Identification of 5HT2A receptor ligands by selective amplification of cells transfected with receptors

• US 7,452,682 B2
• Filed: 05/03/2006
• Issued: 11/18/2008
• 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:
  US11/417,070
• Publication Number:
  US 20060292606A1
• Time in Patent Office: 930 Days
• Field of Search: None
• US Class Current: 435/7.1
• 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 method of determining whether a substance is a ligand of a constitutively active wild-type 5HT2A receptor, wherein said receptor has a basal response, said method comprising:

obtaining a first population of cells comprising said receptor, wherein DNA encoding a marker indicative of the extent of cellular proliferation has been introduced into said first population of cells and wherein the amount of marker in said cells is influenced by the degree to which said receptor is active;

contacting said first population of cells with a test substance which is a potential ligand of said receptor;

comparing the amount of marker in said first population of cells which has been contacted with said test substance to the amount of marker in a second population of cells which has not been contacted with said test substance, said second population of cells comprising said receptor and DNA encoding said marker such that the extent of marker activity in said cells is influenced by the degree to which said receptor is active, wherein said test substance is a ligand of said receptor if the amount of marker in said first population of cells is significantly different than the amount of marker in said second population of cells.

2. The method of claim 1, wherein said comparing step is performed by measuring the amount of said marker in said first population of cells at a time after contacting said first population of cells with said test substance which is prior to the time at which foci resulting from the proliferation of said first population of cells would be visible and by measuring the amount of said marker produced by said second population of cells at the same time as the amount of said marker in said test culture was measured.

3. The method of claim 1, wherein said comparing step is performed within two weeks of the day that said first population of cells was contacted with said substance.

4. The method of claim 1, wherein said comparing step is performed within 4 days of the day that said first population of cells was contacted with said substance.

5. The method of claim 1, further comprising contacting said first population of cells and said second population of cells wit a substance known to be an agonist of said constitutively active wild-type 5HT2A receptor.

6. The method of claim 1, further comprising contacting said first population of cells and said second population of cells with a substance known to be an antagonist of said constitutively active wild-type 5HT2A receptor.

7. The method of claim 1, wherein said first population of cells and said second population of cells are obtained by introducing said DNA encoding said constitutively active wild-type 5HT2A receptor and said DNA encoding said marker into a starting cell culture and dividing said starting cell culture into multiple aliquots.

8. The method of claim 1, wherein said determining step comprises determining whether said substance inhibits said basal receptor response, wherein said substance is an inverse agonist of said receptor if said substance inhibits said basal receptor response of said constitutively active wild-type 5HT2A receptor.

9. The method of claim 1, wherein said second population of cells 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 constitutively active wild-type 5HT2A 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 constitutively active wild-type 5HT2A receptor; and

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