Method of identifying a compound for inhibiting or stimulating human G protein-coupled receptors

• US RE42,190 E1
• Filed: 01/24/2007
• Issued: 03/01/2011
• Est. Priority Date: 11/20/1998
• Status: Expired due to Fees

• Current Assignee: Arena Pharmaceuticals Incorporated
• Original Assignee: Arena Pharmaceuticals Incorporated
• Inventors: Leonard, James N., Chen, Ruoping
• Primary Examiner(s): Landsman; Robert
• Assistant Examiner(s): Chandra; Gyan
• Application Number:
  US11/657,110
• Time in Patent Office: 1,497 Days
• Field of Search: None
• US Class Current: 435/7.21
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/723 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor

Abstract

The invention disclosed in this patent document relates to transmembrane receptors, more particularly to endogenous, human orphan G protein-coupled receptors.

Claims

1. A method for identifying a compound for regulating insulin concentration in the blood of a mammal comprising the steps of:

contacting one or more candidate compounds with a host cell that expresses a receptor comprising the amino acid sequence of SEQ ID NO;

8; and

measuring the ability of the compound or compounds to inhibit or stimulate said receptor, wherein said inhibition or stimulation of said receptor is indicative of a compound for regulating insulin concentration in the blood of a mammal.

2. The method of claim 1 wherein said compound for regulating insulin concentration in the blood of a mammal is a therapeutic for treating diabetes.

3. The method of claim 1 wherein the compound for regulating insulin concentration in the blood of a mammal is selected from agonist, partial agonist, and inverse agonist of the receptor.

4. The method of claim 1 wherein said host cell comprises an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:

8.

5. The method of claim 1 where said host cell is produced by a method comprising:

transfecting a cell with an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO;

8;

wherein said host cell, under appropriate culture conditions, produces a polypeptide comprising said amino acid sequence of SEQ ID NO;

8.

6. A method for identifying a compound for regulating glucose concentration in the blood of a mammal comprising the steps of:

contacting one or more candidate compounds with a host cell that expresses a receptor comprising the amino acid sequence of SEQ ID NO;

8; and

measuring the ability of the compound or compounds to inhibit or stimulate said receptor, wherein said inhibition or stimulation of said receptor is indicative of a compound for regulating glucose concentration in the blood of a mammal.

7. The method of claim 6 wherein said host cell comprises an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:

8.

8. The method of claim 6 where said host cell is produced by a method comprising:

transfecting a cell with an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO;

8;

wherein said host cell, under appropriate culture conditions, produces a polypeptide comprising said amino acid sequence of SEQ ID NO;

8.

9. A method for identifying a compound for regulating glucagon concentration in the blood of a mammal comprising the steps of:

contacting one or more candidate compounds with a host cell that expresses a receptor comprising the amino acid sequence of SEQ ID NO;

8; and

measuring the ability of the compound or compounds to inhibit or stimulate said receptor, wherein said inhibition or stimulation of said receptor is indicative of a compound for regulating glucagon concentration in the blood of a mammal.

10. The method of claim 9 wherein said host cell comprises an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:

8.

11. The method of claim 9 where said host cell is produced by a method comprising:

transfecting a cell with an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO;

8;

wherein said host cell, under appropriate culture conditions, produces a polypeptide comprising said amino acid sequence of SEQ ID NO;

8.

12. A method for identifying a compound for inhibiting or stimulating a receptor comprising:

a) the amino acid sequence of SEQ ID NO;

8;

b) a mutant of SEQ ID NO;

8, wherein lysine is substituted for leucine at amino acid residue 224;

c) an amino acid sequence encoded by a nucleotide sequence that hybridizes to the complete complement of SEQ ID NO;

7 at 42°

C., followed by washing in 0.1×

SSC at 65°

C.;

d) an amino sequence encoded by the nucleotide sequence of SEQ ID NO;

7;

e) a G protein-coupled receptor having at least 95% identity to the amino acid sequence of SEQ ID NO;

8, wherein said G protein-coupled receptor is capable of modulating insulin or glucagon levels;

or

f) a G protein-coupled receptor encoded by a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO;

7, wherein said G protein-coupled receptor is capable of modulating insulin or glucagon levels,

comprising the steps of;

i) contacting one or more candidate compounds with a host cell or membrane thereof,

13. The method of claim 12, wherein the compound is selected from agonist, partial agonist, and inverse agonist of the receptor.

14. The method of claim 13, wherein the compound is an agonist of the receptor.

15. The method of claim 13, wherein the compound is a partial agonist of the receptor.

16. The method of claim 13, wherein the compound is an inverse agonist of the receptor.

17. The method of claim 12, wherein said host cell comprises an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising:

a) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO;

8;

b) a nucleotide sequence encoding a polypeptide comprising a mutant of SEQ ID NO;

8, wherein lysine is substituted for leucine at amino acid residue 224;

c) a nucleotide sequence that hybridizes to the complete complement of SEQ ID NO;

7 at 42°

C., followed by washing in 0.1×

SSC at 65°

C.;

d) the nucleotide sequence of SEQ ID NO;

7;

e) a nucleotide sequence encoding a G protein-coupled receptor having at least 95% identity to the amino acid sequence of SEQ ID NO;

8, wherein said G protein-coupled receptor is capable of modulating insulin or glucagon levels;

or

f) a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO;

7, wherein said nucleotide sequence encodes a G protein-coupled receptor capable of modulating insulin or glucagon levels.

18. The method of claim 12, wherein said host cell is produced by a method comprising:

transfecting a cell with an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising;

a) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO;

8;

b) a nucleotide sequence encoding a polypeptide comprising a mutant of SEQ ID NO;

8, wherein lysine is substituted for leucine at amino acid residue 224;

c) a nucleotide sequence that hybridizes to the complete complement of SEQ ID NO;

7 at 42°

C., followed by washing in 0.1×

SSC at 65°

C.;

d) the nucleotide sequence of SEQ ID NO;

7;

e) a nucleotide sequence encoding a G protein-coupled receptor having at least 95% identity to the amino acid sequence of SEQ ID NO;

8, wherein said G protein-coupled receptor is capable of modulating insulin or glucagon levels;

or

f) a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO;

7, wherein said nucleotide sequence encodes a G protein-coupled receptor capable of modulating insulin or glucagon levels,

wherein said host cell, under appropriate culture conditions, produces a polypeptide comprising;

a) the amino acid sequence of SEQ ID NO;

8;

b) a mutant of SEQ ID NO;

8, wherein lysine is substituted for leucine at amino acid residue 224;

c) an amino acid sequence encoded by a nucleotide sequence that hybridizes to the complete complement of SEQ ID NO;

7 at 42°

C., followed by washing in 0.1×

SSC at 65°

C.;

d) an amino sequence encoded by the nucleotide sequence of SEQ ID NO;

7;

e) a G protein-coupled receptor having at least 95% identity to the amino acid sequence of SEQ ID NO;

8, wherein said G protein-coupled receptor is capable of modulating insulin or glucagon levels;

or

f) a G protein-coupled receptor encoded by a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO;

7, wherein said G protein-coupled receptor is capable of modulating insulin or glucagon levels.

19. The method of claim 12, wherein the receptor comprises the amino acid sequence of SEQ ID NO:

8.

20. The method of claim 12, wherein the receptor is a mutant of SEQ ID NO:

8, wherein lysine is substituted for leucine at amino acid residue 224.

21. The method of claim 12, wherein the ability of the compound or compounds to inhibit or stimulate said receptor is measured by measuring the activity of a second messenger.

22. The method of claim 21, wherein the second messenger is selected from the group consisting of adenyl cyclase and phospholipase C.

23. The method of claim 12, wherein the ability of the compound or compounds to inhibit or stimulate said receptor is measured by measuring the level of a second messenger.

24. The method of claim 23, wherein the second messenger is selected from the group consisting of cAMP, diacyl glycerol, and inositol 1,4,5-triphosphate.

25. The method of claim 12, wherein the ability of the compound or compounds to inhibit or stimulate said receptor is measured by measuring the binding of GTPγ

S to a membrane comprising said G protein-coupled receptor.

26. The method of claim 12, wherein the host cell is a mammalian host cell.

27. The method of claim 12, wherein the host cell is a yeast host cell.

28. The method of claim 12, wherein the host cell comprises a reporter system comprising multiple cAMP responsive elements operably linked to a reporter gene.

29. The method of claim 12, wherein said receptor is a constitutively activated receptor.

30. The method according to claim 12, wherein said method comprises identifying a compound for inhibiting or stimulating a receptor comprising:

a) a G protein-coupled receptor having at least 98% identity to the amino acid sequence of SEQ ID NO;

8, wherein said G protein-coupled receptor is capable of modulating insulin or glucagon levels;

or

b) a G protein-coupled receptor encoded by a nucleotide sequence having at least 98% identity to the nucleotide sequence of SEQ ID NO;

7, wherein said G protein-coupled receptor is capable of modulating insulin or glucagon levels, comprising the steps of;

contacting one or more candidate compounds with a host cell or membrane thereof,

31. The method of claim 17, wherein said host cell comprises an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising:

a) a nucleotide sequence encoding a G protein-coupled receptor having at least 98% identity to the amino acid sequence of SEQ ID NO;

8, wherein said G protein-coupled receptor is capable of modulating insulin or glucagon levels;

or

b) a nucleotide sequence having at least 98% identity to the nucleotide sequence of SEQ ID NO;

7, wherein said nucleotide sequence encodes a G protein-coupled receptor capable of modulating insulin or glucagon levels.

32. The method of claim 18, wherein said host cell is produced by a method comprising:

transfecting a cell with an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising;

a) a nucleotide sequence encoding a G protein-coupled receptor having at least 98% identity to the amino acid sequence of SEQ ID NO;

8, wherein said G protein-coupled receptor is capable of modulating insulin or glucagon levels;

or

b) a nucleotide sequence having at least 98% identity to the nucleotide sequence of SEQ ID NO;

7, wherein said nucleotide sequence encodes a G protein-coupled receptor capable of modulating insulin or glucagon levels,

wherein said host cell, under appropriate culture conditions, produces a polypeptide comprising;

a) a G protein-coupled receptor having at least 98% identity to the amino acid sequence of SEQ ID NO;

8, wherein said G protein-coupled receptor is capable of modulating insulin or glucagon levels;

or

b) a G protein-coupled receptor encoded by a nucleotide sequence having at least 98% identity to the nucleotide sequence of SEQ ID NO;

7, wherein said G protein-coupled receptor is capable of modulating insulin or glucagon levels.