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

• US 7,410,777 B2
• Filed: 09/20/2002
• Issued: 08/12/2008
• Est. Priority Date: 04/14/1997
• Status: Expired due to Fees

• Current Assignee: Arena Pharmaceuticals Incorporated
• Original Assignee: Arena Pharmaceuticals Incorporated
• Inventors: Liaw, Chen W., Chalmers, Derek T., Behan, Dominic P.
• Primary Examiner(s): Nickol; Gary B.
• Assistant Examiner(s): BASI, NIRMAL SINGH
• Application Number:
  US10/251,385
• Publication Number:
  US 20030105292A1
• Time in Patent Office: 2,153 Days
• Field of Search: None
• US Class Current: 435/69.1
• 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, P¹ 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 method for creating a non-endogenous, constitutively active version of an endogenous human G protein coupled receptor (GPCR), said endogenous GPCR comprising a transmembrane 6 region and an intracellular loop 3 region, the method comprising:

(a) selecting an endogenous human GPCR comprising a proline residue in the transmembrane-6 region, wherein the GPCR is not the β

2-adrenergic receptor or the α

_1B-adrenergic receptor;

(b) identifying the endogenous 16^th amino acid residue from the proline residue of step (a), in a carboxy-terminus to amino-terminus direction;

(c) altering the identified amino acid residue of step (b) to a non-endogenous amino acid residue to create a non-endogenous version of the endogenous human GPCR; and

(d) determining if the non-endogenous version of the endogenous human GPCR of step (c) is constitutively active by measuring a difference in an intracellular signal measured for the non-endogenous version as compared with a signal induced by the endogenous human GPCR.

2. The method of claim 1 wherein the amino acid residue that is two residues from said proline residue in the transmembrane 6 region, in a carboxy-terminus to amino-terminus direction, is tryptophan.

3. The method of claim 1 or claim 2 wherein the endogenous 16^th amino acid residue from said proline residue in a carboxy-terminus to amino-terminus direction has been altered to an alanine residue.

4. The method of claim 1 or claim 2 wherein the endogenous 16th amino acid residue from said proline residue in a carboxy-terminus to aminoterminus direction has been altered to an arginine residue.

5. The method of claim 1 or claim 2 wherein the endogenous 16^th amino acid residue from said proline residue in a carboxy-terminus to amino-terminus direction has been altered to an histidine residue.

6. A method for creating a non-endogenous, constitutively active version of an endogenous human G protein coupled receptor (GPCR), said endogenous GPCR comprising a transmembrane 6 region and an intracellular loop 3 region, the method comprising:

(a) providing a polynucleotide;

said polynucleotide encoding an endogenous human GPCR, said endogenous GPCR comprising a transmembrane 6 region and an intracellular loop 3 region, said transmembrane 6 region comprisin a roline residue, wherein the GPCR is not the β

2-adrenergic receptor or the α

_1B-adernergic receptor;

(b) identifying the codon of said polynucleotide corresponding to the endogenous 16^th amino acid residue from said proline residue of said GPCR of step (a), in a carboxy-terminus to amino-terminus direction;

(c) altering said identified codon of step (b) to encode a non-endogenous amino acid residue, to provide a non-endogenous polynucleotide;

(d) expressing said non-endogenous polynucleotide in a host cell, thereby providing a non-endogenous version of the endogenous human GPCR; and

(e) determining if the non-endogenous version of the endogenous human GPCR of step (d) is constitutively active by measuring a difference in an intracellular signal measured for the non-endogenous version as compared with a signal induced by the endogenous human GPCR.

7. The method of claim 6 wherein the amino acid residue that is two residues from said proline residue in the transmembrane 6 region, in a carboxy-terminus to amino-terminus direction, is tryptophan.

8. The method of claim 6 or claim 7 wherein said identified codon of step (b) has been altered to be a codon encoding alanine.

9. The method of claim 6 or claim 7 wherein said identified codon of step (b) has been altered to be a codon encoding arginine.

10. The method of claim 6 or claim 7 wherein said identified codon of step (b) has been altered to be a codon encoding histidine.