Claims
- 1. A method of screening for substances capable of modulating γ-secretase activity comprising the steps:
(a) providing a source of γ-secretase activity, (b) providing a substrate for γ-secretase activity, said substrate comprising a cleavage site corresponding to the naturally occurring cleavage site between amino acid 49 and amino acid 50 of the γ-secretase substrate C99 (SEQ ID NO:2), (c) incubating said source and said substrate with a test substance under conditions allowing enzymatic activity of γ-secretase activity, and (d) determining γ-secretase activity by detecting a C-terminal fragment released from said substrate, wherein said C-terminal fragment is a fragment beginning with a sequence corresponding to the N-terminal sequence of CTFγ/50 (SEQ. ID NO: 1).
- 2. The method according to claim 1, comprising the additional step of comparing said determined γ-secretase activity to the γ-secretase activity obtained upon carrying out said incubation in the absence of said test substance.
- 3. The method according to claim 1, wherein the test substance is screened for an inhibitory effect on γ-secretase activity.
- 4. The method according to claim 2, wherein the test substance is screened for an inhibitory effect on γ-secretase activity.
- 5. The method according to claim 1, wherein the source of γ-secretase activity is a membrane of a human embryonic kidney (HEK) 293 cell.
- 6. The method according to claim 1, wherein said substrate of γ-secretase activity is selected from human wild type βAPP770, wild type βAPP695, swAPP695, C99 and C83.
- 7. The method according to claim 1, wherein said step of determining γ-secretase activity comprises measuring the release of CTFγ/50.
- 8. The method according to claim 7, wherein CTFγ/50 is measured by immunoblotting or ELISA.
- 9. The method according to claim 1, wherein the test substance is a substance showing an inhibitory effect in an Aβ release based assay.
- 10. The method according to claim 1, wherein the method is carried out in a high throughput screening (HTS) format.
- 11. A kit for the identification of substances capable of inhibiting γ-secretase activity comprising:
(a) a source of γ-secretase activity, (b) a substrate for γ-secretase activity, said substrate comprising a cleavage site corresponding to the naturally occurring cleavage site between amino acid 49 and amino acid 50 of the γ-secretase substrate C99 (SEQ ID NO:2), (c) a reaction buffer, and (d) a means for specifically detecting a C-terminal fragment released from said substrate, wherein said C-terminal fragment is a fragment beginning with a sequence corresponding to the N-terminal sequence of CTFγ/50 (SEQ ID NO:1).
- 12. A method of identifying a substance capable of inhibiting γ-secretase activity comprising using the method of claim 1.
- 13. A method of identifying a substance capable of inhibiting γ-secretase activity comprising using the kit of claim 11.
- 14. A substance capable of inhibiting γ-secretase activity, identified using a method according to claim 1.
- 15. A substance capable of inhibiting γ-secretase activity, identified using a kit according to claim 11.
- 16. A pharmaceutical composition comprising a substance according to claim 14.
- 17. A pharmaceutical composition comprising a substance according to claim 15.
- 18. A method of treating a neurodegenerative disease in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a substance according to claim 14.
- 19. A method of treating a neurodegenerative disease in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a substance according to claim 15.
- 20. A polypeptide having the amino acid sequence shown in SEQ ID NO:1.
Priority Claims (1)
Number |
Date |
Country |
Kind |
DE 101 31 899.5 |
Jul 2001 |
DE |
|
RELATED APPLICATIONS
[0001] Benefit of U.S. Provisional Application Serial No. 60/306,123, filed on Jul. 17, 2001 is hereby claimed. Said Provisional Application is herein incorporated by reference in its entirety.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60306123 |
Jul 2001 |
US |