Screening method for GPCR ligands

• US 8,623,606 B2
• Filed: 08/09/2007
• Issued: 01/07/2014
• Est. Priority Date: 08/22/2006
• Status: Expired due to Fees

• Current Assignee: Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e)
• Original Assignee: Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e)
• Inventors: Perez-Balderas, Francisco, Hausch, Felix
• Primary Examiner(s): Li, Ruixiang
• Application Number:
  US12/310,296
• Publication Number:
  US 20090286259A1
• Time in Patent Office: 2,343 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

This invention relates to a method of identifying a compound capable of binding to a target domain of a G-protein coupled receptor comprising the steps of: (a) providing a receptor comprising said target domain and a first group linked to said target domain; (b) bringing into contact said receptor of (a) with a test molecule comprising a second group and said compound linked to each other, wherein said first group binds said second group; and (c) determining, subsequent to the binding of said first group to said second group, whether said compound binds to said target domain.

Claims

1. A method of identifying a compound capable of forming a weak bond with a target domain of a G-protein coupled receptor comprising the steps of:

(a) providing a receptor comprising said target domain and a first group linked to said target domain, wherein said target domain comprises the transmembrane or juxtamembrane domain of said G-protein coupled receptor, and wherein said first group comprises;

(i) the N-terminal domain of said G-protein coupled receptor;

(ii) the N-terminal domain of a different G-protein coupled receptor fragment or analog thereof;

or

(iii) avidin or biotin;

a metal ion or chelator;

a strep-tag or strep-tactin;

glutathion or GST;

tetracystein or diarsen;

(b) bringing into contact said receptor of (a) with a test molecule comprising a second group and said compound linked to each other, wherein said first group binds said second group, wherein said second group comprises a component selected from the group consisting of

(i) the C-terminal part of a natural cognate ligand of said G-protein coupled receptor defined in (a) (i) or (ii) capable of binding said N-terminal domain defined in (a) (i) or (ii); and

(ii) biotin or avidin;

a metal ion or chelator;

a strep-tag or strep-tactin;

glutathion or GST;

tetracystein or diarsen; and

(c) determining, subsequent to the binding of said first group to said second group, whether said compound forms a weak bond to said target domain,

and wherein said compound is a peptide, peptidomimetic or small organic molecule, thereby identifying a compound capable of forming a weak bond with a target domain of a G-protein coupled receptor.

2. The method of claim 1, wherein said first group is linked to at least one of the N-Terminus of said transmembrane domain, the N-terminus of said juxtamembrane domain.

3. The method of claim 1, wherein said G-protein coupled receptor is a class B G-protein coupled receptor.

4. The method of claim 3, wherein said class B G-protein coupled receptor is selected from the group consisting of corticotropin releasing factor receptors CRFR1, CRFR2a, CRFR2b and CRFR2c;

parathyroid hormone receptors PTHR1 and PTHR2;

glucagon receptor;

growth hormone releasing hormone receptor;

glucagon-related peptide receptors GLP 1 R and GLP2R;

gastric inhibitory polypeptide receptor;

secretin receptor;

calcitonin receptor;

calcitonin receptor-like receptor;

vasoactive intestinal peptide receptors VIPR1 and VIPR2; and

adenylate cyclase activating polypeptide 1 receptor.

5. The method of claim 1, wherein said compound is selected from the group consisting of a peptide, a peptidomimetic and a small organic molecule.

6. The method of claim 1, wherein said compound has a molecular weight below 300 Da.

7. The method of claim 1, wherein said receptor is selected from the group consisting of

(i) solubilized;

and

(ii) located in a membrane of a micelle or a membrane preparation.

8. The method of claim 1, wherein said target domain and said first group, and/or wherein said second group and said compound are linked through a linker, said linker is selected from the group consisting of alkyl with 1 to 30 carbon atoms, polyethylene glycol with 1 to 20 ethylene moieties, polyalanine with 1 to 20 residues, caproic acid, substituted or unsubstituted poly-p-phenylene and triazol.