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

Patent metadata

Field	Value
Publication number	US-12092646-B2
Application number	US-202117507492-A
Country	US
Kind code	B2
Filing date	Oct 21, 2021
Priority date	Jul 16, 2010
Publication date	Sep 17, 2024
Grant date	Sep 17, 2024

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.

First claim

What is claimed is:

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.

Assignees

• Vib Vzw
• Univ Brussel Vrije
• Univ Leland Stanford Junior
• Trustees Of Leland Stanford Junior Univ

Inventors

• Steyaert Jan
• Pardon Els
• Rasmussen Soren G F
• Fung Juan Jose
• Kobilka Brian
• Laeremans Toon

Classifications

• G01N33/6857

  Antibody fragments · CPC title

• G01N23/20

  by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials · CPC title

• C07K2317/56

  variable (Fv) region, i.e. VH and/or VL · CPC title

• C07K2317/51

  Complete heavy chain or Fd fragment, i.e. VH + CH1 · CPC title

• G01N33/68

  involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)} · CPC title