Method for detecting glucagon-like peptide-1 antagonists and agonists

• US 5,846,747 A
• Filed: 06/05/1997
• Issued: 12/08/1998
• Est. Priority Date: 03/25/1992
• Status: Expired due to Term

• Current Assignee: Novo Nordisk A/S
• Original Assignee: Novo Nordisk A/S
• Inventors: Knudsen, Liselotte Bjerre, Thorens, Bernard
• Primary Examiner(s): Fitzgerald, David L.
• Application Number:
  US08/869,477
• Time in Patent Office: 551 Days
• Field of Search: 435/7.21, 435/7.1, 435/29
• US Class Current: 506/10
• CPC Class Codes: G01N 2333/605 Glucagons
• CPC Class Codes: G01N 2333/72 for hormones for neuromediators G01N2333/70571
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

The present invention relates to a recombinant glucagon-like peptide-1 (GLP-1) receptor, to a DNA construct which comprises a DNA sequence encoding a GLP-1 receptor, to methods of screening for antagonists of GLP-1 activity, and to the use of the GLP-1 receptor for screening for antagonists of GLP-1 activity.

Claims

1. A method for detecting the presence of a gucagon-like peptide-1 (GLP-1) antagonist, comprising the steps of:

(a) exposing a compound in the presence of GLP-1 agonist to a GLP-1 receptor produced in a heterologous exression system and coupled to a response pathway under conditions md for a time sufficient to allow binding of the compound to the receptor and an associated response through the pathway, wherein the GLP-1 receptor is a GLP-1 tecetor polypeptide having the amino acid sequence of a naturally occurring mammalian GLP-1 receptor encoded by cDNA molecule isolated from a mammalian library and comprisig a nucleotide sequence selected from the group consisting of (i) SEQ ID NO;

1, (ii) SEQ ID NO;

3, and (iii) the sequence of cDNA molecule capable of specifically hybridizing with a probe having the sequence of the complement of SEQ ID NO;

1 or SEQ ID NO;

3 under conditions of reduced stringency, wherein a probe having the sequence of the complement of SEQ ID NO;

1 will specifically identify a cDNA comprising the sequence shown in SEQ ID NO;

3 in a human cDNA library, said receptor polypeptide capable of binding GLP-1 with K_D of less than 100 nM; and

(b) detecting a reduction in the stimulation of the response pathway resulting from the binding of the compound to the GTP-1 receptor, relative to the stimulation of the response pathway by the GLP-1 agonist alone and therefrom determiring the presence of a GLP-1 antagonist.

2. The method of claim 1 wherein the response pathway is a membrane-bound adenylate cyclase response pathway.

3. The method of claim 2 wherein the step of detecting comprises measuring a reduction in cyclic AMP production by the membrane-bound adenylate cyclase response pathway.

4. The method of claim 1 wherein the response pathway includes a luciferase reporter system.

5. The method of claim 1, wherein the GLP-1 receptor polypeptide has the sequence of a GLP-1 receptor of rat or human origin.

6. The method of claim 1, wherein the expression system comprises heterologous DNA comprising the nucleotide sequence shown in SEQ ID NO:

1.

7. A method for detecting the presence of a GLP-1 agonist, comprising the steps of:

(a) exposing a compound to a GLP-1 receptor produced in a heterologous expression system and coupled to a response pathway under conditions and for a time sufficient to allow binding of the compound to the receptor and an associated response through the pathway, wherein the GLP-1 recptor is a GLP-1 receptor polypeptide having the amino acid sequence of a naturally occurring mammalian GLP-1 receptor encoded by cDNA molecule isolated from a mammalian library and comprising a nucleotide sequence selected from the group consisting of (i) SEQ ID NO;

1, (ii) SEQ ID NO;

3, and (iii) the sequence of cDNA molecule capable of specifically hybridizing with a probe having the sequence of the complement of SEQ NO;

1 or SEQ ID NO;

3 under conditions of reduced stringency, wherein a probe having the sequence of the complement of SEQ ID NO;

1 will specifically identify a cDNA comprising the sequence shown in SEQ ID NO;

3 in a human cDNA library, said receptor polypeptide capable of binding GLP-1 with K_D of less than 100 nM; and

(b) detecting stimulation of the response pathway relative to the non stimulated pathway.

8. The method of claim 7 wherein the response pathway is a membrane-bound adenylate cyclase response pathway.

9. The method of claim 8 wherein the step of detecting comprises measuring a production in cyclic AMP production by the membrane.

10. The method of claim 7 wherein the response pathway includes a luciferase reporter system.

11. The metlod of claim 7, wherein the GLP-1 receptor polypeptide has the sequence of a GLP-1 receptor of rat or human origin.

12. The method of claim 7, wherein the expression system comprises heterologous DNA comprising the nucleotide sequence shown in SEQ ID NO:

1.