigen-binding fragment thereof is selected from the group consisting of a Fab, a Fab′, a F(ab′)2, an Fd, a single-chain Fvs (scFv), a single-chain antibody, a disulfide-linked Fv (dsFv), a fragment comprising a VL domain and a fragment comprising a VH domain. 
     
     
         47 . The method of  claim 45 , wherein the protein scaffold is selected from the group consisting of an alphabody, a protein A, a protein G, a designed ankyrin-repeat domain (DARPin), a fibronectin-type III repeat, an anticalin, a knottin, and an engineered CH2 domain. 
     
     
         48 . The method of  claim 43 , wherein the protein binding domain binds an intracellular domain of the GPCR. 
     
     
         49 . The method of  claim 43 , wherein the active conformational state of the GPCR comprises the cytoplasmic end of transmembrane segment 6 (TM6) being moved outward and away from the core of the GPCR as compared to the GPCR when not bound to the protein binding domain. 
     
     
         50 . The method of  claim 43 , wherein the protein binding domain increases thermostability of the GPCR in the active conformational state as compared to thermostability of the GPCR when not bound to the protein binding domain. 
     
     
         51 . The method of  claim 43 , wherein the GPCR is a mammalian protein, a plant protein, a microbial protein, a viral protein, or an insect protein. 
     
     
         52 . The method of  claim 43 , wherein the GPCR is a human protein. 
     
     
         53 . The method of  claim 52 , wherein the GPCR is selected from the group consisting of a GPCR of the glutamate family of GPCRs, a GPCR of the rhodopsin family of GPCRs, a GPCR of the adhesion family of GPCRs, a GPCR of the frizzled/taste2 family of GPCRs, and a GPCR of the secretin family of GPCRs. 
     
     
         54 . The method of  claim 53 , wherein the GPCR is a GPCR of the rhodopsin family of GPCRs. 
     
     
         55 . The method of  claim 54 , wherein the GPCR is an adrenergic receptor, a muscarinic receptor, or an angiotensin receptor. 
     
     
         56 . The method of  claim 43 , wherein the protein binding domain is a nanobody. 
     
     
         57 . A method for identifying a test compound that binds to an inactive conformational state of a GPCR, the method comprising:
 (i) providing a GPCR and a protein binding domain that binds to an inactive conformational state of the GPCR;   (ii) providing a test compound; and   (iii) evaluating whether the test compound binds to the inactive conformational state of the GPCR.   
     
     
         58 . The method of  claim 57 , further comprising:
 (iv) selecting a compound that binds to the inactive conformational state of the GPCR.   
     
     
         59 . The method of  claim 57 , wherein the test compound is selected from the group consisting of a polypeptide, a peptide, a small molecule, a natural product, a peptidomimetic, a nucleic acid, a lipid, a lipopeptide, a carbohydrate, an antibody or an antigen-binding fragment thereof, a heavy chain antibody (hcAb), a single domain antibody (sdAb), a minibody, a variable domain derived from a camelid heavy chain antibody, a variable domain of an immunoglobulin new antigen receptor (V NAR ), and a protein scaffold. 
     
     
         60 . The method of  claim 59 , wherein the antigen-binding fragment thereof is selected from the group consisting of a Fab, a Fab′, a F(ab′)2, an Fd, a single-chain Fvs (scFv), a single-chain antibody, a disulfide-linked Fv (dsFv), a fragment comprising a VL domain and a fragment comprising a VH domain. 
     
     
         61 . The method of  claim 59 , wherein the protein scaffold is selected from the group consisting of an alphabody, a protein A, a protein G, a designed ankyrin-repeat domain (DARPin), a fibronectin-type III repeat, an anticalin, a knottin, and an engineered CH2 domain. 
     
     
         62 . The method of  claim 57 , wherein the protein binding domain binds an intracellular domain of the GPCR. 
     
     
         63 . The method of  claim 57 , wherein the inactive conformational state of the GPCR comprises the cytoplasmic end of transmembrane segment 6 (TM6) remaining in its inactive state proximal to the core of the GPCR, compared to the active state in which TM6 is outward and distal to the core of the GPCR. 
     
     
         64 . The method of  claim 57 , wherein the protein binding domain increases thermostability of the GPCR in the inactive conformational state as compared to thermostability of the GPCR when not bound to the protein binding domain. 
     
     
         65 . The method of  claim 57 , wherein the GPCR is a mammalian protein, a plant protein, a microbial protein, a viral protein, or an insect protein. 
     
     
         66 . The method of  claim 57 , wherein the GPCR is a human protein. 
     
     
         67 . The method of  claim 66 , wherein the GPCR is selected from the group consisting of a GPCR of the glutamate family of GPCRs, a GPCR of the rhodopsin family of GPCRs, a GPCR of the adhesion family of GPCRs, a GPCR of the frizzled/taste2 family of GPCRs, and a GPCR of the secretin family of GPCRs. 
     
     
         68 . The method of  claim 67 , wherein the GPCR is a GPCR of the rhodopsin family of GPCRs. 
     
     
         69 . The method of  claim 68 , wherein the GPCR is an adrenergic receptor, a muscarinic receptor, or an angiotensin receptor.