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

Patent metadata

Field	Value
Publication number	US-9453065-B2
Application number	US-201113810652-A
Country	US
Kind code	B2
Filing date	Jul 18, 2011
Priority date	Jul 16, 2010
Publication date	Sep 27, 2016
Grant date	Sep 27, 2016

Abstract

The disclosure relates to the field of GPCR structure biology and signaling. In particular, it 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, it 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 hereof 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, also encompassed are the diagnostic, prognostic and therapeutic usefulness of these protein binding domains for GPCR-related diseases.

First claim

The invention claimed is:

1. A complex comprising:
a nanobody able to specifically bind to and stabilize an active conformational state of a beta 2 adrenergic receptor (β 2 AR), wherein the nanobody is able to enhance the affinity of the β 2 AR for an agonist, and
β 2 AR in an active conformational state.

2. The complex according to claim 1 further comprising:
a receptor ligand.

3. The complex of claim 1 , wherein said receptor ligand is selected from the group consisting of a small molecule, a protein, a peptide, a protein scaffold, a nucleic acid, an ion, a carbohydrate, an antibody, and any suitable fragment thereof.

4. The complex of claim 1 , wherein said complex is in a solubilized form or immobilized to a solid support.

5. The complex of claim 1 , wherein said complex is crystalline.

6. A crystalline form of a complex comprising:
(i) a nanobody able to specifically bind to and stabilize an active conformational state of a beta 2 adrenergic receptor (β 2 AR), wherein the nanobody is able to enhance the affinity of the β 2 AR for an agonist,
(ii) a β 2 AR in an active conformational state, and
(iii) a ligand.

7. The complex of claim 1 , wherein the nanobody is able to specifically bind to an agonist-bound β 2 AR.

8. The complex of claim 1 , wherein the nanobody is able to increase the thermostability of an active conformational state of a β 2 AR upon binding.

9. The complex of claim 1 , wherein the β 2 AR is a mammalian protein, a plant protein, a microbial protein, a viral protein, or an insect protein.

10. The complex of claim 1 , wherein the β 2 AR is a human protein.

11. The complex of claim 1 , wherein the nanobody mimics the binding of a G protein to the active conformational state of the β 2 AR.

12. The complex of claim 1 , wherein the nanobody shows G protein-like behavior.

13. The complex of claim 1 , wherein the nanobody is bound to the G protein binding site of the β 2 AR.

14. The complex of claim 1 , wherein the β 2 AR, is in a conformation where the cytoplasmic end of transmembrane segment 6 (TM6) is moved outward and away from the core of the β 2 AR as compared to the β 2 AR when not bound to the nanobody.

15. The complex of claim 1 , wherein the complex is in a cellular composition.

16. The complex of claim 1 , wherein the nanobody binds to the β 2 AR in a pocket in the β 2 AR formed by amino acids from transmembrane segments 3, 5, 6, and 7.

17. The complex of claim 16 , wherein the third complementary determining region of the nanobody is bound to the pocket.

18. A complex comprising:
a nanobody able to specifically bind to and stabilize an active conformational state of a beta 2 adrenergic receptor (β 2 AR), wherein the nanobody is able to enhance the affinity of the β 2 AR for an agonist, and
a β 2 AR in an active conformational state, and
wherein the nanobody is bound in the G protein binding site of the β 2 AR;
wherein the β 2 AR is in a conformation where the cytoplasmic end of transmembrane segment 6 (TM6) is moved outward and away from the core of the β 2 AR as compared to the β 2 AR when not bound to the nanobody; and
wherein the β 2 AR has enhanced affinity for an agonist as compared to the β 2 AR when not bound to the nanobody.

Assignees

• Steyaert Jan
• Pardon Els
• Rasmussen Soren
• Fung Juan
• Kobilka Brian
• Laeremans Toon
• Vib Vzw
• Univ Brussel Vrije
• Univ Leland Stanford Junior

Inventors

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

Classifications

• A61P37/06

  Immunosuppressants, e.g. drugs for graft rejection · CPC title

• A61P9/00

  Drugs for disorders of the cardiovascular system · CPC title

• A61P31/04

  Antibacterial agents · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61P25/00

  Drugs for disorders of the nervous system · CPC title