Methods for screening for binding partners of G-protein coupled receptors

Patent metadata

Field	Value
Publication number	US-10126313-B2
Application number	US-201614989744-A
Country	US
Kind code	B2
Filing date	Jan 6, 2016
Priority date	Dec 20, 2007
Publication date	Nov 13, 2018
Grant date	Nov 13, 2018

Abstract

A method of producing a conformational specific binding partner of a GPCR, the method comprising: a) providing a mutant GPCR of a parent GPCR, wherein the mutant GPCR has increased stability in a particular conformation relative to the parent GPCR; b) providing a test compound; c) determining whether the test compound binds to the mutant GPCR when residing in a particular conformation; and d) isolating a test compound that binds to the mutant GPCR when residing in the particular formation. Methods of producing GPCRs with increased stability relative to a parent GPCR are also disclosed.

First claim

The invention claimed is:

1. A mutant human G-protein coupled receptor (GPCR) with increased conformational stability in a particular conformation compared to its parent human GPCR in the same particular conformation, which particular conformation is selected from an agonist conformation and an antagonist conformation, produced by a method comprising:
(a) providing one or more mutants of a parent human GPCR;
(b) contacting the one or more mutants and the parent human GPCR with a ligand of a particular class, wherein:
(i) an agonist ligand is used to select for one or more mutants with increased conformational stability in the agonist conformation; and
(ii) an antagonist ligand is used to select for one or more mutants with increased conformational stability in the antagonist conformation;

(c) determining whether the one or more mutant GPCRs has increased stability with respect to binding the selected ligand compared to the stability of the parent human GPCR with respect to binding the selected ligand, and
(d) selecting the one or more mutants that exhibit increased stability compared to the parent human GPCR with respect to binding the selected ligand.

2. A mutant human G-protein coupled receptor (GPCR) with increased conformational stability in an agonist conformation compared to its parent human GPCR in an agonist conformation, produced by a method comprising:
(a) providing one or more mutants of a parent human GPCR;
(b) contacting the one or more mutants and the parent human GPCR with an agonist ligand;
(c) determining whether the one or more mutant GPCRs has increased stability with respect to binding the agonist ligand compared to the parent human GPCR with respect to binding the agonist ligand, and
(d) selecting those mutants that have an increased stability compared to the parent human GPCR with respect to binding of the agonist ligand.

3. The mutant human GPCR of claim 2 , wherein the one or more mutants each differs from its parent human GPCR by one or more point mutations.

4. The mutant human GPCR of claim 2 , wherein the mutant human GPCR has increased conformational thermostability relative to its parent human GPCR.

5. The mutant human GPCR of claim 2 , wherein the mutant human GPCR is selected from the group consisting of: a mutant adenosine receptor, a mutant β-adrenergic receptor, a mutant neurotensin receptor, a mutant muscarinic acid receptor, a mutant 5-hydroxytryptamine receptor, a mutant adrenoceptor, a mutant anaphylatoxin receptor, a mutant angiotensin receptor, a mutant apelin receptor, a mutant bombesin receptor, a mutant bradykinin receptor, a mutant cannabinoid receptor, a mutant chemokine receptor, a mutant cholecystokinin receptor, a mutant dopamine receptor, a mutant endothelin receptor, a mutant free fatty acid receptor, a mutant bile acid receptor, a mutant galanin receptor, a mutant motilin receptor, a mutant ghrelin receptor, a mutant glycoprotein hormone receptor, a mutant GnRH receptor, a mutant histamine receptor, a mutant KiSS1-derived peptide receptor, a mutant leukotriene and lipoxin receptor, a mutant lysophospholipid receptor, a mutant melanin-concentrating hormone receptor, a mutant melanocortin receptor, a mutant melatonin receptor, a mutant neuromedin U receptor, a mutant neuropeptide receptor, a mutant N-formylpeptide family receptor, a mutant nicotinic acid receptor, a mutant opiod receptor, a mutant opsin-like receptor, a mutant orexin receptor, a mutant P2Y receptor, a mutant peptide P518 receptor, a mutant platelet-activating factor receptor, a mutant prokineticin receptor, a mutant prolactin-releasing peptide receptor, a mutant prostanoid receptor, a mutant protease-activated receptor, a mutant relaxin receptor, a mutant somatostatin receptor, a mutant SPC/LPC receptor, a mutant tachykinin receptor, a mutant trace amino receptor, a mutant thryotropin-releasing hormone receptor, a mutant urotensin receptor, a mutant vasopressin/oxytocin receptor, a mutant orphan GPCR, a mutant calcitonin receptor, a mutant corticotropin releasing factor receptor, a mutant glucagon receptor, a mutant parathyroid receptor, a mutant VIP/PACAP receptor, a mutant LNB7TM receptor, a mutant GABA receptor, a mutant metabotropic glutamate receptor, and a mutant calcium sensor receptor.

6. The mutant human GPCR of claim 2 , wherein the mutant human GPCR is a muscarinic receptor.

7. A mutant human G-protein coupled receptor (GPCR) with increased conformational stability in an antagonist conformation compared to its parent human GPCR in an antagonist conformation, produced by a method comprising:
(a) providing one or more mutants of a parent human GPCR;
(b) contacting the one or more mutants and the parent human GPCR with an antagonist ligand;
(c) determining whether the one or more mutant GPCRs has increased stability with respect to binding the antagonist ligand compared to the parent human GPCR with respect to binding the antagonist ligand, and
(d) selecting those mutants that have an increased stability compared to the parent human GPCR with respect to binding of the antagonist ligand.

8. The mutant human GPCR of claim 7 , wherein the one or more mutants each differs from its parent human GPCR by one or more point mutations.

9. The mutant human GPCR of claim 7 , wherein the mutant human GPCR has increased conformational thermostability relative to its parent human GPCR.

10. The mutant human GPCR of claim 7 , wherein the mutant human GPCR is selected from the group consisting of: a mutant adenosine receptor, a mutant β-adrenergic receptor, a mutant neurotensin receptor, a mutant muscarinic acid receptor, a mutant 5-hydroxytryptamine receptor, a mutant adrenoceptor, a mutant anaphylatoxin receptor, a mutant angiotensin receptor, a mutant apelin receptor, a mutant bombesin receptor, a mutant bradykinin receptor, a mutant cannabinoid receptor, a mutant chemokine receptor, a mutant cholecystokinin receptor, a mutant dopamine receptor, a mutant endothelin receptor, a mutant free fatty acid receptor, a mutant bile acid receptor, a mutant galanin receptor, a mutant motilin receptor, a mutant ghrelin receptor, a mutant glycoprotein hormone receptor, a mutant GnRH receptor, a mutant histamine receptor, a mutant KiSS1-derived peptide receptor, a mutant leukotriene and lipoxin receptor, a mutant lysophospholipid receptor, a mutant melanin-concentrating hormone receptor, a mutant melanocortin receptor, a mutant melatonin receptor, a mutant neuromedin U receptor, a mutant neuropeptide receptor, a mutant N-formylpeptide family receptor, a mutant nicotinic acid receptor, a mutant opiod receptor, a mutant opsin-like receptor, a mutant orexin receptor, a mutant P2Y receptor, a mutant peptide P518 receptor, a mutant platelet-activating factor receptor, a mutant prokineticin receptor, a mutant prolactin-releasing peptide receptor, a mutant prostanoid receptor, a mutant protease-activated receptor, a mutant relaxin receptor, a mutant somatostatin receptor, a mutant SPC/LPC receptor, a mutant tachykinin receptor, a mutant trace amino receptor, a mutant thryotropin-releasing hormone receptor, a mutant urotensin receptor, a mutant vasopressin/oxytocin receptor, a mutant orphan GPCR, a mutant calcitonin receptor, a mutant corticotropin releasing factor receptor, a mutant glucagon receptor, a mutant parathyroid receptor, a mutant VIP/PACAP receptor, a mutant LNB7TM receptor, a mutant GABA receptor, a mutant metabotropic glutamate receptor, and a mutant calcium sensor receptor.

11. The mutant human GPCR of claim 7 , wherein the mutant human GPCR is a muscarinic receptor.

12. A mutant human G-protein coupled receptor (GPCR) with increased conformational stability in a particular conf

Assignees

• Heptares Therapeutics Ltd

Inventors

• Weir Malcolm Peter
• Henderson Richard
• Tate Christopher Gordon
• Magnani Francesca
• Serrano-Vega Maria Josefa
• Shibata Yoko
• Warne Antony Johannes

Classifications

• G01N2500/00

  Screening for compounds of potential therapeutic value · CPC title

• G01N2333/726

  G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH · CPC title

• C07K14/705

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title

• G01N33/566

  using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds} · CPC title

• G01N33/54366

  Apparatus specially adapted for solid-phase testing · CPC title