Identification of ligands by selective amplification of cells transfected with receptors

• US 5,707,798 A
• Filed: 07/12/1994
• Issued: 01/13/1998
• Est. Priority Date: 07/13/1993
• Status: Expired due to Term

• Current Assignee: Acadia Pharmaceuticals Inc.
• Original Assignee: Novo Nordisk A/S
• Inventors: Brann, Mark Robert
• Primary Examiner(s): ULM, JOHN D
• Application Number:
  US08/273,669
• Time in Patent Office: 1,281 Days
• Field of Search: 435/6, 435/7.1, 435/7.2, 436/501
• 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 of detecting a substance which is a ligand of a receptor, said method comprising,

(a) incubating a cell culture which comprises cells transfected with DNA coding for said receptor which increases or decreases cell amplification in response to a ligand, said transfected cells also comprising a transfected marker of cell amplification, and which said cell culture also comprises nontransfected cells which do not comprise said transfected marker, with a test substance which is a potential agonist or antagonist of the receptor for a time sufficient to permit amplification of said transfected cells, and

(b) determining the level of amplification of said transfected cells by measuring the level of the marker in the cell culture.

2. A method according to claim 1, wherein any agonist activity of the test substance is determined by an enhanced effect of the receptor on amplification of the transfected cells relative to the nontransfected cells.

3. A method according to claim 1, wherein any antagonist activity of the test substance is determined by inhibition of the effect of the receptor on amplification of the transfected cells relative to the nontransfected cells.

4. A method according to claim 1, wherein the transfected marker is transfected receptor DNA, transcribed receptor mRNA, an enzyme, a binding protein or an antigen.

5. A method according to claim 1, wherein the transfected marker is said DNA coding for said receptor or mRNA encoded thereby and its presence is determined by DNA amplification and/or hybridization techniques.

6. A method according to claim 1, wherein the marker is an enzyme selected from the group consisting of phosphatases, β

-galactosidase, urease, glucose oxidase, carbonic anhydrase, acetylcholinesterase, glucoamylase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, β

-glucosidase, proteases, pyruvate decarboxylase, esterases, luciferase, alcohol dehydrogenase, and peroxidases.

7. A method according to claim 1 wherein the marker is acid or alkaline phosphatase.

8. A method according to claim 1 wherein the marker is horseradish peroxidase.

9. A method according to claim 1, wherein the transfected cells are transfected with DNA encoding a single receptor.

10. A method according to claim 9, wherein the DNA encodes a tyrosine kinase receptor.

11. A method according to claim 9, wherein the DNA encodes a G-protein coupled receptor.

12. A method according to claim 9, wherein the DNA encodes a tyrosine kinase activating receptor.

13. A method according to claim 12, wherein the test substance is incubated with the transfected cells in the presence of an agonist of receptor stimulation of cell amplification.

14. A method according to claim 9, wherein the DNA encodes a tyrosine phosphatase receptor.

15. A method according to claim 9, wherein the DNA encodes a transcription factor.

16. A method according to claim 9, wherein the DNA encodes asteroid receptor.

17. A method according to claim 9, wherein the DNA encodes an oncogene.

18. A method according to claim 9, wherein the DNA encodes a lipid-gated ion channel.

19. A method according to claim 9, wherein the DNA encodes a voltage-gated ion channel.

20. A method according to claim 9, wherein the DNA encodes a chimeric receptor composed of a ligand-binding region of one receptor and a signal transducing region of another receptor.

21. A method of detecting a substance which is a ligand of a receptor, said method comprising

(a) transfecting a cell culture with DNA encoding said receptor which increases or decreases cell amplification in response to a ligand and with DNA encoding a marker enzyme,

(b) 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,

(c) incubating each aliquot with one or more test substances which are potential agonists or antagonists for a period of time sufficient to permit cell amplification, and

(d) determining any increase or decrease in cell amplification by measuring marker enzyme activity in each aliquot.

22. A method according to claim 21, wherein the cells in the cell culture are transfected with DNAs encoding two or more distinct receptors, each transfected cell expressing an individual receptor.

23. A method according to claim 21, wherein the DNA encodes a tyrosine kinase receptor.

24. A method according to claim 21, wherein the DNA encodes a G-protein coupled receptor.

25. A method according to claim 21, wherein the DNA encodes a tyrosine kinase activating receptor.

26. A method according to claim 21, wherein the DNA encodes a tyrosine phosphatase receptor.

27. A method according to claim 21, wherein the DNA encodes a transcription factor.

28. A method according to claim 21, wherein the DNA encodes asteroid receptor.

29. A method according to claim 21, wherein the DNA encodes an oncogene.

30. A method according to claim 21, wherein the DNA encodes a ligand-gated ion channel.

31. A method according to claim 21, wherein the DNA encodes a voltage-gated ion channel.

32. A method according to claim 21, wherein the DNA encodes a chimeric receptor composed of a ligand-binding region of one receptor and a signal transducing region of another receptor.

33. A method according to claim 21, wherein any agonist activity of one or more test substances is determined by an enhanced effect of the receptor on amplification of the transfected cells relative to nontransfected cells.

34. A method according to claim 21, wherein any antagonist activity of one or more test substances is determined by an enhanced effect of the receptor on amplification of the transfected cells relative to nontransfected cells.

35. A method according to claim 34, wherein one or more test substances are incubated with the transfected cells in the presence of an agonist of receptor stimulation of cell amplification.

36. A method according to claim 21, wherein the marker is an enzyme selected from the group consisting of β

-galactosidase, alkaline phosphatase, firefly luciferase, and alcohol dehydrogenase.

37. A method according to claim 21, wherein cells which express a given receptor DNA express a marker which is distinguishable from a marker expressed by cells transfected with another receptor DNA.

38. A method of detecting a substance which is a ligand of a receptor, said method comprising

(a) transfecting a cell culture with DNA encoding said receptor which increases or decreases cell amplification in response to a ligand and with DNA encoding a marker enzyme,

(b) 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,

(c) incubating each aliquot with varying concentrations of test substance which is a potential agonist or antagonist for a period of time sufficient to permit cell amplification, and

(d) determining any change in cell amplification by measuring marker enzyme activity in each aliquot.

39. A method for detecting a substance which is a ligand of a receptor, said method comprising

(a) transfecting a cell culture with DNAs encoding two or more distinct receptors, each transfected cell expressing an individual receptor, and with DNA encoding a marker enzyme,

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

(c) incubating each aliquot with one or more test substances which act as potential agonists or antagonists of the receptors for a period of time sufficient to permit cell amplification,

(d) determining any change in cell amplification by measuring marker enzyme activity in each aliquot, identifying active ligands by their ability to alter cell amplification, and

(e) identifying which receptor is activated by the ligand.

40. A method according to claim 39 in which the receptor activated by the ligand is identified by identifying which receptor is activated by the ligand by assaying the receptor DNA and/or mRNA by PEA amplification and/or hybridization techniques.

41. A method according to claim 39 in which the receptor activated by the ligand is identified by identifying which receptor is activated by the ligand by adding a substrate for each individual marker enzyme followed by assay.