# US 8,785,135 B2 · Mutant G-protein coupled receptors and methods for selecting them

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- Number: US 8,785,135 B2
- Document ID: US8785135B2
- Status: Granted
- Kind: B2
- Application number: 12/450,358
- Filed: 2008-03-20
- Published: 2010-07-29
- Granted: 2014-07-22
- Earliest claimed: 2007-03-22
- Assignees: Heptares Therapeutics Limited
- Applicants: Richard Henderson; Christopher Gordon Tate; Francesca Magnani; Maria Josefa Serrano-Vega; Yoko Shibata; Antony Johannes Warne; Malcolm Peter Weir
- Inventors: Richard Henderson; Christopher Gordon Tate; Francesca Magnani; Maria Josefa Serrano-Vega; Yoko Shibata; Antony Johannes Warne; Malcolm Peter Weir
- Web page: https://patentgrep.com/patent/US8785135B2

## CPC

- C07K 14/723: {G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor}
- C12N 15/1034: {Isolating an individual clone by screening libraries}
- 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}
- G01N 2333/726: G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention relates in some aspects to mutant G protein coupled receptors (GPCRs) and methods for selecting those with increased stability. In certain aspects, the invention relates to the selection and preparation of mutant GPCRs which have increased stability under a particular condition compared to their respective parent proteins.

## Claims

### Claim 1

A method for selecting a G-protein coupled receptor (GPCR) with increased conformational thermostability in a particular conformation compared to its parent GPCR in the same particular conformation, which particular conformation is selected from an agonist conformation and an antagonist conformation, the method comprising
(a) preparing one or more mutants of a parent GPCR, wherein the one or more mutants each differs from its parent GPCR by one or more point mutations and wherein the one or more mutants and the parent GPCR are provided in a solubilised form obtained by solubilising the one or more mutants and the parent GPCR from a membrane using a detergent;
(b) contacting the one or more mutants and the parent 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 by binding to the one or more mutants; and
(ii) an antagonist ligand is used to select for one or more mutants with increased conformational stability in the antagonist conformation by binding to the one or more mutants;
(c) exposing to heat the one or more mutants and the parent GPCR, wherein the one or more mutants and the parent GPCR are bound to the ligand;
(d) measuring the stability of binding of the one or more mutants and the parent GPCR to the ligand by measuring the ability of the one or more mutants and the parent GPCR to retain ligand binding capacity following exposure to heat; and
(e) selecting the one or more mutants that exhibit increased stability of binding to the ligand following exposure to heat.

### Claim 2 (depends on 1)

The method of claim 1 wherein the ligand is from the agonist class of ligands and the particular conformation in which the GPCR resides in step (d) is the agonist conformation.

### Claim 3 (depends on 1)

The method of claim 1 wherein the binding affinity of the mutant for the ligand is substantially the same or greater than the binding affinity of the parent for the ligand.

### Claim 4 (depends on 1)

The method of claim 1 wherein the method is repeated for one or more rounds, with the selected mutants having increased conformational stability in step (a) representing the parent GPCR in a subsequent round of the method.

### Claim 5 (depends on 1)

The method of claim 1 wherein the ligand is any one of a full agonist, a partial agonist, an inverse agonist and an antagonist.

### Claim 6 (depends on 1)

The method of claim 1 wherein the ligand is a polypeptide which binds to the GPCR.

### Claim 7 (depends on 1)

The method of claim 1 wherein in step (b) two or more ligands are used, the presence of each causes the GPCR to reside in the same particular conformation.

### Claim 8 (depends on 1)

The method of claim 1 wherein a mutant GPCR is selected which has reduced ability to bind a ligand of a different class to the ligand used in step (b) compared to its parent.

### Claim 9 (depends on 1)

The method of claim 1 wherein the GPCR is any one of a β-adrenergic receptor, an adenosine receptor and a neurotensin receptor.

### Claim 10 (depends on 1)

A method for preparing a mutant GPCR, the method comprising
(a) carrying out the method of claim 1,
(b) identifying the position or positions of the mutated amino acid residue or residues in the mutant GPCR or GPCRs which has been selected for-increased conformational stability, and
(c) synthesising a mutant GPCR which contains a replacement amino acid at one or more of the positions identified.

### Claim 11 (depends on 10)

The method of claim 10 wherein the mutant GPCR contains a plurality of mutations compared to the parent GPCR.

### Claim 12 (depends on 10)

The method according to claim 10 wherein it is determined whether the selected or prepared mutant GPCR is able to couple to a G protein.

### Claim 13 (depends on 10)

The method according to claim 10 wherein it is determined whether the selected or prepared mutant GPCR is able to bind a plurality of ligands of the same class as the selecting ligand with a comparable spread or rank order of affinity as the parent GPCR.

### Claim 14 (depends on 1)

The method according to claim 1 wherein it is determined whether the selected or prepared mutant GPCR is able to couple to a G protein.

### Claim 15 (depends on 1)

The method according to claim 1 wherein it is determined whether the selected or prepared mutant GPCR is able to bind a plurality of ligands of the same class as the ligand used in step (b) with a comparable spread or rank order of affinity as the parent GPCR.

### Claim 16 (depends on 1)

A method as claimed in claim 1 wherein the ligand is detectably labeled.

### Claim 17 (depends on 16)

A method as claimed in claim 16 wherein the ligand is fluorescently labeled.

### Claim 18 (depends on 1)

A method as claimed in claim 1 wherein determining whether the or each mutant GPCR has increased conformational stability in step (d) comprises the use of fluorescence resonance energy transfer (FRET).