Claims
- 1. A composition comprising;
a polypeptide forming a hetero-dimer with one processed mammalian caspase-9 monomer (SEQ ID NO:1), said polypeptide having a surface groove from BIR3 (SEQ ID NO:2), or variant thereof, said variant having at least 90% sequence identity with (SEQ ID NO:2) for binding to the mammalian initiator caspase-9, said surface groove including amino acid residues P325, G326, H343, and L344.
- 2. The composition of claim 1 wherein said polypeptide is a variant of a BIR3 surface groove of c-IAP1 (SEQ ID NO:14) or a variant thereof.
- 3. The composition of claim 1 wherein said polypeptide is a variant of BIR3 surface groove of c-IAP2 (SEQ ID NO:15) or a variant thereof.
- 4. The composition of claim 1 wherein said polypeptide is the BIR3 surface groove of XIAP (SEQ ID NO 3).
- 5. The composition of claim 1 wherein the polypeptide includes the BIR-2 (SEQ ID NO: R) repeat or the BIR-1 (SEQ ID NO:20) repeat unit.
- 6. The composition of claim 1 wherein BIR3 (SEQ ID NO: 2) binds to the protein-protein recognition interface of the caspase-9 (SEQ ID NO:1).
- 7. The composition of claim 1 wherein said polypeptide includes one or more zinc ions.
- 8. The composition of claim 1 wherein said polypeptide inhibits activation of procaspase-3 (SEQ ID NO: 10) through inhibition of the mammalian caspase-9 (SEQ ID NO:1).
- 9. The composition of claim 1 wherein the BIR3 (SEQ ID NO:2) domain of said polypeptide bonds to the caspase-9 small subunit (SEQ ID NO:9).
- 10. The composition of claim 1 wherein said polypeptide forms a catalytically inactive complex with the mammalian caspase-9.
- 11. The composition of claim 1 including pharmaceutically-acceptable salts of said polypeptide or variants thereof.
- 12. The composition of claim 1 and a pharmaceutically acceptable excipient.
- 13. A composition comprising;
a polypeptide forming a 1:1 complex with a processed mammalian caspase-9 (SEQ ID NO:1), said polypeptide having a surface groove from BIR3 (SEQ ID NO:2) for binding to the mammalian caspase-9, said polypeptide having one or more point mutations of surface groove amino acid residues P325, G326, H343.
- 14. The composition of claim 13 wherein polypeptide is the BIR3 of XIAP (SEQ ID NO:3) or variants and salts thereof.
- 15. The composition of claim 13 wherein said polypeptide is a purified and isolated form of XIAP (SEQ ID NO:13).
- 16. The composition of claim 13 wherein said complex activates procaspase-3 (SEQ ID NO:10).
- 17. The composition of claim 13 wherein said polypeptide is a modified c-IAP1 (SEQ ID NO:14).
- 18. The composition of claim 13 wherein said polypeptide is a modified c-IAP2 (SEQ ID NO:15).
- 19. The composition of claim 13 further comprising an excipient.
- 20. A method of inhibiting the activity of caspase-9 comprising:
combining processed mammalian caspase-9 (SEQ ID NO:1) with a composition that includes a polypeptide forming a 1:1 complex with said mammalian caspase-9, said polypeptide having a surface groove from BIR3 (SEQ ID NO:2) for binding to the mammalian caspase-9 and said surface groove including amino acid residues P325, G326, H343, and L344.
- 21. The method of claim 20 wherein the caspase-9 is in one or more cells.
- 22. The method of claim 20 wherein the caspase-9 present within cells of a mammal subject individual.
- 23. The method of claim 20 wherein the composition includes an excipient.
- 24. A method of inhibiting effector caspase activity comprising:
combining a mixture of effector caspase with mammalian caspase-9 (SEQ ID NO:1) with a composition that includes a polypeptide forming a 1:1 complex with said mammalian caspase-9, said polypeptide having a surface groove from BIR3 (SEQ ID NO:2) for binding to the mammalian caspase-9 and said surface groove including amino acid residues P325, G326, H343, and L344.
- 25. The method of claim 24 wherein the effector caspase is procaspase-3 (SEQ ID NO:10).
- 26. A method of making procaspase-9 zymogen comprising:
co-expressing the catalytic subunit of caspase-9 in a first vector with a BIR3 domain of XIAP in a second vector in Escherichia coli.
- 27. The method of claim 26 wherein said first vector is pET-21b.
- 28. The method of claim 26 wherein said second vector is pBB75.
- 29. The method of claim 26 wherein said Escherichia coli is strain BL21(DE3)
- 30. The method of claim 26 further comprising purification of said mixture.
- 31. A composition comprising;
an isolated polypeptide or variant thereof, said variant having at least 90% sequence identity with BIR3 (SEQ ID NO:2), said polypeptide forming a heterodimer complex with a mammalian caspase -9 (SEQ ID NO:1) and having a surface groove from BIR3 (SEQ ID NO:2) for binding to mammalian initiator caspase, said surface groove including amino acid residues P325, G326, H343, and L344.
- 32. A composition comprising;
a polypeptide forming a hetero-dimer with an apoptosome-activated caspase-9 (SEQ ID NO:7), said polypeptide having a surface groove from BIR3 (SEQ ID NO:2), or variant thereof, said variant having at least 90% sequence identity with SEQ ID NO:2 for binding to the apoptosome-activated caspase-9 (SEQ ID NO:7), said surface groove including amino acid residues P325, G326, H343, and L344.
- 33. A composition comprising;
a polypeptide forming a hetero-dimer with one mammalian caspase-9 monomer (SEQ ID NO:1), said polypeptide having a surface groove from BIR3 (SEQ ID NO:2), or variant thereof, said variant having at least 90% sequence identity with (SEQ ID NO:2) for binding to the mammalian initiator caspase-9, said surface groove including amino acid residues P325, G326, and L344.
- 34. The composition of claim 33 wherein said polypeptide is a variant of BIR3 surface groove of c-IAP1 (SEQ ID NO:14).
- 35. The composition of claim 33 wherein said polypeptide is a variant of BIR3 surface groove of c-IAP2 (SEQ ID NO:15).
- 36. The composition of claim 33 wherein said polypeptide is the BIR3 surface groove of XIAP (SEQ ID NO 3) or variant thereof.
- 37. The composition of claim 33 wherein the polypeptide includes the BIR-2 (SEQ ID NO: R) repeat or the BIR-1 (SEQ ID NO:20) repeat unit.
- 38. The composition of claim 33 wherein BIR3 (SEQ ID NO:2) binds to the protein-protein recognition interface of the caspase-9 (SEQ ID NO:1).
- 39. The composition of claim 33 wherein said polypeptide includes one or more zinc ions.
- 40. The composition of claim 33 wherein said polypeptide inhibits activation of procaspase-3 (SEQ ID NO: 21) through inhibition of an initiator caspase.
- 41. The composition of claim 33 wherein the BIR3 (SEQ ID NO: 2) domain of said polypeptide bonds to the caspase-9 small subunit (SEQ ID NO:9) of said caspase-9.
- 42. The composition of claim 33 wherein said polypeptide forms a catalytically inactive complex with the initiator caspase.
- 43. An isolated nucleic acid molecule at least 90% identical to a nucleic acid molecule selected from the group consisting of:
a nucleic acid molecule consisting of a nucleotide sequence encoding the amino acid sequence of caspase-9 F404D (SEQ ID NO: 25) wherein said caspase-9 F404D inhibits apoptosis; a nucleic acid molecule consisting of a nucleotide sequence encoding caspase-9 ΔS. (amino acid residues 139 to 315 and 331 to 416 of SEQ ID NO:23 ) wherein said caspase-9 ΔS activates apoptosis; and a nucleic acid molecule consisting of a nucleotide sequence encoding caspase-9 ΔL (amino acid residues 139 to 315 and 339 to 416 of SEQ ID NO:24) wherein said caspase-9 ΔL inhibits apoptosis.
- 44. A vector comprising the nucleic acid molecule of claim 43.
- 45. A host transformed with the vector of claim 44.
- 46. A method for making a caspase-9 polypeptide, comprising:
(a) inserting a nucleic acid molecule of claim 1 into a vector; (b) transforming a host with said vector; and (c) culturing said host under conditions to induce expression of the caspase-9 polypeptide (SEQ ID NO:23), (SEQ ID NO:24), or (SEQ ID NO:25) or variants thereof having at least 90% of the sequence identity with said polypeptides.
- 47. A composition comprising:
an initiator caspase specific binding agent having a caspase-9 or apoptosome activated caspase-9 recognition binding sequence and caspase-9 inhibiting amino acid residues Pro325, Gly326,His343, and Leu344 in BIR3 of XIAP, wherein the specific binding agent forms a heterodimer complex with an initiator caspase to inhibit its catalytic activity with an procaspase-3.
- 48. The composition of claim 47 wherein the specific binding agent is a peptidomimetic of the BIR3 domain of XIAP.
- 49. The composition of claim 47 wherein the specific binding agent is a polypeptide and variants thereof that are functionally equivalent to the caspase-9 inhibiting amino acid residues Pro325, Gly326,His343, and Leu344 in BIR3 of XIAP.
- 50. A composition comprising:
an initiator caspase specific binding agent having a caspase-9 or apoptosome activated caspase-9 recognition binding sequence and including point mutations of the caspase-9 inhibiting amino acid residues functionally equivalent to Pro325, Gly326,His343, and Leu344 in BIR3 of XIAP wherein the specific binding agent forms a heterodimer complex with an initiator caspase to modify its catalytic activity.
- 51. The composition of claim 50 wherein the specific binding agent is a peptidomimetic of the point mutated BIR3 domain of XIAP
- 52. The composition of claim 50 wherein the specific binding agent is a polypeptide.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S. Provisional Application Serial No. 60/443,590 filed Jan. 30, 2003 the contents of which are incorporated herein by reference in their entirety.
GOVERNMENT INTERESTS
[0002] The United States Government may have certain rights to this invention pursuant to work funded by Grant No. CA90269.
Provisional Applications (1)
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Number |
Date |
Country |
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60443950 |
Jan 2003 |
US |