SCREENING METHOD FOR GPCR LIGANDS

• US 20090286259A1
• Filed: 08/09/2007
• Published: 11/19/2009
• Est. Priority Date: 08/22/2006
• Status: Active Grant

• 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: Hausch, Felix, Perez-Balderas, Francisco
• Granted Patent: US 8,623,606 B2
• Time in Patent Office: Days
• US Class Current: 435/7.200
• 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 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

(b) bringing into contact said receptor of (a) with a test molecule comprising a

(c) determining, subsequent to the binding of said first group to said second.

2. The method of claim 1, wherein said target domain comprises the transmembrane or juxtamembrane domain of said G-protein coupled receptor or a fragment or analog thereof.

3. The method of claim 2, wherein said first group is linked to the extracellular part and/or the N-terminus of said transmembrane or juxtamembrane domain or a fragment or analog thereof.

4. The method of claim 3, wherein said first group comprises:

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

(ii) the N-terminal domain of a different G-protein coupled receptor or a 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.

5. The method of claim 4, 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 claim 4 (i) or (ii) or a fragment or analog thereof capable of binding said N-terminal domain defined in claim 4 (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.

6. The method of claim 1, wherein said target domain comprises the extracellular or N-terminal domain of said G-protein coupled receptor or a fragment or analog thereof.

7. The method of claim 6, wherein said first group comprises a component selected from the group consisting of

(i) the transmembrane or juxtamembrane domain of said G-protein coupled receptor

(ii) the transmembrane or juxtamembrane domain of a different G-protein coupled

8. The method of claim 7, wherein said second group comprises the N-terminal part of a natural cognate ligand of said G-protein coupled receptor or a fragment or analog thereof capable of binding and/or activating said transmembrane or juxtamembrane domain or fragment or analog thereof defined in claim 7.

9. The method of claim 8, wherein said G-protein coupled receptor is a class B G-protein coupled receptor.

10. The method of claim 9, 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 GLP1 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.

11. The method of claim 10, wherein said compound is selected from the group consisting of a peptide, a peptidomimetic and a small organic molecule.

12. The method of claim 10, wherein said compound has a molecular weight below 300 Da.

13. The method of claim 10, further comprising the step of

(d) optimising the binding of said compound to said target domain, wherein said

14. The method of claim 10, further comprising the steps of

(e) bringing into contact of said receptor defined in any one of claims 1 (a), 2 to 4, 6,

(f) determining whether said compound being devoid of said second group binds to

15. The method of claim 14, wherein said determining in step (c) or step (f) is effected by determining displacement of a labelled reference compound known to bind the target domain.

16. The method of claim 14, wherein said determining in step (c) or step (f) is effected by determining whether a conformational or functional change of said target domain occurs, said change being indicative of a compound binding to said target domain.

17. The method of claim 14, wherein said receptor comprises the C-terminal domain of said G-protein coupled receptor or of a different G-protein coupled receptor or a fragment of said C-terminal domain, wherein said fragment is capable of initiating signal transduction.

18. The method of claim 17, wherein said determining in said steps (c) and/or (f) is effected by determining

(i) the formation of a secondary messenger;

(ii) the displacement of a GTP analog;

(iii) covalent modification of said receptor such as phosphorylation;

or

(iv) recruiting of an interaction partner of said receptor such as arrestin or a G-protein.

19. The method of claim 18, wherein said secondary messenger is selected from the group consisting of cAMP, inositol trisphosphate and Ca²⁺.

20. The method of claim 19, wherein

(i) said receptor comprises (an) additional group(s) sensitive to said conformational

(ii) said determining in said steps (c) and/or (f) is effected by determining a parameter of said additional group(s).

21. The method of claim 20, wherein said additional group(s) is/are selected from groups detectable by spectroscopic means, and wherein said parameter is the absorption and/or emission characteristic of said additional group(s).

22. The method of claim 21, wherein said additional group(s) is/are a FRET pair or a BRET pair.

23. The method of claim 22, wherein said receptor is located in the membrane of a cell.

24. The method of claim 23, wherein said additional group is selected from the group consisting of

(i) a tag capable of binding a cognate antibody;

and

(ii) an enzyme fused to said target domain.

25. The method of claim 24, wherein said determining in said steps (c) and/or (T) is effected by determining cellular dynamics.

26. The method of claim 25, wherein said cellular dynamics is selected from

(i) arrestin translocation;

and

(v) internalisation of the receptor.

27. The method of claim 3, wherein said receptor is selected from the group consisting of

(i) solubilized;

and

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

28. The method of claim 27, wherein the linker between said target domain and said first group and/or the linker between said second group and said compound 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.

31. The kit of claim 29, further comprising the receptor of claim 28.

34. A method of producing the test molecule of claim 1 comprising reacting the compound of claim 32 and a compound selected from peptide, peptidomimetic and small organic molecule, wherein said peptide, peptidomimetic and small organic molecule carries a second reactive group.

42. The kit of claim 30, further comprising the receptor of claim 28.

43. A method of producing the test molecule of claim 1 comprising reacting the compound of claim 33 and a compound selected from peptide, peptidomimetic and small organic molecule, wherein said peptide, peptidomimetic and small organic molecule carries a second reactive group.

45. The method of claim 34, wherein said first and/or said second reactive group is selected from the group consisting of thiol, amine, alkyne, azide, aldehyde, activated ester, phosphor ylide, hydroxylamine, alpha-halogen carboxamide/carboxylester, alpha, beta-unsaturated carbonyls, activated disulfides, Michael acceptor, Michael donor, diene and dienophile.

29. A kit comprising a library of test molecules, wherein each test molecule comprises:

(i) a peptide comprising or consisting of the sequence of any one of SEQ ID NO;

1 to 3, or a sequence having at least 70% sequence identity to said sequence of any

(iii) the 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

30. A kit comprising a library of test molecules, wherein each test molecule comprises:

(i) a peptide consisting of the sequence of any one of secretin, VIP, PACAP, adrenomedullin, calcitonin, amylin or CGRP, wherein between 1 and 10 N-terminal amino acids are deleted from said peptide, or a sequence having at least 70% sequence identity to the truncated peptide, or an analog thereof, linked to (H) a member of a library of compounds;

wherein

(iii) the 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

32. A compound comprising or consisting of.

(i) a peptide comprising or consisting of the sequence of any one of SEQ ID

(ii) a first reactive group, wherein

(iii) the 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

35. The compound of claim 32, wherein said first and/or said second reactive group is selected from the group consisting of thiol, amine, alkyne, azide, aldehyde, activated ester, phosphorylide, hydroxylamine, alpha-halogen carboxamide/carboxylester, alpha, beta-unsaturated carbonyls, activated disulfides, Michael acceptor, Michael donor, diene and dienophile.

33. A compound comprising:

(i) a peptide consisting of the sequence of any one of secretin, VIP, PACAP, adrenomedullin, calcitonin, amylin or CGRP, wherein between 1 and 10 N-terminal amino acids are deleted from said peptide, or a sequence having at least 70% sequence identity to the truncated peptide, or an analog thereof, linked to

(ii) a first reactive group;

wherein

(iii) the 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

44. The compound of claim 33, wherein said first and/or said second reactive group is selected from the group consisting of thiol, amine, alkyne, azide, aldehyde, activated ester, phosphor ylide, hydroxylamine, alpha-halogen carboxamide/carboxylester, alpha, beta-unsaturated carbonyls, activated disulfides, Michael acceptor, Michael donor, diene and dienophile.

36. A chimeric G-protein coupled receptor comprising:

(i) a class A or a class C G-protein coupled receptor or a fragment thereof

(ii) the N-terminal domain of a class B G-protein coupled receptor or a fragment of

37. A membrane preparation comprising the chimeric G-protein coupled receptor of claim 36.

38. A nucleic acid encoding the chimeric G-protein coupled receptor of claim 36.

39. A vector comprising the nucleic acid of claim 38.

40. A cell transformed with the vector of claim 39.

41. A cell transformed with the nucleic acid of claim 38.