Novel death associated proteins, and THAP1 and PAR4 pathways in apoptosis control

• US 20030186337A1
• Filed: 12/10/2002
• Published: 10/02/2003
• Est. Priority Date: 12/18/2001
• Status: Active Grant

• Current Assignee: Centre National De La Recherche Scientifique, Endocube S.A.S.
• Original Assignee: Centre National De La Recherche Scientifique, Endocube S.A.S.
• Inventors: Roussigne, Myriam, Amalric, Francois, Girard, Jean-Philippe, Clouaire, Thomas, Kossida, Sophia
• Granted Patent: US 7,572,886 B2
• Time in Patent Office: Days
• US Class Current: 435/7.23
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 15/12 for climacteric disorders
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/06 : Antianaemics
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/4747 : Apoptosis related proteins
• CPC Class Codes: C07K 2319/30 : Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The invention relates to genes and proteins of the THAP (THanatos (death)-Associated Protein) family comprising a THAP domain, and their use in diagnostics, treatment of disease, and in the identification of molecules for the treatment of disease. The invention also relates to the Par4 protein and SLC chemokine pathways, including the interaction of Par4 and SLC with THAP family proteins, and the recruitment and localization of Par4 to PML nuclear bodies.

Claims

1. A method of identifying a test compound that modulates THAP-mediated activities comprising:

contacting a THAP-family polypeptide or a biologically active fragment thereof with a test compound, wherein said THAP-family polypeptide comprises an amino acid sequence having at least 30% amino acid identity to an amino acid sequence of SEQ ID NO;

1; and

determining whether said test compound selectively modulates the activity of said THAP-family polypeptide or biologically active fragment thereof, wherein a determination that said test compound selectively modulates the activity of said polypeptide indicates that said test compound is a candidate modulator of THAP-mediated activities.

2. The method of claim 1, wherein the THAP-family polypeptide comprises the amino acid sequence of SEQ ID NO:

1, or a biologically active fragment thereof.

3. The method of claim 1, wherein the THAP-family polypeptide comprises the amino acid sequence of SEQ ID NO:

2, or a biologically active fragment thereof.

4. The method of claim 1, wherein the THAP-family polypeptide comprises the amino acid sequence of SEQ ID NO:

3, or a biologically active fragment thereof.

5. The method of claim 1, wherein the THAP-family polypeptide comprises the amino acid sequence of SEQ ID NO:

4, or a biologically active fragment thereof.

6. The method of claim 1, wherein the THAP-family polypeptide comprises the amino acid sequence of SEQ ID NO:

5, or a biologically active fragment thereof.

7. The method of claim 1, wherein the THAP-family polypeptide comprises the amino acid sequence of SEQ ID NO:

6, or a biologically active fragment thereof.

8. The method of claim 1, wherein the THAP-family polypeptide comprises the amino acid sequence of SEQ ID NO:

7, or a biologically active fragment thereof.

9. The method of claim 1, wherein the THAP-family polypeptide comprises the amino acid sequence of SEQ ID NO:

8, or a biologically active fragment thereof.

10. The method of claim 1, wherein the THAP-family polypeptide comprises the amino acid sequence of SEQ ID NO:

9, or a biologically active fragment thereof.

11. The method of claim 1, wherein the THAP-family polypeptide comprises the amino acid sequence of SEQ ID NO:

10, or a biologically active fragment thereof.

12. The method of claim 1, wherein the THAP-family polypeptide comprises the amino acid sequence of SEQ ID NO:

11, or a biologically active fragment thereof.

13. The method of claim 1, wherein the THAP-family polypeptide comprises the amino acid sequence of SEQ ID NO:

12, or a biologically active fragment thereof.

14. The method of claim 1, wherein the THAP-family polypeptide comprises the amino acid sequence of SEQ ID NO:

13, or a biologically active fragment thereof.

15. The method of claim 1, wherein the THAP-family polypeptide comprises the amino acid sequence of SEQ ID NO:

14 or a biologically active fragments thereof.

16. The method of claim 1, wherein the THAP-family polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs:

15-114 or a biologically active fragments thereof.

17. The method of claim 1, wherein said THAP-mediated activity is selected from the group consisting of interaction with a THAP-family target protein, binding to a nucleic acid, binding to PAR-4, binding to SLC, binding to PML, binding to a polypeptide found in PML-NBs, localization to PML-NBs, targeting a THAP-family target protein to PML-NBs, and inducing apoptosis

18. The method of claim 17, wherein said THAP-mediated activity is binding to PAR-4.

19. The method of claim 17, wherein said THAP-mediated activity is binding to SLC.

20. The method of claim 17, wherein said THAP-mediated activity is inducing apoptosis.

21. The method of claim 17, wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:

140-159.

22. The method of claim 1, wherein said amino acid identity is determined using an algorithm selected from the group consisting of XBLAST with the parameters, score=50 and wordlength=3, Gapped BLAST with the default parameters of XBLAST, and BLAST with the defaul parameters of XBLAST.

23. An isolated or purified THAP domain polypeptide consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs:

1-2, amino acids 1-89 of SEQ ID NOs;

3-5, amino acids 1-90 of SEQ ID NOs;

6-9, amino acids 1-92 of SEQ ID NO;

10, amino acids 1-90 of SEQ ID NOs;

11-14 and homologs having at least 30% amino acid identity to any aforementioned sequence, wherein said polypeptide binds to a nucleic acid.

24. The isolated or purified THAP domain polypeptide of claim 23 consisting essentially of SEQ ID NO:

1.

25. The isolated or purified THAP domain polypeptide of claim 23, wherein said amino acid identity is determined using an algorithm selected from the group consisting of XBLAST with the parameters, score=50 and wordlength=3, Gapped BLAST with the default parameters of XBLAST, and BLAST with the defaul parameters of XBLAST.

26. The isolated or purified THAP domain polypeptide of claim 23, wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:

140-159.

27. An isolated or purified nucleic acid which encodes the THAP domain polypeptide of claim 23 or a complement thereof.

28. An isolated or purified PAR4-binding domain polypeptide consisting essentially of an amino acid sequence selected from the group consisting of amino acids 143-192 of SEQ ID NO:

3, amino acids 132-181 of SEQ ID NO;

4, amino acids 186-234 of SEQ ID NO;

5, SEQ ID NO;

15 and homologs having at least 30% amino acid identity to any aforementioned sequence, wherein said polypeptide binds to PAR4.

29. The isolated or purified PAR4-binding domain of claim 28 consisting essentially of SEQ ID NO:

15.

30. The isolated or purified PAR4-binding domain of claim 28 consisting essentially of amino acids 143-193 of SEQ ID NO:

3.

31. The isolated or purified PAR4-binding domain of claim 28 consisting essentially of amino acids 132-181 of SEQ ID NO:

4.

32. The isolated or purified PAR4-binding domain of claim 28 consisting essentially of amino acids 186-234 of SEQ ID NO:

5.

33. The isolated or purified PAR4-binding domain polypeptide of claim 28, wherein said amino acid identity is determined using an algorithm selected from the group consisting of XBLAST with the parameters, score=50 and wordlength=3, Gapped BLAST with the default parameters of XBLAST, and BLAST with the defaul parameters of XBLAST.

34. An isolated or purified nucleic acid which encodes the PAR4-binding domain polypeptide of claim 28 or a complement thereof.

35. An isolated or purified SLC-binding domain polypeptide consisting essentially of an amino acid sequence selected from the group consisting of amino acids 143-213 of SEQ ID NO:

3 and homologs thereof having at least 30% amino acid identity, wherein said polypeptide binds to SLC.

36. The isolated or purified SLC-binding domain polypeptide of claim 35, wherein said amino acid identity is determined using an algorithm selected from the group consisting of XBLAST with the parameters, score=50 and wordlength=3, Gapped BLAST with the default parameters of XBLAST, and BLAST with the defaul parameters of XBLAST.

37. An isolated or purified nucleic acid which encodes the SLC-binding domain polypeptide of claim 35 or a complement thereof.

38. A fusion protein comprising an Fc region of an immunoglobulin fused to a polypeptide comprising an amino acid sequence selected from the group consisting of amino acids 143-213 of SEQ ID NO:

3 and homologs thereof having at least 30% amino acid identity.

39. An oligomeric THAP protein comprising a plurality of THAP polypeptides, wherein each THAP polypeptide comprises an amino acid sequence selected from the group consisting of amino acid 143-213 of SEQ ID NO:

3 and homologs thereof having at least 30% amino acid identity.

40. A medicament comprising an effective amount of a THAP1 polypeptide or an SLC-binding fragment thereof, together with a pharmaceutically acceptable carrier.

41. An isolated or purified THAP dimerization domain polypeptide consisting essentially of an amino acid sequence selected from the group consisting of amino acids 143 and 192 of SEQ ID NO:

3 and homologs thereof having at least 30% amino acid identity, wherein said polypeptide binds to a THAP-family polypeptide.

42. The isolated or purified THAP dimerization domain polypeptide of claim 41, wherein said amino acid identity is determined using an algorithm selected from the group consisting of XBLAST with the parameters, score=50 and wordlength=3, Gapped BLAST with the default parameters of XBLAST, and BLAST with the defaul parameters of XBLAST.

43. An isolated or purified nucleic acid which encodes the THAP dimerization domain polypeptide of claim 41 or a complement thereof.

44. An expression vector comprising a promoter operably linked to a nucleic acid having a nucleotide sequence selected from the group consisting of SEQ ID NOs:

160-175 and portions thereof comprising at least 18 consecutive nucleotides.

45. The expression vector of claim 44, wherein said promoter is a promoter which is not operably linked to said nucleic acid selected from the group consisting of SEQ ID NOs.:

160-175 in a naturally occurring genome.

46. A host cell comprising the expression vector of claim 44.

47. An expression vector comprising a promoter operably linked to a nucleic acid encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:

1-114 and portions thereof comprising at least 18 consecutive nucleotides.

48. The expression vector of claim 47, wherein said promoter is a promoter which is not operably linked to said nucleic acid selected from the group consisting of SEQ ID NOs.:

160-175 in a naturally occurring genome.

49. A host cell comprising the expression vector of claim 47.

50. A method of identifying a candidate inhibitor of a THAP-family polypeptide, a candidate inhibitor of apoptosis, or a candidate compound for the treatment of a cell proliferative disorder, said method comprising:

contacting a THAP-family polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

1-114 or a fragment comprising a span of at least 6 contiguous amino acids of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

1-114 with a test compound; and

determining whether said compound selectively binds to said polypeptide, wherein a determination that said compound selectively binds to said polypeptide indicates that said compound is a candidate inhibitor of a THAP-family polypeptide, a candidate inhibitor of apoptosis, or a candidate compound for the treatment of a cell proliferative disorder.

51. A method of identifying a candidate inhibitor of apoptosis, a candidate compound for the treatment of a cell proliferative disorder, or a candidate inhibitor of a THAP-family polypeptide of SEQ ID NOs:

1-114 or a fragment comprising a span of at least 6 contiguous amino acids of a polypeptide according to SEQ ID NOs;

1-114, said method comprising;

contacting said THAP-family polypeptide with a test compound; and

determining whether said compound selectively inhibits at least one biological activity selected from the group consisting of interaction with a THAP-family target protein, binding to a nucleic acid sequence, binding to PAR-4, binding to SLC, binding to PML, binding to a polypeptide found in PML-NBs, localization to PML-NBs, targeting a THAP-family target protein to PML-NBs, and inducing apoptosis, wherein a determination that said compound selectively inhibits said at least one biological activity of said polypeptide indicates that said compound is a candidate inhibitor of a THAP-family polypeptide, a candidate inhibitor of apoptosis, or a candidate compound for the treatment of a cell proliferative disorder.

52. A method of identifying a candidate inhibitor of apoptosis, a candidate compound for the treatment of a cell proliferative disorder, or a candidate inhibitor of a THAP-family polypeptide of SEQ ID NOs:

1-114 or a fragment comprising a span of at least 6 contiguous amino acids of a polypeptide according to SEQ ID NOs;

1-114, said method comprising;

contacting a cell comprising said THAP-family polypeptide with a test compound; and

determining whether said compound selectively inhibits at least one biological activity selected from the group consisting of interaction with a THAP-family target protein, binding to a nucleic acid sequence, binding to PAR-4, binding to SLC, binding to PML, binding to a polypeptide found in PML-NBs, localization to PML-NBs, targeting a THAP-family target protein to PML-NBs, and inducing apoptosis, wherein a determination that said compound selectively inhibits said at least one biological activity of said polypeptide indicates that said compound is a candidate inhibitor of a THAP-family polypeptide, a candidate inhibitor of apoptosis, or a candidate compound for the treatment of a cell proliferative disorder.

53. A method of identifying a candidate modulator of THAP-family activity, said method comprising:

providing a THAP-family polypeptide of SEQ ID NOs;

1-114 or, a fragment comprising a span of at least 6 contiguous amino acids of a polypeptide according to SEQ ID NOs;

1-114; and

providing a THAP-family target polypeptide or a fragment thereof; and

determining whether a test compound selectively modulates the ability of said THAP-family polypeptide to bind to said THAP-family target polypeptide, wherein a determination that said test compound selectively modulates the ability of said THAP-family polypeptide to bind to said THAP-family target polypeptide indicates that said compound is a candidate modulator of THAP-family activity.

54. The method of claim 53, wherein said THAP-family polypeptide is provided by a first expression vector comprising a nucleic acid encoding a THAP-family polypeptide of SEQ ID NOs:

1-114 or, a fragment comprising a contiguous span of at least 6 contiguous amino acids of a polypeptide according to SEQ ID NOs;

1-114, and wherein said THAP-family target polypeptide is provided by a second expression vector comprising a nucleic acid encoding a THAP-family target polypeptide, or a fragment thereof.

55. The method of claim 53, wherein said THAP-family activity is apoptosis activity.

56. The method of claim 53, wherein said THAP-family target protein is PAR-4.

57. The method of claim 53, wherein said THAP-family polypeptide is a THAP-1, THAP-2 or THAP-3 protein and said THAP-family target protein is PAR-4.

58. The method of claim 53, wherein said THAP-family target protein is SLC.

59. A method of modulating apoptosis in a cell comprising modulating the activity of a THAP-family protein.

60. The method of claim 59, wherein said THAP-family protein is selected from the group consisting of SEQ ID NOs:

1-114.

61. The method of claim 59, wherein modulating the activity of a THAP-family protein comprises modulating the interaction of a THAP-family protein and a THAP-family target protein.

62. The method of claim 59, wherein modulating the activity of a THAP-family protein comprises modulating the interaction of a THAP-family protein and a PAR4 protein.

63. A method of identifying a candidate activator of a THAP-family polypeptide, a candidate activator of apoptosis, or a candidate compound for the treatment of a cell proliferative disorder, said method comprising:

contacting a THAP-family polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

1-98 or a fragment comprising a span of at least 6 contiguous amino acids of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

1-98 with a test compound; and

determining whether said compound selectively binds to said polypeptide, wherein a determination that said compound selectively binds to said polypeptide indicates that said compound is a candidate activator of a THAP-family polypeptide, a candidate activator of apoptosis, or a candidate compound for the treatment of a cell proliferative disorder.

64. A method of identifying a candidate activator of apoptosis, a candidate compound for the treatment of a cell proliferative disorder, or a candidate activator of a THAP-family polypeptide of SEQ ID NOs:

1-98 or a fragment comprising a span of at least 6 contiguous amino acids of a polypeptide according to SEQ ID NOs;

1-98, said method comprising;

contacting said THAP-family polypeptide with a test compound; and

determining whether said compound selectively activates at least one biological activity selected from the group consisting of interaction with a THAP-family target protein, binding to a nucleic acid sequence, binding to PAR-4, binding to SLC, binding to PML, binding to a polypeptide found in PML-NBs, localization to PML-NBs, targeting a THAP-family target protein to PML-NBs, and inducing apoptosis, wherein a determination that said compound selectively activates said at least one biological activity of said polypeptide indicates that said compound is a candidate activator of a THAP-family polypeptide, a candidate activator of apoptosis, or a candidate compound for the treatment of a cell proliferative disorder.

65. A method of identifying a candidate activator of apoptosis, a candidate compound for the treatment of a cell proliferative disorder, or a candidate activator of a THAP-family polypeptide of SEQ ID NOs:

1 to 98 or a fragment comprising a span of at least 6 contiguous amino acids of a polypeptide according to SEQ ID NOs;

1-98, said method comprising;

contacting a cell comprising said THAP-family polypeptide with a test compound; and

determining whether said compound selectively activates at least one biological activity selected from the group consisting of interaction with a THAP-family target protein, binding to a nucleic acid sequence, binding to PAR-4, binding to SLC, binding to PML, binding to a polypeptide found in PML-NBs, localization to PML-NBs, targeting a THAP-family target protein to PML-NBs, and inducing apoptosis, wherein a determination that said compound selectively activates said at least one biological activity of said polypeptide indicates that said compound is a candidate activator of a THAP-family polypeptide, a candidate activator of apoptosis, or a candidate compound for the treatment of a cell proliferative disorder.

66. A method of ameliorating a condition associated with the activity of SLC in an individual comprising administering a polypeptide comprising the SLC binding domain of a THAP-family protein to said individual.

67. The method of claim 66, wherein said polypeptide comprises a fusion protein comprising an Fc region of an immunoglobulin fused to a polypeptide comprising an amino acid sequence selected from the group consisting of amino acids 143-213 of SEQ ID NO:

3 and homologs thereof having at least 30% amino acid identity.

68. The method of claim 66, wherein said polypeptide comprises an oligomeric THAP protein comprising a plurality of THAP polypeptides, wherein each THAP polypeptide comprises an amino acid sequence selected from the group consisting of amino acid 143-213 of SEQ ID NO:

3 and homologs thereof having at least 30% amino acid identity.

69. A method of modulating angiogenesis in an individual comprising modulating the activity of a THAP-family protein in said individual.

70. The method of claim 69, wherein said THAP-family protein is selected from the group consisting of SEQ ID NOs:

1-114.

71. The method of claim 69, wherein said modulation is inhibition.

72. The method of claim 69, wherein said modulation is induction.

73. A method of reducing cell death in an individual comprising inhibiting the activity of a THAP-family protein in said individual.

74. The method of claim 73, wherein said THAP-family protein is selected from the group consisting of SEQ ID NOs:

1-114.

75. The method according to claim 73, wherein the activity of said THAP-family protein is inhibited in the CNS.

76. A method of reducing inflammation or an inflammatory disorder in an individual comprising modulating the activity of a THAP-family protein in said individual.

77. The method of claim 76, wherein said THAP-family protein is selected from the group consisting of SEQ ID NOs:

1-114.

78. A method of reducing the extent of cancer in an individual comprising modulating the activity of a THAP-family protein in said individual.

79. The method of claim 78, wherein said THAP-family protein is selected from the group consisting of SEQ ID NOs:

1-114.

80. The method of claim 78, wherein increasing the activity of said THAP family protein induces apoptosis, inhibits cell division, inhibits metastatic potential, reduces tumor burden, increases sensitivity to chemotherapy or radiotherapy, kills a cancer cell, inhibits the growth of a cancer cell, kills an endothelial cell, inhibits the growth of an endothelial cell, inhibits angiogenesis, or induces tumor regression.