Identification of ligands by selective amplification of cells transfected with receptors

• US 7,491,503 B2
• Filed: 05/03/2006
• Issued: 02/17/2009
• Est. Priority Date: 10/07/1999
• Status: Expired due to Fees

• Current Assignee: Acadia Pharmaceuticals Inc.
• Original Assignee: Acadia Pharmaceuticals Inc.
• Inventors: Brann, Mark R.
• Primary Examiner(s): Ulm; John D
• Application Number:
  US11/417,083
• Publication Number:
  US 20060286610A1
• Time in Patent Office: 1,021 Days
• Field of Search: None
• US Class Current: 435/7.21
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: 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
• CPC Class Codes: G01N 33/942 Serotonin, i.e. 5-hydroxy-tryptamine

Abstract

The invention is directed to a method for identifying substances acting as ligands for transfected receptors by using transfected markers to measure receptor/ligand interactions. The present invention also relates to a method of identifying compounds which act as inverse agonists of the 5-HT2A receptor, the method comprising contacting a constitutively active 5-HT2A receptor with at least one test compound and determining any decrease in the amount of basal activity of the receptor so as to identify a test compound which is an inverse agonist of the 5-HT2A receptor. Such inverse agonists may be used in the treatment of schizophrenia and related psychoses.

Claims

1. A cell culture comprising:

cells comprising DNA encoding a constitutively active wild-type 5-HT2A receptor and DNA encoding a marker, wherein said marker is different from said receptor and is selected from the group consisting of an enzyme which produces a detectable end product, a binding protein and an antigen that may be visualized with labeled antibodies, wherein said DNA encoding said marker has been introduced into said cell, wherein the amount of said marker in said cell culture is indicative of the number of cells expressing said receptor, wherein the rate of transcription of said DNA encoding said marker is not influenced by the extent to which said receptor has been activated and wherein exposure of said receptor to a ligand results in an increase or decrease in the proliferation rate of said first population of cells; and

a substance being evaluated to determine whether it is a ligand of said receptor.

2. The cell culture of claim 1, wherein said receptor has an elevated basal or constitutive receptor response.

3. The cell culture of claim 1, wherein said cell culture further comprises cells selected from the group consisting of:

(a) cells which do not comprise DNA encoding said marker;

(b) cells which do not comprise DNA encoding said receptor;

(c) cells which comprise DNA encoding said receptor but not DNA encoding said marker;

(d) cells which comprise DNA encoding said marker but not DNA encoding said receptor; and

(e) a combination of two or more of the cells recited in (a)-(d).

4. The cell culture of claim 1, wherein said marker is an enzyme which produces a detectable end product.

5. The cell culture of claim 1, wherein said marker is a binding protein.

6. The cell culture of claim 1, wherein said marker is an antigen that may be visualized with labeled antibodies.

7. A plurality of cell cultures, each cell culture comprising:

cells comprising DNA encoding a constitutively active wild-type 5-HT2A receptor and DNA encoding a marker, wherein said marker is different from said receptor and is selected from the group consisting of an enzyme which produces a detectable end product, a binding protein and an antigen that may be visualized with labeled antibodies, wherein said DNA encoding said marker has been introduced into said cell, wherein the amount of said marker in said cell culture is indicative of the number of cells expressing said receptor, wherein the rate of transcription of said DNA encoding said marker is not influenced by the extent to which said receptor has been activated and wherein exposure of said receptor to a ligand results in an increase or decrease in the proliferation rate of said first population of cells; and

a substance being evaluated to determine whether it is a ligand of said receptor, wherein the substance being evaluated to determine whether it is a ligand of said receptor is different in at least two of said plurality of cell cultures.

8. The cell culture of claim 7, wherein said receptor has an elevated basal or constitutive receptor response.

9. The cell culture of claim 7, wherein said cell culture further comprises cells selected from the group consisting of:

(a) cells which do not comprise DNA encoding said marker;

(b) cells which do not comprise DNA encoding said receptor;

(c) cells which comprise DNA encoding said receptor but not DNA encoding said marker;

(d) cells which comprise DNA encoding said marker but not DNA encoding said receptor; and

(e) a combination of two or more of the cells recited in (a)-(d).

10. The cell culture of claim 7, wherein said marker is an enzyme which produces a detectable end product.

11. The cell culture of claim 7, wherein said marker is a binding protein.

12. The cell culture of claim 7, wherein said marker is an antigen that may be visualized with labeled antibodies.