Constitutively activated human G protein coupled receptors

• US 8,440,391 B2
• Filed: 11/26/2003
• Issued: 05/14/2013
• Est. Priority Date: 10/13/1998
• Status: Expired due to Fees

• Current Assignee: Arena Pharmaceuticals Incorporated (Pfizer Inc.)
• Original Assignee: Arena Pharmaceuticals Incorporated (Pfizer Inc.)
• Inventors: Chen, Ruoping, Liaw, Chen W., Lowitz, Kevin P., Chalmers, Derek T., Behan, Dominic P.
• Primary Examiner(s): Li, Ruixiang
• Application Number:
  US10/723,955
• Publication Number:
  US 20040110238A1
• Time in Patent Office: 3,457 Days
• Field of Search: None
• US Class Current: 435/4
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/723 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Abstract

The invention disclosed in this patent document relates to transmembrane receptors, more particularly to a human G protein-coupled receptor for which the endogenous ligand is unknown (“orphan GPCR receptors”), and most particularly to mutated (non-endogenous) versions of the human GPCRs for evidence of constitutive activity.

Claims

1. A method of screening for a compound that increases cAMP levels in peripheral blood leukocytes, comprising:

(a) contacting a candidate compound with a G protein-coupled receptor (GPCR) that is present on the surface of a recombinant host cell or isolated membrane thereof, wherein said GPCR comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO;

82; and

(b) determining that said candidate compound is an agonist of said GPCR; and

(c) determining if said agonist increases cAMP levels in a peripheral blood leukocyte.

2. The method of claim 1, wherein said determining step (b) comprises:

determining if said candidate compound is a partial agonist of said GPCR.

3. The method of claim 1, wherein said determining step (b) and/or said determining step (c) comprises detecting cAMP.

4. The method of claim 3, wherein said detecting cAMP employs ELISA using an anti-cAMP antibody.

5. The method of claim 3, wherein the recombinant host cell comprises a reporter system comprising multiple cAMP responsive elements operably linked to a reporter gene.

6. The method of claim 3, wherein said detecting cAMP comprises detecting an increase in intracellular cAMP accumulation.

7. The method of claim 1, wherein said determining step (b) comprises using [35S]GTPγ

S to monitor G protein coupling to a membrane comprising said GPCR.

8. The method of claim 1, wherein said GPCR comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

82.

9. The method of claim 1, wherein said GPCR comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

82.

10. The method of claim 1, wherein said GPCR comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:

82.

11. The method of claim 1, wherein the GPCR comprises one or more of the following amino acid substitutions:

P43A, K97N or I130F, relative to SEQ ID NO;

82.

12. The method of claim 1, wherein said GPCR is constitutively active.

13. The method of claim 1, wherein the GPCR comprises the following amino acid substitution:

I225K, relative to SEQ ID NO;

82.

14. The method of claim 1, wherein the method further comprises formulating said agonist as a pharmaceutical.

15. The method of claim 1, wherein the GPCR forms part of a fusion protein with a G protein.

16. The method of claim 1, wherein the host cell is a mammalian host cell.

17. The method of claim 1, wherein the host cell is a yeast host cell.

18. The method of claim 1, wherein the recombinant host cell comprises an expression vector which comprises a nucleic acid encoding said GPCR.

19. The method of claim 1, wherein said method further comprises determining whether said agonist increases cAMP levels in a peripheral blood leukocyte.

20. The method of claim 19, wherein the peripheral blood leukocyte is a human peripheral blood leukocyte.