Bioluminescent caspase assay compounds
A sensitive bioluminescent assay to detect proteases including caspases is provided which employs an aminoluciferin or a carboxy-terminal protected derivative thereof covalently linked via a peptide bond to a substrate for a caspase or an aminoluciferin or a carboxy-terminal protected derivative thereof covalently linked via a peptide bond to a peptide substrate comprising aspartate that is specifically cleaved by a protease specific for the substrate.
1. A compound comprising aminoluciferin or a carboxy-terminal protected derivative thereof covalently linked via a peptide bond to a substrate for a caspase, wherein the compound is a substrate for a caspase and a prosubstrate for a beetle luciferase, wherein the substrate comprises X 1 -X 2 -X 3 -D, wherein X 1 is Y, D, L, V, I, A, W, or P; X 2 is V or E; and X 3 is any amino acid.
2. The compound of claim 1 wherein the substrate comprises DEVD (SEQ ID NO:1).
3. The compound of claim 1 wherein the substrate comprises YVAD (SEQ ID NO:2).
4. The compound of claim 1 wherein the substrate comprises LEHD (SEQ ID NO:3).
5. The compound of claim 1 wherein X 3 is A, V, H, I, or T.
6. The compound of claim 1 which is a carboxy-terminal protected derivative of aminoluciferin.
7. The compound of claim 6 wherein the carboxy-terminus is protected as a (C 1 -C 6 )alkyl ester.
8. The compound of claim 6 , wherein the carboxy-terminus is protected as a methyl, ethyl, propyl, or tert-butyl ester.
9. The compound of claim 6 wherein the N-terminus of the substrate is modified to prevent degradation by aminopeptidases.
10. The compound of claim 9 wherein the N-terminus of the substrate is modified with an amino protecting group.
11. The compound of claim 9 wherein the carboxy-terminus is protected by coupling to a solid support.
12. The compound of claim 1 wherein the substrate is DEVD (SEQ ID NO:1).
13. The compound of claim 1 wherein the substrate is X 1 -X 2 -X 3 -D, wherein X 1 is Y, D, L, V, I, A, W, or P; X 2 is V or E; and X 3 is any amino acid.