Identification of ligands by selective amplification of cells transfected with receptors

• US 20050244862A1
• Filed: 03/16/2005
• Published: 11/03/2005
• Est. Priority Date: 10/07/1999
• Status: Active Grant

• Current Assignee: Mark R. Brann
• Original Assignee: Mark R. Brann
• Inventors: Brann, Mark R.
• Granted Patent: US 7,425,420 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 receptor which increases or decreases the rate of cellular proliferation in response to a ligand, said method comprising:

obtaining a test cell culture which comprises a first population of cells and a second population of cells, wherein DNA coding for said receptor and also DNA encoding a marker indicative of the extent of cellular proliferation has been introduced into said first population of cells such that the amount of said marker produced by said first population of cells is influenced by the degree to which said receptor is active, and wherein the amount of marker produced by said second population of cells is not influenced by the degree to which said receptor is active or wherein said second population of cells does not comprise DNA encoding said marker;

contacting said test cell culture with a test substance which is a potential ligand of said receptor;

obtaining a control cell culture, said control cell culture comprising cells comprising said DNA coding for said receptor and said DNA encoding said marker, wherein said control cell culture has not been contacted with said test substance;

determining whether the extent of proliferation of said first population of cells in said test cell culture is significantly greater or significantly less than the extent of proliferation of said cells in said control cell culture by comparing the amount of marker activity produced by said first population of cells in said test culture to the amount of marker activity produced by said cells in said control culture, wherein said test substance is an agonist of said receptor if the extent of proliferation of said first population of cells in said test culture is significantly greater than the extent of proliferation of said cells in said control culture and wherein said test substance is an antagonist or inverse agonist of said receptor if the extent of proliferation of said first population of cells in said test culture is significantly less than the extent of proliferation of said cells in said control culture.

2. The method of claim 1, wherein said determining step is performed by measuring the amount of said marker produced by said test culture at a time after contacting said test cell culture 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 control cell culture at the same time as the amount of said marker in said test culture was measured.

3. The method of claim 1, wherein said determining step is performed within two weeks of the day that said test cell culture was contacted with said substance.

4. The method of claim 1, wherein said determining step is performed within 4 days of the day that said test cell culture was contacted with said substance.

5. The method of claim 1, further comprising contacting said test cell culture and said control cell culture with a substance known to be an agonist of said receptor.

6. The method of claim 1, further comprising contacting said test cell culture and said cell control culture with a substance known to be an antagonist of said receptor.

7. The method of claim 1, wherein said test cell culture and said control cell culture are obtained by introducing said DNA encoding said 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 receptor is the 5-HT2A receptor.

9. The method of claim 1, wherein said receptor has an elevated basal or constitutive receptor response and wherein said determining step comprises determining whether said substance inhibits said elevated basal or constitutive receptor response, wherein said substance is an inverse agonist of said receptor if said substance inhibits said elevated or constitutive receptor response of said receptor.

10. The method of claim 1, wherein said second population of cells 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).

11. A method of determining whether a substance is a ligand of a receptor which increases or decreases the rate of cellular proliferation in response to a ligand, said method comprising,

obtaining a test cell culture which comprises a first population of cells and a second population of cells, wherein DNA coding for said receptor and also DNA encoding a marker indicative of the extent of cellular proliferation has been introduced into said first population of cells such that the amount of said marker produced by said first population of cells is influenced by the degree to which said receptor is active, and wherein the amount of said marker produced by said second population of cells is not influenced by the degree to which said receptor is active or wherein said second population of cells does not comprise DNA encoding said marker;

contacting said test cell culture with a test substance which is a potential ligand of said receptor; and

determining whether the test substance confers a competitive advantage or a competitive disadvantage on the cells in said first population of said test culture relative to the cells in said second population of said test culture by measuring the amount of said marker produced by said test culture.

12. The method of claim 11, wherein said determining step is performed by measuring the amount of said marker produced by said test culture at a time after contacting said test cell culture 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.

13. The method of claim 11, wherein said determining step is performed within two weeks of the day that said test cell culture was contacted with said substance.

14. The method of claim 11, wherein said determining step is performed within 4 days of the day that said test cell culture was contacted with said substance.

15. The method of claim 11, further comprising contacting said test cell culture with a substance known to be an agonist of said receptor.

16. The method of claim 11, further comprising contacting said test cell culture with a substance known to be an antagonist of said receptor.

17. The method of claim 11, wherein said receptor is the 5-HT2A receptor.

18. The method of claim 11, wherein said receptor has an elevated basal or constitutive receptor response and wherein said determining step comprises determining whether said substance inhibits said elevated basal or constitutive receptor response, wherein said substance is an inverse agonist of said receptor if said substance inhibits said elevated or constitutive receptor response of said receptor.

19. The method of claim 11, wherein said second population of cells 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).

20. A method of determining whether a substance is a ligand of a receptor which increases or decreases the rate of cellular proliferation in response to a ligand, said method comprising:

obtaining a test cell culture which comprises a first population of cells and a second population of cells, wherein DNA coding for said receptor and also DNA encoding a marker indicative of the extent of cellular proliferation has been introduced into said first population of cells such that the amount of said marker produced by said first population of cells is influenced by the degree to which said receptor is active, wherein the transcription of said DNA encoding said marker is not under the control of said receptor, and wherein the amount of marker in said second population of cells is not influenced by the degree to which said receptor is active or wherein said second population of cells does not comprise said DNA encoding said marker;

contacting said test cell culture with a test substance which is a potential ligand of said receptor;

obtaining a control cell culture, said control cell culture comprising cells comprising said DNA coding for said receptor and said DNA encoding said marker, wherein said control cell culture has not been contacted with said test substance;

determining whether the extent of proliferation of said first population of cells in said test cell culture is significantly greater or significantly less than the extent of proliferation of said cells in said control cell culture by comparing the amount of marker activity produced by said first population of cells in said test culture to the amount of marker activity produced by said cells in said control culture, wherein said test substance is an agonist of said receptor if the extent of proliferation of said first population of cells in said test culture is significantly greater than the extent of proliferation of said cells in said control culture and wherein said test substance is an antagonist or inverse agonist of said receptor if the extent of proliferation of said first population of cells in said test culture is significantly less than the extent of proliferation of said cells in said control culture.

21. The method of claim 20, wherein said receptor is the 5-HT2A receptor.

22. The method of claim 20, wherein said receptor has an elevated basal or constitutive receptor response and wherein said determining step comprises determining whether said substance inhibits said elevated basal or constitutive receptor response, wherein said substance is an inverse agonist of said receptor if said substance inhibits said elevated or constitutive receptor response of said receptor.

23. The method of claim 20, wherein said second population of cells 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).

24. A cell culture comprising:

a first population of cells into which DNA encoding a receptor has been introduced and into which DNA encoding a marker whose amount corresponds to the number of cells in said first population of cells has been introduced such that the amount of said marker produced by said first population of cells is influenced by the degree to which said receptor is active, wherein the transcription of said DNA encoding said marker is not under the control of said receptor 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;

a second population of cells in which the amount of marker is not influenced by the degree to which said receptor is active or into which DNA encoding said marker has not been introduced; and

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

25. The cell culture of claim 24, wherein said receptor is the 5-HT2A receptor.

26. The cell culture of claim 24, wherein said receptor has an elevated basal or constitutive receptor response.

27. The cell culture of claim 24, wherein said second population of cells 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).

28. A method of determining whether a substance is a ligand of a receptor which increases or decreases the rate of cellular proliferation in response to a ligand, said method comprising:

obtaining a test cell culture which comprises a first population of cells and a second population of cells, wherein DNA coding for a plurality of receptors and also DNA encoding a marker indicative of the extent of cellular proliferation has been introduced into said first population of cells such that the amount of said marker produced by said first population of cells is influenced by the degree to which said plurality of receptors are active, and wherein the amount of said marker produced by said second population of cells is not influenced by the degree to which said plurality of receptors are active or wherein said second population of cells does not comprise said DNA encoding said marker;

contacting said test cell culture with a test substance which is a potential ligand of at least one of said plurality of receptors;

obtaining a control cell culture, said control cell culture comprising cells comprising said DNA coding for said plurality of receptors and said DNA encoding said marker, wherein said control cell culture has not been contacted with said test substance;

determining whether the extent of proliferation of cells in said first population of cells in said test cell culture is significantly greater or significantly less than the extent of proliferation of said cells in said control cell culture by comparing the amount of marker activity produced by said first population of cells in said test culture to the amount of marker activity produced by said cells in said control culture, wherein said test substance is an agonist of at least one of said plurality of receptors if the extent of proliferation of some cells in said first population of cells in said test culture is significantly greater than the extent of proliferation of said cells in said control culture and wherein said test substance is an antagonist or inverse agonist of said receptor if the extent of proliferation of cells in said first population of cells in said test culture is significantly less than the extent of proliferation of said cells in said control culture.

29. The method of claim 28, wherein DNA encoding a plurality of markers has been introduced into said cells comprising said DNA encoding said plurality of receptors such that cells comprising DNA encoding a given receptor comprise DNA encoding a different marker than cells comprising DNA encoding the other receptors.

30. The method of claim 28, wherein said determining step is performed by measuring the amount of said marker produced by said test culture at a time after contacting said test cell culture 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 control cell culture at the same time as the amount of said marker in said test culture was measured.

31. The method of claim 28, wherein said determining step is performed within two weeks of the day that said test cell culture was contacted with said substance.

32. The method of claim 28, wherein said determining step is performed within 4 days of the day that said test cell culture was contacted with said substance.

33. The method of claim 28, further comprising contacting said test cell culture and said control cell culture with a substance known to be an agonist of at least one of said plurality of receptors.

34. The method of claim 28, further comprising contacting said test cell culture and said cell control culture with a substance known to be an antagonist of at least one of said plurality of receptors.

35. The method of claim 28, wherein said test cell culture and said control cell culture are obtained by introducing DNA encoding said plurality of receptors and DNA encoding said marker into a starting cell culture and dividing said starting cell culture into multiple aliquots.

36. The method of claim 28, wherein one of said plurality of receptors is the 5-HT2A receptor.

37. The method of claim 28, wherein at least one of said plurality of receptors has an elevated basal or constitutive receptor response and wherein said determining step comprises determining whether said substance inhibits said elevated basal or constitutive receptor response of said at least one of said plurality of receptors, wherein said substance is an inverse agonist of said at least one of said plurality of receptors if said substance inhibits said elevated or constitutive receptor response of said at least one of said plurality of receptors.

38. The method of claim 28, wherein said second population of cells 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 any of said plurality of receptors;

(c) cells which comprise DNA encoding at least one of said plurality of receptors but not DNA encoding said marker;

(d) cells which comprise DNA encoding said marker but not DNA encoding any of said plurality of receptors; and

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

40. The method of claim 38, wherein DNA encoding a plurality of markers has been introduced into said cells comprising said DNA encoding said plurality of receptors such that cells comprising DNA encoding a given receptor comprise DNA encoding a different marker than cells comprising DNA encoding the other receptors.

41. The method of claim 38, wherein said determining step is performed by measuring the amount of said marker produced by said test culture at a time after contacting said test cell culture 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.

42. The method of claim 38, wherein said determining step is performed within two weeks of the day that said test cell culture was contacted with said substance.

43. The method of claim 38, wherein said determining step is performed within 4 days of the day that said test cell culture was contacted with said substance.

44. The method of claim 38, further comprising contacting said test cell culture with a substance known to be an agonist of at least one of said plurality of receptors.

45. The method of claim 38, further comprising contacting said test cell culture with a substance known to be an antagonist of at least one of said plurality of receptors.

46. The method of claim 38, wherein one of said plurality of receptors is the 5-HT2A receptor.

47. The method of claim 38, wherein at least one of said plurality of receptors has an elevated basal or constitutive receptor response and wherein said determining step comprises determining whether said substance inhibits said elevated basal or constitutive receptor response of said at least one of said plurality of receptors, wherein said substance is an inverse agonist of said at least one of said plurality of receptors if said substance inhibits said elevated or constitutive receptor response of said at least one of said plurality of receptors.

48. The method of claim 38, wherein said second population of cells 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 any of said plurality of receptors;

(c) cells which comprise DNA encoding at least one of said plurality of receptors but not DNA encoding said marker;

(d) cells which comprise DNA encoding said marker but not DNA encoding any of said plurality of receptors; and

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

39. A method of determining whether a substance is a ligand of a receptor which increases or decreases the rate of cellular proliferation in response to a ligand, said method comprising,

obtaining a test cell culture which comprises a first population of cells and a second population of cells, wherein DNA coding for a plurality of receptors and also DNA encoding a marker indicative of the extent of cellular proliferation has been introduced into said first population of cells such that the amount of said marker produced by said first population of cells is influenced by the degree to which said plurality of receptors are active, and wherein the amount of said marker in said said second population of cells is not influenced by the degree to which said plurality of receptors are active or wherein said second population of cells does not comprise said DNA encoding said marker;

contacting said test cell culture with a test substance which is a potential ligand of at least one of said plurality of receptors; and

determining whether the test substance confers a competitive advantage or a competitive disadvantage on cells in said first population of said test culture which express at least one of said plurality of receptors relative to the cells in said second population of said test culture by measuring the amount of said marker produced by said test culture.

49. A method of determining whether a substance is a ligand of a receptor which increases or decreases the rate of cellular proliferation in response to a ligand, said method comprising:

obtaining a test cell culture which comprises a first population of cells and a second population of cells, wherein DNA coding for a plurality of receptors and also DNA encoding a marker indicative of the extent of cellular proliferation has been introduced into said first population of cells such that the amount of said marker produced by said first population of cells is influenced by the degree to which said plurality of receptors are active, wherein the transcription of said DNA encoding said marker is not under the control of any of said plurality of receptors, and wherein the amount of said marker produced by said second population of cells is not influenced by the degree to which said plurality of receptors are active or wherein said second population of cells does not comprise said DNA encoding said marker;

contacting said test cell culture with a test substance which is a potential ligand of at least one of said plurality of receptors;

obtaining a control cell culture, said control cell culture comprising cells comprising said DNA coding for said plurality of receptors and said DNA encoding said marker, wherein said control cell culture has not been contacted with said test substance;

determining whether the extent of proliferation of said first population of cells in said test cell culture is significantly greater or significantly less than the extent of proliferation of said cells in said control cell culture by comparing the amount of marker activity produced by said first population of cells in said test culture to the amount of marker activity produced by said cells in said control culture, wherein said test substance is an agonist of at least one of said plurality of receptors if the extent of proliferation of said first population of cells in said test culture is significantly greater than the extent of proliferation of said cells in said control culture and wherein said test substance is an antagonist or inverse agonist of at least one of said plurality of receptors if the extent of proliferation of said first population of cells in said test culture is significantly less than the extent of proliferation of said cells in said control culture.

50. The method of claim 49, wherein one of said plurality of receptors is the 5-HT2A receptor.

51. The method of claim 49, wherein at least one of said plurality of receptors has an elevated basal or constitutive receptor response and wherein said determining step comprises determining whether said substance inhibits said elevated basal or constitutive receptor response of said at least one of said plurality of receptors, wherein said substance is an inverse agonist of said at least one of said plurality of receptors if said substance inhibits said elevated or constitutive receptor response of said at least one of said plurality of receptors.

52. The method of claim 49, wherein said second population of cells 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 any of said plurality of receptors;

(c) cells which comprise DNA encoding at least one of said plurality of receptors but not DNA encoding said marker;

(d) cells which comprise DNA encoding said marker but not DNA encoding any of said plurality of receptors; and

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

53. A cell culture comprising:

a first population of cells into which a DNA encoding plurality of receptors has been introduced and into which DNA encoding a marker whose amount corresponds to the number of cells in said first population of cells has been introduced such that the amount of said marker produced by said first population of cells is influenced by the degree to which said plurality of receptors are acive, wherein the transcription of said DNA encoding said marker is not under the control of said receptor 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;

a second population of cells in which the amount of said marker is not influenced by the degree to which said plurality of receptors are active or into which DNA encoding said marker has not been introduced; and

a substance being evaluated to determine whether it is a ligand of at least one of said plurality of receptors.

54. The cell culture of claim 53, wherein one of said plurality of receptors is the 5-HT2A receptor.

55. The cell culture of claim 53, wherein at least one of said plurality of receptors has an elevated basal or constitutive receptor response.

56. The cell culture of claim 53, wherein said second population of cells 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 any of said plurality of receptors;

(c) cells which comprise DNA encoding at least one of said plurality of receptors but not DNA encoding said marker;

(d) cells which comprise DNA encoding said marker but not DNA encoding any of said plurality of receptors; and

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

57. A cell culture comprising:

a first population of cells into which a DNA encoding plurality of receptors has been introduced and into which DNA encoding a plurality of markers whose amount corresponds to the number of cells in said first population of cells has been introduced such that the amount of said marker produced by said first population of cells is influenced by the degree to which said plurality of receptors are active, wherein the transcription of said DNA encoding said plurality of markers is not under the control of any of said plurality of receptors 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;

a second population of cells in which the amount of said marker is not influenced by the degree to which said plurality of receptors are active or into which DNA encoding said marker has not been introduced; and

a substance being evaluated to determine whether it is a ligand of at least one of said plurality of receptors.

58. The cell culture of claim 57, wherein one of said plurality of receptors is the 5-HT2A receptor.

59. The cell culture of claim 57, wherein at least one of said plurality of receptors has an elevated basal or constitutive receptor response.

60. The cell culture of claim 57, wherein said second population of cells 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 any of said plurality of receptors;

(c) cells which comprise DNA encoding at least one of said plurality of receptors but not DNA encoding said marker;

(d) cells which comprise DNA encoding said marker but not DNA encoding any of said plurality of receptors; and

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