IP Library › Patent Application 12269980
Patent Application
App. No. 12/269,980

METHOD OF IDENTIFYING COMPOUNDS THAT MODULATE CASPASE 9 ACTIVITY VIA USE OF A KOSMOTROPIC SALT

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Patent No.
US None
App. No.
12/269,980
Abstract

The present invention discloses methods for activating Caspase 9 in such a way that it can be used in assays to discover modulators of Caspase 9.

Claims (24)

1 . A method for identifying a compound that modulates Caspase 9 activity, comprising:

a) contacting a sample containing Caspase 9 or a fragment with enzymatic activity thereof with a test compound and a Kosmotropic agent; and

b) detecting the activity of Caspase 9 or the fragment, wherein a change in activity indicates a compound which modulates Caspase 9 activity.

2 . The method of claim 1 , wherein said modulation comprises inhibition of Caspase 9 activity.

3 . The method of claim 1 , wherein said activity is detected using a binding assay.

4 . The method of claim 1 , wherein said activity is detected by determining the turnover of a substrate.

5 . The method of claim 4 , wherein said substrate comprises a site cleaved by Caspase 9 selected from the group consisting of a polypeptide, oligopeptide, and a peptide.

6 . The method of claim 5 , wherein the substrate comprises a polypeptide selected from the group consisting of LEHD (SEQ ID NO: 5), WEHD (SEQ ID NO: 4), DEVD (SEQ ID NO: 1), ZEVD, and YVAD (SEQ ID NO: 7).

7 . The method of claim 4 , wherein said substrate is a fluorogenic substrate.

8 . The method of claim 7 , wherein said fluorogenic substrate comprises a labeled peptide selected from the group consisting of LEHD-AMC (SEQ ID NO: 5), WEHD-AMC (SEQ ID NO: 4), DEVD-AMC (SEQ ID NO: 1), ZEVD-AMC, YVAD-AMC (SEQ ID NO: 7), LEHD-AFC (SEQ ID NO: 5), WEHD-AFC (SEQ ID NO: 4), DEVD-AFC (SEQ ID NO: 1), ZEVD-AFC, and YVAD-AFC (SEQ ID NO: 7).

9 . The method of claim 1 , wherein said sample comprises a cell lysate.

10 . The method of claim 1 , wherein said sample comprises a fragment of Caspase 9 with enzymatic activity.

11 . The method of claim 1 , wherein the agent is a Hofmeister series ion.

12 . The method of claim 1 , wherein the agent is a sodium salt selected from the group consisting of sulfate, phosphate, acetate, and citrate.

13 . The method of claim 1 , wherein the agent is an ammonium salt selected from the group consisting of sulfate, phosphate, acetate, and citrate.

14 . The method of claim 12 wherein the agent is citrate.

15 . The method of claim 13 wherein the agent is citrate.

16 . A method for identifying an inhibitor or enhancer of Caspase 9 processing, comprising:

a) contacting a sample containing proCaspase 9 or a cleavagae site containing fragment thereof with protein capable of processing proCaspase 9 and at least one candidate inhibitor or candidate enhancer; and

b) detecting the presence of large and small Caspase 9 subunits, and therefrom determining the level of proCaspase 9 processing, wherein a decrease in processing indicates the presence of caspase processing inhibitor, and wherein an increase in processing indicates the presence of a caspase processing enhancer.

17 . The method of claim 16 , wherein the protein capable of processing proCaspase 9 is CPP32.

18 . The method of claim 16 , wherein the protein capable of processing proCapase 9 is granzyme B.

19 . The method of claim 16 , wherein the detection of the large and small subunits of Caspase 9 is by gel electrophoresis.

20 . The method of claim 16 , wherein the sample further comprises citrate.

Assignments (1)
CHANGE OF NAME Recorded Jun 16, 2010
From: WYETH
To: WYETH LLC
Reel/Frame 024541/0922 →