Identification of ligands by selective amplification of cells transfected with receptors

• US 5,955,281 A
• Filed: 11/07/1997
• Issued: 09/21/1999
• Est. Priority Date: 07/13/1993
• Status: Expired due to Term

• Current Assignee: Acadia Pharmaceuticals Inc.
• Original Assignee: Acadia Pharmaceuticals Inc.
• Inventors: Brann, Mark Robert
• Primary Examiner(s): ULM, JOHN D
• Application Number:
  US08/965,947
• Time in Patent Office: 683 Days
• Field of Search: 435/6, 435/7.1, 435/72, 435/172.3, 435/252.3, 435/320.1, 435/455
• US Class Current: 435/6.14
• CPC Class Codes: C07K 14/70571 for neuromediators, e.g. serotonin receptor, dopamine receptor
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Q 2334/10 p-Nitrophenol derivatives
• CPC Class Codes: G01N 2333/48 Nerve growth factor [NGF]
• CPC Class Codes: G01N 2333/70571 for neuromediators, e.g. serotonin receptor, dopamine receptor
• CPC Class Codes: G01N 2500/10 involving cells
• 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 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/5017 for testing neoplastic activity
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways
• 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 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• 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 invention is directed to a method for identifying substances acting as ligands for transfected receptors by using transfected markers to measure receptor/ligand interactions.

Claims

1. A method for detecting a mutant form of a receptor or mutant form of a signal transducing protein associated with said receptor, which mutant form mediates cell amplification in the absence of an agonist of said receptor and which activation of non-mutant form of said receptor or non-mutant form of said signal transducing protein mediates cell amplification in the presence of, but not the absence of, an effective concentration of said agonist, comprising

(a) providing a library of DNAs expected to comprise at least one DNA sequence coding for the mutant form of said receptor or mutant form of a signal transducing protein;

(b) transfecting the DNAs of step (a) into cells, said transfected cells also comprising a transfected marker of cell amplification;

(c) incubating a cell culture which comprises the transfected cells of step (b) and which also comprises non-transfected cells which do not comprise said transfected marker (i) in the absence of an agonist of said receptor or (ii) in the presence of a lower concentration of said agonist than the effective concentration required for the activation of the non-mutant form of said receptor or a non-mutated form of said signal transducing protein for a period of time sufficient to permit cell amplification of said transfected cells; and

(d) determining the level of cell amplification of said transfected cells by measuring the level of marker in cell culture, increased levels of marker indicating the presence of a mutant form of said receptor or signal transducing protein in said cell culture, which mutant form mediates cell amplification in the absence of an agonist of the receptor or in the presence of a lower concentration of said agonist than the effective concentration required for the activation of the non-mutant form of said receptor or a non-mutated form of said signal transducing protein.

2. The method according to claim 1, wherein the mutant form is associated with a disease state.

3. The method according to claim 1, wherein the mutant form is mutated m5 nicotinic acetylcholine receptor.

4. The method according to claim 1, wherein the DNA transfected into the cell comprises an oncogene.

5. The method according to claim 1, wherein the transfected marker is DNA coding for said receptor or mRNA transcribed thereby, DNA coding for an enzyme, DNA coding for a binding protein or DNA coding for an antigen.

6. The method according to claim 1, wherein the transfected marker is DNA coding for an enzyme selected from the group consisting of phosphatases, beta-galactosidase, urease, glucose oxidase, carbonic anhydrase, acetylcholinestsse, glucoamylase, malate dehydrogenase, glucose-6-phosphate dehydrogenasce, beta-glucosidase, proteases, pyruvate decarboxylase, esterases, luciferase, alcohol dehydrogenase, and peroxidases.

7. The method according to claim 1, wherein the transfected marker is DNA coding for an acid phosphatase or alkaline phosphatase.

8. The method according to claim 1, wherein the transfected marker is DNA coding for horseradish peroxidase.

9. The method according to claim 1, wherein the transfected marker is DNA coding for beta-galactosidase.

10. A method for detecting a mutant form of a receptor or mutant form of a signal transducing protein associated with said rector, which mutant form mediates cell amplification in the absence of an agonist of said receptor and which activation of non-mutant form of said receptor or non-mutant form of said signal transducing protein mediates cell amplification in the presence of, but not absence of, an effective concentration of said agonist, comprising

(a) providing a library of DNAs coding for a given receptor or signal transducing protein associated with said receptor, said library being expected to comprise at least one DNA sequence coding for the mutant form of said receptor or mutant form of said signal transducing protein;

(b) transfecting the DNAs of step (a) into cell culture, said transfected cell culture also comprising a transfected DNA coding for a marker enzyme of cell amplification;

(c) dividing the cell culture of step (a), the cell culture comprising transfected cells comprising said marker enzyme and nontransfected cells not comprising said marker enzyme into several identical aliquots,

(d) incubating each aliquot with one or more test substances for a period of time sufficient to permit cell amplification of said transfected cells and hereby distinguish between the mutant and wild-type form of the protein, and

(e) determining any change in cell amplification by measuring marker enzyme activity in each aliquot, increased levels of marker indicating the presence of a mutant form of said receptor or signal transducing protein in said cell culture, which mutant form mediates cell amplification in the absence of an agonist of the receptor.

11. The method according to claim 10, wherein the marker enzyme is select from the group consisting of phosphatases, beta-galactosidase, urease, glucose oxidase, carbonic anhydrase, acetylcholinesterase, glucoamylase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, beta-glucosidase, proteases, pyruvate decarboxylase, esterases, luciferase, alcohol dehydrogease, and peroxidases.

12. The method according to claim 10, wherein the mutant form of the receptor is a mutated m5-receptor, mutated m5 muscarinic acetylcholine receptor.

13. The method according to claim 10, wherein the marker enzyme is beta-galactosidase.