Methods for identifying modulators of apoptosis

• US 20030175819A1
• Filed: 11/27/2002
• Published: 09/18/2003
• Est. Priority Date: 11/28/2001
• Status: Active Grant

• Current Assignee: Burnham Institute For Medical Research
• Original Assignee: Burnham Institute For Medical Research
• Inventors: Reed, John C., Guo, Bin
• Granted Patent: US 7,422,862 B2
• Time in Patent Office: Days
• US Class Current: 435/7.2
• CPC Class Codes: G01N 2510/00 Detection of programmed cell death, i.e. apoptosis
• 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 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5079 Mitochondria
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• 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

Abstract

The invention provides a method of identifying an effective compound that modulates the binding of Humanin to Bax or Bid. The invention also provides a method of identifying an effective compound that modulates an activity of Bax or Bid. In addition, the invention provides a method of identifying a Humanin-like compound that binds to Bax or Bid or modulates an activity of Bax or Bid, or inhibits the apoptotic activity of Bax or Bid. The invention further provides an isolated polypeptide containing a mitochondrial-derived form of Humanin (SEQ ID NO:3) or a functional fragment thereof where the fragment contains the methionine at position 16 of SEQ ID NO:3.

Claims

1. A method of identifying an effective compound that modulates the binding of Humanin to Bax, comprising the steps of:

(a) contacting said Humanin with said Bax under conditions suitable to form a Humanin-Bax complex;

(b) contacting said Humanin-Bax complex with a candidate compound; and

(c) determining the ability of said candidate compound to modulate the binding of said Humanin to said Bax, wherein modulation of the binding of said Humanin to said Bax indicates that said candidate compound is an effective compound that modulates the binding of said Humanin to said Bax.

2. The method of claim 1, wherein said contacting is performed in vitro.

3. The method of claim 1, wherein said contacting is performed in vivo.

4. The method of claim 1, wherein said effective compound increases the binding of said Humanin to said Bax.

5. The method of claim 1, wherein said effective compound decreases the binding of said Humanin to said Bax.

6. The method of claim 1, wherein said candidate compound is selected from the group consisting of:

a polypeptide, peptidomimetic, non-peptidyl compound, carbohydrate, lipid, a synthetic compound, a natural product, an antibody or antibody fragment, a small organic molecules, a small inorganic molecule, and a nucleotide sequence.

7. The method of claim 1, wherein said candidate compound is a small organic molecule.

8. The method of claim 1, wherein said candidate compound is a polypeptide.

9. The method of claim 1, wherein said candidate compound is a Humanin-like compound.

10. The method of claim 1, wherein said binding is determined using a method selected from the group consisting of:

a two-hybrid assay, co-immunoprecipitation assay, co-localization assay, scintillation proximity assay (SPA), UV or chemical cross-linking, biomolecular interaction analysis (BIA), mass spectrometry (MS), nuclear magnetic resonance (NMR), and fluorescence polarization assays (FPA).

11. The method of claim 10, wherein said binding is determined using a yeast two-hybrid assay.

12. A method for identifying a Humanin-like compound that binds to Bax, comprising the steps of:

(a) contacting said Humanin-like compound with said Bax, under conditions suitable to form a complex; and

(b) determining the ability of said Humanin-like compound to bind said Bax.

13. The method of claim 12, wherein said contacting is performed in vitro.

14. The method of claim 12, wherein said contacting is performed in vivo.

15. The method of claim 12, wherein said ability to bind said Bax is determined using a method selected from the group consisting of:

a two-hybrid assay, co-immunoprecipitation assay, co-localization assay, scintillation proximity assay (SPA), UV or chemical cross-linking, biomolecular interaction analysis (BIA), mass spectrometry (MS), nuclear magnetic resonance (NMR), and fluorescence polarization assays (FPA).

16. The method of claim 15, wherein said ability to bind said Bax is determined using a yeast two-hybrid assay.

17. A method for identifying a Humanin-like compound that inhibits the apoptotic activity of Bax, comprising the steps of:

(a) expressing Bax in a cell, wherein said expression results in the death of said cell;

(b) exposing the cell to a Humanin-like compound; and

(c) detecting the survival of said cell, wherein survival of said cell indicates that said Humanin-like compound binds to said Bax.

18. The method of claim 17, wherein said cell is a yeast cell.

19. A method for identifying a Humanin-like compound that binds to Bax, comprising the steps of:

a) contacting Humanin with said Bax under conditions suitable to form a Humanin-Bax complex;

(b) contacting said Humanin-Bax complex with said Humanin-like compound; and

(c) determining the ability of said Humanin-like compound to bind to said Bax.

20. The method of claim 19, wherein said contacting is performed in vitro.

21. The method of claim 19, wherein said binding of said Humanin-like compound to said Bax is determined using a method selected from the group consisting of:

a two-hybrid assay, co-immunoprecipitation assay, co-localization assay, scintillation proximity assay (SPA), UV or chemical cross-linking, biomolecular interaction analysis (BIA), mass spectrometry (MS), nuclear magnetic resonance (NMR), and fluorescence polarization assays (FPA).

22. The method of claim 19, wherein said Humanin is labeled.

23. A method for identifying a Humanin-like compound that modulates an activity of Bax, comprising the steps of:

(a) measuring an activity of said Bax;

(b) contacting said Bax with said Humanin-like compound under conditions suitable to form a Humanin-like compound/Bax complex;

(c) determining the amount of activity of said Bax when bound to said Humanin-like compound and

(d) comparing the amount of activity of from step (a) with the amount of activity from step (c) wherein a change in the activity of Bax indicates that said Humain-like compound modulates an activity of Bax.

24. The method of claim 23, wherein the activity of Bax is increased by said Humanin-like compound.

25. The method of claim 23, wherein the activity of Bax is decreased by said Humanin-like compound.

26. The method of claim 23, wherein said contacting is performed in vitro.

27. The method of claim 23, wherein said contacting is performed in vivo.

28. The method of claim 23, wherein the activity of Bax is selected from the group consisting of:

induction of cell death, ability to form a pore, ability to form dimers with Bcl-2 related polypeptides, ability to homodimerize or self-associate, ability to integrate into membranes, ability to translocate from cytosol to mitochondria upon activation, and ability to activate a caspase.

29. A method of identifying an effective compound that modulates an activity of Bax, comprising the steps of:

(a) contacting Humanin with said Bax under conditions suitable to form a Humanin-Bax complex;

(b) measuring an activity of said Bax;

(c) contacting said Humanin-Bax complex with a candidate compound;

(d) determining the amount of activity of said Bax in the presence of said candidate compound; and

(e) comparing the amount of activity from step (b) with the amount of activity from step (d), wherein modulation of an activity of Bax indicates that said candidate compound is an effective compound that modulates an activity of Bax.

30. The method of claim 29, wherein said activity of Bax is increased by said effective compound.

31. The method of claim 29, wherein said activity of Bax is decreased by said effective compound.

32. The method of claim 29, wherein said activity of said Bax is selected from the group consisting of:

induction of cell death, ability to form a pore, ability to form dimers with Bcl-2 related polypeptides, ability to homodimerize or self-associate, ability to integrate into membranes, ability to translocate from cytosol to mitochondria upon activation, and ability to activate a caspase.

33. The method of claim 29, wherein said candidate compound is selected from the group consisting of:

a polypeptide, peptidomimetic, non-peptidyl compound, carbohydrate, lipid, a synthetic compound, a natural product, an antibody or antibody fragment, a small organic molecules, a small inorganic molecule, and a nucleotide sequence.

34. The method of claim 29, wherein said effective compound is a small organic molecule.

35. The method of claim 29, wherein said effective compound is a polypeptide.

36. The method of claim 29, wherein said contacting is performed in vitro.

37. The method of claim 29, wherein said contacting is performed in vivo.

38. A method of identifying an effective compound that modulates the binding of Humanin to Bid, comprising the steps of:

(a) contacting said Humanin with said Bid under conditions suitable to form a Humanin-Bid complex;

(b) contacting said Humanin-Bid complex with a candidate compound; and

(c) determining the ability of said candidate compound to modulate the binding of said Humanin to said Bid, wherein modulation of the binding of said Humanin to said Bid indicates that said candidate compound is an effective compound that modulates the binding of said Humanin to said Bid.

39. The method of claim 38, wherein said contacting is performed in vitro.

40. The method of claim 38, wherein said contacting is performed in vivo.

41. The method of claim 38, wherein said effective compound increases the binding of said Humanin to said Bid.

42. The method of claim 38, wherein said effective compound decreases the binding of said Humanin to said Bid.

43. The method of claim 38, wherein said candidate compound is selected from the group consisting of:

a polypeptide, peptidomimetic, non-peptidyl compound, carbohydrate, lipid, a synthetic compound, a natural product, an antibody or antibody fragment, a small organic molecules, a small inorganic molecule, and a nucleotide sequence.

44. The method of claim 38, wherein said candidate compound is a small organic molecule.

45. The method of claim 38, wherein said candidate compound is a polypeptide.

46. The method of claim 38, wherein said candidate compound is a Humanin-like compound.

47. The method of claim 38, wherein said binding is determined using a method selected from the group consisting of:

a two-hybrid assay, co-immunoprecipitation assay, co-localization assay, scintillation proximity assay (SPA), UV or chemical cross-linking, biomolecular interaction analysis (BIA), mass spectrometry (MS), nuclear magnetic resonance (NMR), and fluorescence polarization assays (FPA).

48. The method of claim 47, wherein said binding is determined using a yeast two-hybrid assay.

49. A method for identifying a Humanin-like compound that binds to Bid, comprising the steps of:

(a) contacting said Humanin-like compound with said Bid, under conditions suitable to form a complex; and

(b) determining the ability of said Humanin-like compound to bind said Bid.

50. The method of claim 49, wherein said contacting is performed in vitro.

51. The method of claim 49, wherein said contacting is performed in vivo.

52. The method of claim 49, wherein said ability to bind said Bid is determined using a method selected from the group consisting of:

a two-hybrid assay, co-immunoprecipitation assay, co-localization assay, scintillation proximity assay (SPA), UV or chemical cross-linking, biomolecular interaction analysis (BIA), mass spectrometry (MS), nuclear magnetic resonance (NMR), and fluorescence polarization assays (FPA).

53. The method of claim 52, wherein said ability to bind said Bid is determined using a yeast two-hybrid assay.

54. A method for identifying a Humanin-like compound that inhibits the apoptotic activity of Bid, comprising the steps of:

(a) expressing Bid in a cell, wherein said expression results in the death of said cell;

(b) exposing the cell to a Humanin-like compound; and

(c) detecting the survival of said cell, wherein survival of said cell indicates that said Humanin-like compound binds to said Bid.

55. The method of claim 54, wherein said cell is a yeast cell.

56. A method for identifying a Humanin-like compound that binds to Bid, comprising the steps of:

(a) contacting Humanin with said Bid under conditions suitable to form a Humanin-Bid complex;

(b) contacting said Humanin-Bid complex with said Humanin-like compound; and

(c) determining the ability of said Humanin-like compound to bind to said Bid.

57. The method of claim 56, wherein said contacting is performed in vitro.

58. The method of claim 56, wherein said binding of said Humanin-like compound to said Bid is determined using a method selected from the group consisting of:

a two-hybrid assay, co-immunoprecipitation assay, co-localization assay, scintillation proximity assay (SPA), UV or chemical cross-linking, biomolecular interaction analysis (BIA), mass spectrometry (MS), nuclear magnetic resonance (NMR), and fluorescence polarization assays (FPA).

59. The method of claim 56, wherein said Humanin is labeled.

60. A method for identifying a Humanin-like compound that modulates an activity of Bid, comprising the steps of:

(a) measuring an activity of said Bid;

(b) contacting said Bid with said Humanin-like compound under conditions suitable to form a Humanin-like compound/Bid complex;

(c) determining the amount of activity of said Bid when bound to said Humanin-like compound and

(d) comparing the amount of activity of from step (a) with the amount of activity from step (c) wherein a change in the activity of Bid indicates that said Humain-like compound modulates an activity of Bid.

61. The method of claim 60, wherein the activity of Bid is increased by said Humanin-like compound.

62. The method of claim 60, wherein the activity of Bid is decreased by said Humanin-like compound.

63. The method of claim 60, wherein said contacting is performed in vitro.

64. The method of claim 60, wherein said contacting is performed in vivo.

65. The method of claim 60, wherein the activity of Bid is selected from the group consisting of:

induction of cell death, ability to form a pore, ability to form dimers with Bcl-2 related polypeptides, ability to trigger Bax activation, ability to integrate into membranes, ability to translocate from cytosol to mitochondria upon activation, and ability to activate a caspase.

66. A method of identifying an effective compound that modulates an activity of Bid, comprising the steps of:

(a) contacting Humanin with said Bid under conditions suitable to form a Humanin-Bid complex;

(b) measuring an activity of said Bid;

(c) contacting said Humanin-Bid complex with a candidate compound;

(d) determining the amount of activity of said Bid in the presence of said candidate compound; and

(e) comparing the amount of activity from step (b) with the amount of activity from step (d), wherein modulation of an activity of Bid indicates that said candidate compound is an effective compound that modulates an activity of Bid.

67. The method of claim 66, wherein said activity of Bid is increased by said effective compound.

68. The method of claim 66, wherein said activity of Bid is decreased by said effective compound.

69. The method of claim 66, wherein said activity of said Bid is selected from the group consisting of:

induction of cell death, ability to form a pore, ability to form dimers with Bcl-2 related polypeptides, ability to trigger Bax activation, ability to integrate into membranes, ability to translocate from cytosol to mitochondria upon activation, and ability to activate a caspase.

70. The method of claim 66, wherein said candidate compound is selected from the group consisting of:

a polypeptide, peptidomimetic, non-peptidyl compound, carbohydrate, lipid, a synthetic compound, a natural product, an antibody or antibody fragment, a small organic molecules, a small inorganic molecule, and a nucleotide sequence.

71. The method of claim 66, wherein said effective compound is a small organic molecule.

72. The method of claim 66, wherein said effective compound is a polypeptide.

73. The method of claim 66, wherein said contacting is performed in vitro.

74. The method of claim 66, wherein said contacting is performed in vivo.

75. An isolated polypeptide comprising the amino acid sequence designated as SEQ ID NO:

3, or a functional fragment thereof, wherein said fragment comprises the methionine at position 16 of SEQ ID NO;

3.

76. The isolated polypeptide of claim 75, wherein one or more non-natural amino acids have been substituted for one or more amino acids.

77. The isolated polypeptide of claim 75, wherein said isolated polypeptide comprises a targeting molecule.