# US 7,611,848 B2 · Method for selecting allosteric regulators of class III G protein-coupled receptors

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- Number: US 7,611,848 B2
- Document ID: US7611848B2
- Status: Granted
- Kind: B2
- Application number: 10/557,986
- Filed: 2004-05-11
- Published: 2008-03-06
- Granted: 2009-11-03
- Earliest claimed: 2003-05-22
- Assignees: Centre National de la Recherche Scientifique (CNRS)
- Inventors: Florence Gaven; Cyril Goudet; Jean-Philippe Pin; Laurent Prezeau
- Web page: https://patentgrep.com/patent/US7611848B2

## CPC

- G01N 33/6896: {Neurological disorders, e.g. Alzheimer's disease}
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/18: Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
- A61P 25/24: Antidepressants
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 25/30: for treating abuse or dependence
- 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 2333/70571: for neuromediators, e.g. serotonin receptor, dopamine receptor
- G01N 2333/726: G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
- G01N 2500/02: Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

A method for identifying and selecting a compound acting as an allosteric regulator of a GPCR-III is described. A method comparing the activity level of a recombinant GPCR-III (GPCR-III-Δ), essentially depleted of the extracellular domain of the corresponding native GPCR-III, in the presence of a given compound, with the activity level of the same GPCR-III-Δ is described. A method of identifying positive or negative allosteric regulators of GPCRs-III is described.

## Claims

### Claim 1

A method for identifying and selecting a compound that acts as a positive allosteric regulator of a class III G protein-coupled receptor (GPCR-III) selected from the group consisting of:
glutamate-activated receptors;
GABA-activated receptors;
putative pheromone receptors; and
taste receptors, said method comprising at least the following steps:
a) producing a recombinant GPCR-III (GPCR-III-Δ) essentially depleted of the extracellular domain of the corresponding native GPCR-III;
b) bringing said compound into contact with said GPCR-III-Δ in the absence of an agonist of said GPRC-III;
c) measuring the level N1 of activity of said GPCR-III-Δ in the presence of said compound;
d) comparing the level N1 measured in step c) with the level N0 of activity of the GPCR-III-Δ in the absence of said compound; and
e) if N1 is significantly different from N0, identifying and selecting said positive allosteric regulator,
where said positive allosteric regulator has no effect on the GPCR-III, if GPCR-III is not simultaneously activated by an agonist of GPCR-III, but behaves like an agonist or an inverse agonist of said GPCR-III-Δ.

### Claim 2 (depends on 1)

The method of claim 1, wherein said GPCR-III-Δ is completely depleted of the extracellular domain of said native GPCR-III.

### Claim 3 (depends on 1)

The method of claim 1, wherein said GPCR-III-Δ comprises a heptahelical membrane domain.

### Claim 4 (depends on 3)

The method of claim 3, wherein step a) comprises at least the following substeps:
a1) PCR amplification of a nucleic acid encoding said membrane domain of said GPCR-III-Δ;
a2) cloning of the amplification product into a recombinant expression vector;
a3) transfection of said recombinant vector into a recombinant host cell; and
a4) production of said GPCR-III-Δ by said recombinant host cell.

### Claim 5 (depends on 1)

The method of claim 1, wherein said GPCR-III-Δ comprises a targeting signal and a heptahelical membrane domain.

### Claim 6 (depends on 5)

The method of claim 5, wherein said targeting signal is a signal peptide.

### Claim 7 (depends on 6)

The method of claim 6, wherein said signal peptide is that of said corresponding native GPCR-III.

### Claim 8 (depends on 5)

The method of claim 5,
wherein said step a) comprises at least the following substeps:
a1) PCR amplification of a nucleic acid encoding said membrane domain of said GPCR-III-Δ;
a2) cloning, into a recombinant expression vector, of the amplification product downstream of a nucleic acid encoding said targeting signal;
a3) transfection of said recombinant vector into a recombinant host cell; and
a4) production of said GPCR-III-A by said recombinant host cell.

### Claim 9 (depends on 4)

The method of claim 4, wherein said recombinant host cell is either a eukaryotic or prokaryotic cell.

### Claim 10 (depends on 1)

The method of claim 1, wherein said GPCR-III is a glutamate-activated receptor.

### Claim 11 (depends on 10)

The method of claim 10, wherein said glutamate-activated receptor is selected from the group consisting of mGlu1, mGlu2 and mGlu5 receptors.

### Claim 12 (depends on 1)

The method of claim 1, wherein said GPCR-III is a GABA-activated receptor.

### Claim 13 (depends on 12)

The method of claim 12, wherein said GABA-activated receptor is the GB2 subunit.