Non-endogenous, constitutively activated human G protein-coupled receptors

• US 20030105292A1
• Filed: 09/20/2002
• Published: 06/05/2003
• Est. Priority Date: 04/14/1997
• Status: Active Grant

• Current Assignee: Arena Pharmaceuticals Incorporated
• Original Assignee: Arena Pharmaceuticals Incorporated
• Inventors: Liaw, Chen W., Chalmers, Derek T., Behan, Dominic P.
• Granted Patent: US 7,410,777 B2
• Time in Patent Office: Days
• US Class Current: 530/350
• 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: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07D 231/12 with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
• CPC Class Codes: C07D 231/16 Halogen atoms or nitro radicals
• CPC Class Codes: C07D 409/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/70571 for neuromediators, e.g. serotonin receptor, dopamine receptor
• CPC Class Codes: C07K 14/723 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• 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

Disclosed herein are constitutively activated, non-endogenous versions of endogenous human G protein-coupled receptors comprising (a) the following amino acid sequence region (C-terminus to N-terminus orientation) and/or (b) the following nucleic acid sequence region (3′ to 5′ orientation) transversing the transmembrane-6 (TM6) and intracellular loop-3 (IC3) regions of the GPCR:

(a) P¹ AA₁₅X

and/or

(b) p^codon (AA-codon)₁₅ X_codon,

respectively. In a most preferred embodiment, P′ and P^codon are endogenous proline and an endogenous nucleic acid encoding region encoding proline, respectively, located within TM6 of the non-endogenous GPCR; AA₁₅ and (AA-codon)₁₅ are 15 endogenous amino acid residues and 15 codons encoding endogenous amino acid residues, respectively; and X and X_codon are non-endogenous lysine and a non-endogenous nucleic acid encoding region encoding lysine, respectively, located within IC3 of the non-endogenous GPCR. Because it is most preferred that the non-endogenous human GPCRs which incorporate these mutations are incorporated into mammalian cells and utilized for the screening of candidate compounds, the non-endogenous human GPCR incorporating the mutation need not be purified and isolated per se (i.e., these are incorporated within the cellular membrane of a mammalian cell), although such purified and isolated non-endogenous human GPCRs are well within the purview of this disclosure.

Claims

1. A constitutively active, non-endogenous version of an endogenous human G protein-coupled receptor (GPCR) comprising the following amino acid residues (carboxy-terminus to amino-terminus orientation) transversing the transmembrane-6 (TM6) and intracellular loop-3 (IC3) regions of the non-endogenous GPCR:

P¹ AA₁₅X

wherein;

(1) P¹ is an amino acid residue located within the TM6 region of the non-endogenous GPCR, where P¹ is selected from the group consisting of (i) the endogenous GPCR proline residue, and (ii) a non-endogenous amino acid residue other than proline;

(2) AA₁₅ are 15 amino acid residues selected from the group consisting of (a) the 15 endogenous amino acid residues of the endogenous GPCR, (b) 15 non-endogenous amino acid residues, and (c) a combination of 15 amino acid residues, the combination comprising at least one endogenous amino acid residue of the endogenous GPCR and at least one non-endogenous amino acid residue, excepting that none of the 15 endogenous amino acid residues that are positioned within the TM6 region of the GPCR is proline; and

(3) X is a non-endogenous amino acid residue located within the IC3 region of said non-endogenous GPCR.

2. The non-endogenous human GPCR of claim 1 wherein P¹ is the endogenous proline residue.

3. The non-endogenous human GPCR of claim 1 wherein P¹ is a non-endogenous amino acid residue other than a proline residue.

4. The non-endogenous human GPCR of claim 1 wherein AA15 are the 15 endogenous amino acid residues of the endogenous GPCR.

5. The non-endogenous human GPCR of claim 1 wherein X is selected from the group consisting of lysine, hisitidine, arganine and alanine residues, excepting that when the endogenous amino acid in position X of said endogenous human GPCR is lysine, X is selected from the group consisting of histidine, arginine and alanine.

6. The non-endogenous human GPCR of claim 1 wherein X is a lysine residue, excepting that when the endogenous amino acid in position X of said endogenous human GPCR is lysine, X is an amino acid other than lysine.

7. The non-endogenous human GPCR of claim 4 wherein X is a lysine residue, excepting that when the endogenous amino acid in position X of said endogenous human GPCR is lysine, X is an amino acid other than lysine.

8. The non-endogenous, human GPCR of claim 1 wherein P¹ is a proline residue and X is a lysine residue, excepting that when the endogenous amino acid in position X of said endogenous human GPCR is lysine, X is an amino acid other than lysine.

9. A host cell comprising the non-endogenous human GPCR of claim 1.

10. The material of claim 9 wherein said host cell is of mammalian origin.

11. The non-endogenous human GPCR of claim 1 in a purified and isolated form.

12. A nucleic acid sequence encoding a constitutively active, non-endogenous version of an endogenous human G protein-coupled receptor (GPCR) comprising the following nucleic acid sequence region transversing the transmembrane-6 (TM6) and intracellular loop-3 (IC3) regions of the GPCR:

3′

-P^codon (AA-codon)₁₅ X_codon-5′

wherein;

(1) p^codon is a nucleic acid encoding region within the TM6 region of the non-endogenous GPCR, where p^codon encodes an amino acid selected from the group consisting of (i) the endogenous GPCR proline residue, and (ii) a non-endogenous amino acid residue other than proline;

(2) (AA-codon)₁₅ are 15 codons encoding 15 amino acid residues selected from the group consisting of (a) the 15 endogenous amino acid residues of the endogenous GPCR, (b) 15 non-endogenous amino acid residues, and (c) a combination of 15 amino acid residues, the combination comprising at least one endogenous amino acid residue of the endogenous GPCR and at least one non-endogenous amino acid residue, excepting that none of the 15 endogenous amino acid residues that are positioned within the TM6 region of the GPCR is proline; and

(3) X_condon is a nucleic acid encoding region residue located within the IC3 region of said non-endogenous human GPCR, where X_codon encodes a non-endogenous amino acid.

13. The nucleic acid sequence of claim 12 wherein P^codon encodes an endogenous proline residue.

14. The nucleic acid sequence of claim 12 wherein P^codon encodes a non-endogenous amino acid residue other than a proline residue.

15. The nucleic acid sequence of claim 12 wherein X_codon encodes a non-endogenous amino acid selected from the group consisting of lysine, histidine, arginine and alanine, excepting that when the endogenous amino acid in position X of said endogenous human GPCR is lysine, X_codon encodes an amino acid selected from the group consisiting of histidine, arginine and alanine.

16. The nucleic acid sequence of claim 13 wherein X_codon encodes a non-endogenous lysine amino acid excepting that when the endogenous amino acid in position X of said endogenous human GPCR is lysine, X_codon encodes an amino acid selected from the group consisiting of histidine, arginine and alanine.

17. The nucleic acid sequence of claim 12 wherein X_codon is selected from the group consisting of AAA, AAG, GCA, GCG, GCC and GCU.

18. The nucleic acid sequence of claim 12 wherein X_codon is selected from the group consisting of AAA and AAG.

19. The nucleic acid sequence of claim 12 wherein P^codon is selected from the group consisting of CCA, CCC, CCG and CCU, and X_codon is selected from the group consisting of AAA and AAG.

20. A vector comprising the nucleic acid sequence of claim 12.

21. A plasmid comprising the nucleic acid sequence of claim 12.

22. A host cell comprising the nucleic acid sequence of claim 21.

23. The nucleic acid sequence of claim 12 in a purified and isolated form.