Control of apoptosis by controlling the propensity of ceramide channel formation
The present invention relates to a novel target to control the apoptotic process, and to the use of this target to identify compounds capable of affecting the apoptotic process, The invention also relates to the use of such identified compounds in the treatment of cancer, stroke, neurodegenerative diseases, viral diseases and other diseases and conditions involving apoptosis.
1. A method for identifying an apoptotic effector compound, wherein said method comprises the steps of:
(A) incubating a phospholipid membrane under conditions sufficient to permit the formation of ceramide channels through said membrane;
(B) incubating said phospholipid membrane in the presence of a candidate apoptotic effector compound; and
(C) determining whether the presence of said candidate apoptotic effector compound affects the formation or stability of said ceramide channels, relative to the extent of such formation or stability observed in the absence of said candidate apoptotic effector compound;
wherein a compound that affects the formation or stability of said ceramide channels is an apoptotic effector compound.
2. The method of claim 1 , wherein said step (B) is performed prior to performing said step (A).
3. The method of claim 1 , wherein said conditions (A) sufficient to permit the formation of ceramide channels comprise providing a ceramide to said phospholipid membrane.
4. The method of claim 3 , wherein said ceramide is C 16 -ceramide.
5. The method of claim 1 , wherein said phospholipid membrane is of defined composition and is a planar membrane or a liposome membrane.
6. The method of claim 5 , wherein said conditions (A) sufficient to permit the formation of ceramide channels comprise providing an agent selected from the group consisting of: nitrous oxide, etoposide, staurosporine, daunorubicin and dexamethasone.
7. The method of claim 1 , wherein said phospholipid membrane is a membrane of a mitochondrion, or a cellular membrane of a yeast or mammalian cell.
8. The method of claim 7 , wherein said conditions (A) sufficient to permit the formation of ceramide channels comprise providing an agent selected from the group consisting of: TNF-alpha, interleukin-1, nitrous oxide, etoposide, staurosporine, daunorubicin and dexamethasone.
9. The method of claim 7 , wherein said conditions (A) sufficient to permit the formation of ceramide channels comprise subjecting said membrane to ionizing radiation, hypoxia or heat.
10. The method of claim 7 , wherein said conditions (A) sufficient to permit the formation of ceramide channels comprise subjecting said cell to serum withdrawal.
11. The method of claim 7 , wherein said phospholipid membrane is a membrane of a mitochondrion.
12. The method of claim 11 , wherein said mitochondrion lacks Bcl-2 proteins.
13. The method of claim 7 , wherein said phospholipid membrane is a cellular membrane of a yeast or mammalian cell.
14. The method of claim 13 , wherein said cell is Bax or Bak deficient.
15. The method of claim 13 , wherein said cell is a mammalian cell.
16. The method of claim 1 , wherein said candidate apoptotic effector compound inhibits the formation or stability of said ceramide channels, relative to the extent of such formation or stability observed in the absence of said candidate apoptotic effector compound.
17. The method of claim 16 , wherein said candidate apoptotic effector compound inhibits the formation of ceramide channels but does not affect the stability of formed ceramide channels, relative to the extent of such formation or stability observed in the absence of said candidate apoptotic effector compound.
18. The method of claim 1 , wherein said candidate apoptotic effector compound induces the formation of, or enhances the stability of, said ceramide channels, relative to the extent of such formation or stability observed in the absence of said candidate apoptotic effector compound.
19. The method of claim 1 , wherein said candidate apoptotic effector compound inhibits the formation induces the formation of, or enhances the stability of, said ceramide channels, relative to the extent of such formation or stability observed in the absence of said candidate apoptotic effector compound.
20. A method of measuring the apoptotic effect of an apoptotic effector compound said method comprising the steps of:
(A) incubating a phospholipid membrane under conditions sufficient to permit the formation of ceramide channels through said membrane;
(B) additionally incubating said phospholipid membrane in the presence of said apoptotic effector compound; and
(C) measuring said compound's apoptotic effect by determining the affect of said compound on the formation or stability of said ceramide channels, relative to the extent of such formation or stability observed in the absence of said compound.