Bioluminescent protease assay with modified aminoluciferin or derivatives thereof
A sensitive bioluminescent assay to detect proteases, including caspases and proteases that specifically cleave a peptide substrate having aspartate, is provided. In one embodiment, the assay 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 luminescent assay method to detect one or more caspases, comprising:
a) providing a mixture comprising a sample suspected of having one or more caspases, luciferase and an amino-modified aminoluciferin or a carboxy-terminal protected derivative thereof, wherein the modification is the covalent linkage of a substrate for the caspase to the amino group of aminoluciferin or the derivative thereof via a peptide bond, and wherein if present in the sample the caspase cleaves the substrate at the peptide bond; and
b) detecting luminescence in the mixture, thereby detecting the presence or amount of one or more caspases in the sample, wherein the luminescent assay is more sensitive than a corresponding assay with a conjugate comprising a fluorophore covalently linked to the substrate or a functional equivalent thereof.
2. A luminescent assay method to detect a protease that specifically cleaves a peptide substrate comprising aspartate, comprising:
a) providing a mixture comprising a sample suspected of having one or more aspartate-specific proteases, luciferase and an amino-modified aminoluciferin or a carboxy-terminal protected derivative thereof, wherein the modification is the covalent linkage of a peptide substrate comprising aspartate to the amino group of aminoluciferin or the derivative thereof via a peptide bond, and wherein if present in the sample the protease cleaves the substrate at the peptide bond; and
b) detecting luminescence in the mixture, thereby detecting the presence or amount of one or more asparatate-specific proteases in the sample, wherein the luminescent assay is more sensitive than a corresponding assay with a conjugate comprising a fluorophore covalently linked to the substrate or a functional equivalent thereof.
3. The method of claim 1 or 2 further comprising correlating luminescence with caspase or aspartate-specific protease concentration or activity.
4. The method of claim 1 or 2 which detects a caspase other than caspase 3 or caspase 7.
5. The method of claim 1 or 2 which detects caspase 3 or caspase 7.
6. The method of claim 1 or 2 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.
7. The method of claim 6 wherein X 3 is A, V, H, I, or T.
8. The method of claim 1 or 2 wherein the substrate comprises DEVD (SEQ ID NO:1).
9. The method of claim 1 or 2 wherein the substrate comprises YVAD (SEQ ID NO:2).
10. The method of claim 1 or 2 wherein the substrate comprises LEHD (SEQ ID NO:3).
11. The method of claim 5 which detects at least 0.2 microunits of caspase.
12. The method of claim 1 or 2 which is at least 2 times more sensitive than a corresponding assay with a conjugate comprising rhodamine-110 covalently linked to the substrate.
13. The method of claim 1 or 2 wherein the sample is a cell lysate.
14. The method of claim 13 wherein the cells are treated with an apoptosis inducing agent prior to lysis.
15. The method of claim 1 or 2 wherein the sample comprises intact cells.
16. The method of claim 15 wherein the cells are treated with an apoptosis inducing agent.
17. The method of claim 1 or 2 wherein the luciferase is a thermostable luciferase.
18. The method of claim 1 wherein the carboxy-terminal protected derivative thereof is a compound of formula (I):
wherein R is a peptide that is a substrate for caspase, which is linked to the remainder of the compound of formula (I) through its C-terminus forming a peptide (amide) bond; and R′ is H or a suitable carboxy protecting group, or a suitable salt thereof.
19. The method of claim 2 wherein the carboxy-terminal protected derivative thereof is a compound of formula (I):
wherein R is a peptide that is linked to the remainder of the compound of formula (I) through an aspartate group at the C-terminus of the peptide forming a peptide bond; and R′ is H or a suitable carboxy protecting group, or a suitable salt thereof.
20. The method of claim 18 or 19 wherein R 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.
21. The method of claim 20 wherein X 3 is A, V, H, I, or T.
22. The method of claim 20 wherein R comprises DEVD (SEQ ID NO:1).
23. The method of claim 20 wherein R comprises YVAD (SEQ ID NO:2).
24. The method of claim 20 wherein R comprises LEHD (SEQ ID NO:3).
25. The method of claim 18 or 19 wherein R′ is (C 1 -C 6 )alkyl, phenyl or benzyl.
26. The method of claim 18 or 19 wherein R′ is (C 1 -C 6 )alkyl.
27. The method of claim 18 or 19 wherein R′ is methyl, ethyl, propyl, or tert-butyl.