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

12092,646

17/507492

October 21, 2021

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.

VIB VZW (Ghent, BE); Vrije Universiteit Brussel (Brussels, BE); The Board of Trustees of the Leland Stanford Junior University (Palo Alto, CA, US)

Trustees of Leland Stanford Junior University (Palo Alto, CA, US), VIB VZW (Ghent, BE), Vrije Universiteit Brussel (Brussels, BE)

1. A method of identifying compounds capable of binding to a conformation stabilized GPCR, the method comprising: determining that a protein binding domain binds to an intracellular epitope of the GPCR and, when bound to the GPCR, stabilizes a conformation of the GPCR; determining that the protein binding domain stabilized GPCR has increased affinity for a ligand of the GPCR as compared to the GPCR in the absence of the protein binding domain; forming a complex comprising the protein binding domain and the GPCR and wherein the protein binding domain bound-GPCR has increased affinity for the ligand; contacting the complex with a test compound, and determining whether the test compound binds to the GPCR in the complex; wherein the protein binding domain is a nanobody or is derived from a camelid antibody and comprises four framework regions and three complementary determining regions.

2. The method according to claim 1 , wherein the complex is on the surface of a cell.

3. The method according to claim 1 , wherein the complex is in a cell.

4. The method according to claim 1 , further comprising determining that the test compound has higher affinity for the GPCR in the complex as compared to the GPCR alone.

5. The method according to claim 1 , further comprising determining that the protein binding domain enhances the affinity of the GPCR for an agonist.

6. The method according to claim 1 , wherein the intracellular epitope is comprised in, located at, or overlaps with the G protein binding site of the GPCR.

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