Non-endogenous, constitutively activated human protein-coupled receptors

• US 6,555,339 B1
• Filed: 10/13/1998
• Issued: 04/29/2003
• Est. Priority Date: 04/14/1997
• Status: Expired due to Term

• Current Assignee: Arena Pharmaceuticals Incorporated
• Original Assignee: Arena Pharmaceuticals Incorporated
• Inventors: Behan, Dominic P., Liaw, Chen W., Chalmers, Derek T.
• Primary Examiner(s): Eyler, Yvonne
• Assistant Examiner(s): Basi, Nirmal S.
• Application Number:
  US09/170,496
• Time in Patent Office: 1,659 Days
• Field of Search: 702/19, 702/20, 702/22, 536/23.1, 536/24.3, 435/69.1, 435/320.1, 435/325, 435/252.3, 435/172.3, 435/68.1, 530/333, 530/300, 530/350
• 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 are constitutively activated, non-endogenous versions of endogenous human GPCRs comprising the following amino acid sequence region (C-terminus to N-terminus orientation) and/or the following nucleic acid sequence region (3′ to 5′ orientation) transversing the transmembrane-6 (TM6) and intracellular loop-3 (IC3) regions of the GPCR:

P¹AA₁₅X  (a)

and/or

P^codon(AA-codon)₁₅X_codon,  (b)

respectively. In a 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 non-endogenous nucleic acid region encoding lysine, respectively, located within IC3 of the non-endogenous GPCR. The purified and isolated non-endogenous human GPCRs having these mutations, and the receptors incorporated into mammalian cells, are well within the present 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 consisting essentially of:

(a) selecting an endogenous human GPCR comprising a proline residue in the transmembrane-6 region;

(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 only 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.