Bioluminescent assays using cyanobenzothiazole compounds
View Patent ↗The invention provides methods that employ derivatives of 2-cyano-6-hydroxy- or 2-cyano-6-amino-benzothiazole, for example, in a bioluminogenic reaction. The invention further provides methods for detecting or determining the presence of molecules and/or enzymes, the modulator activity of such molecules, and/or the activity of such enzymes. The methods are adaptable to high-throughput format.
1. An in vitro method of detecting a non-luciferase enzyme in a cell comprising:
(a) contacting cells with a substrate for the non-luciferase enzyme, the substrate being a derivative of a 2-cyano-6-substituted benzothiazole under conditions which allow for a reaction between the enzyme and the substrate;
(b) adding a luciferase reaction mixture to the contacted cells, wherein the luciferase reaction mixture comprises D-cysteine and a firefly luciferase; and
(c) measuring bioluminescence.
2. The method of claim 1 , wherein the derivative is a compound of formula (I)
wherein
R 1 is H, OH, OR x or NR x R y ;
R 2 is (C 1 -C 3 )alkyl, trifluoromethyl, amino, nitro, or halo;
n is 0, 1, 2, or 3;
R x is (i) (C 1 -C 10 )alkylaryl wherein the aryl is optionally substituted with one to five groups selected from the group consisting of halo, hydroxy, amino groups, amino acids, peptides and esters, or (ii) (C 1 -C 10 )alkyl, wherein the alkyl is optionally substituted with one to five groups selected from the group consisting of alkoxy, hydroxy, halo, and amino; and
R y is hydrogen or (C 1 -C 10 )alkyl.
3. The method of claim 1 , wherein the derivative is selected from the group consisting of:
4. The method of claim 1 wherein the non-luciferase enzyme comprises UDP glucuronosyl transferase (UGT), glutathione transferase (GST), cytochrome P450 (CYP450), flavin monoamine oxidase (FMO), histone deacetylase (HDAC), or a protease.
5. The method of claim 1 , wherein the luciferase reaction mixture further comprises an esterase.
6. The method of claim 1 , wherein the cells are hepatocytes.
7. The method of claim 1 , wherein the cells are further contacted with a test compound prior to or simultaneously with step (a).
8. The method of claim 7 , wherein the test compound is selected from the group consisting of an inhibitor of the non-luciferase enzyme, an inducer for the non-luciferase enzyme, a substrate for the non-luciferase enzyme, and an activator of the non-luciferase enzyme.
9. An in vitro method of detecting a non-luciferase enzyme in a cell comprising:
(a) contacting cells with a substrate for the non-luciferase enzyme, the substrate being a derivative of a 2-cyano-6-substituted benzothiazole, under conditions which allow for a reaction between the enzyme and the substrate, in a first reaction vessel to form an incubation mixture;
(b) transferring at least a portion of the incubation mixture to a second reaction vessel;
(c) adding a luciferase reaction mixture, wherein the luciferase reaction mixture comprises D-cysteine and a firefly luciferase, to the second reaction vessel; and
(d) measuring bioluminescence.
10. The method of claim 9 , wherein the derivative is a compound of formula (I)
wherein
R 1 is H, OH, OR x or NR x R y ;
R 2 is (C 1 -C 3 )alkyl, trifluoromethyl, amino, nitro, or halo;
n is 0, 1, 2, or 3;
R x is (i) (C 1 -C 10 )alkylaryl wherein the aryl is optionally substituted with one to five groups selected from the group consisting of halo, hydroxy, amino groups, amino acids, peptides and esters, or (ii) (C 1 -C 10 )alkyl, wherein the alkyl is optionally substituted with one to five groups selected from the group consisting of alkoxy, hydroxy, halo, and amino; and
R y is hydrogen or (C 1 -C 10 )alkyl.
11. The method of claim 9 , wherein the derivative is selected from the group consisting of:
12. The method of claim 9 , wherein the non-luciferase enzyme comprises UGT, GST, CYP450, FMO, HDAC, or a protease.
13. The method of claim 9 , wherein the luciferase reaction mixture further comprises an esterase.
14. The method of claim 9 , wherein the cells are hepatocytes.
15. The method of claim 9 , wherein the cells are further contacted with a test compound prior to or simultaneously with step (a).
16. The method of claim 15 , wherein the test compound is selected from the group consisting of an inhibitor of the non-luciferase enzyme, an inducer for the non-luciferase enzyme, a substrate for the non-luciferase enzyme, and an activator of the non-luciferase enzyme.
17. An in vitro method of screening for modulators of a non-luciferase enzyme comprising:
(a) contacting cells with a test compound;
(b) adding a substrate for the non-luciferase enzyme, the substrate being a derivative of a 2-cyano-6-substituted benzothiazole, under conditions which allow for a reaction between the substrate and the non-luciferase enzyme, to form a mixture;
(c) adding a luciferase reaction mixture, wherein the luciferase reaction mixture comprises D-cysteine and a firefly luciferase, to the mixture; and
(d) measuring bioluminescence; wherein the bioluminescence is compared to that from cells not contacted with a test compound.
18. The method of claim 17 , wherein the derivative is a compound of formula (I)
wherein
R 1 is H, OH, OR x or NR x R y ;
R 2 is (C 1 -C 3 )alkyl, trifluoromethyl, amino, nitro, or halo;
n is 0, 1, 2, or 3;
R x is (i) (C 1 -C 10 )alkylaryl wherein the aryl is optionally substituted with one to five groups selected from the group consisting of halo, hydroxy, amino groups, amino acids, peptides and esters, or (ii) (C 1 -C 10 )alkyl, wherein the alkyl is optionally substituted with one to five groups selected from the group consisting of alkoxy, hydroxy, halo, and amino; and
R y is hydrogen or (C 1 -C 10 )alkyl.
19. The method of claim 17 , wherein the derivative is selected from the group consisting of:
20. The method of claim 17 , wherein the non-luciferase enzyme comprises UGT, GST, CYP450, FMO, HDAC, or a protease.
21. The method of claim 17 , wherein the luciferase reaction mixture further comprises an esterase.
22. The method of claim 17 , wherein the cells are hepatocytes.
23. An in vitro method of detecting more than one non-luciferase enzyme in a cell comprising:
a) contacting cells with more than one bioluminogenic substrate wherein one of the substrates is a derivative of a 2-cyano-6-substituted benzothiazole under conditions which allow for a reaction between the enzymes and the substrates in a first reaction vessel to form a reaction mixture;
b) transferring a portion of the incubation mixture to a second and third reaction vessel;
c) adding to one of the reaction vessels a luciferase reaction mixture comprising D-cysteine and a firefly luciferase and adding to the other reaction vessel luciferase reaction mixture comprising a firefly luciferase without D-cysteine; and
d) measuring bioluminescence in both reaction vessels.
24. The method of claim 23 , wherein the derivative is a compound of formula (I)
wherein
R 1 is H, OH, OR x or NR x R y ;
R 2 is (C 1 -C 3 )alkyl, trifluoromethyl, amino, nitro, or halo;
n is 0, 1, 2, or 3;
R x is (i) (C 1 -C 10 )alkylaryl wherein the aryl is optionally substituted with one to five groups selected from the group consisting of halo, hydroxy, amino groups, amino acids, peptides and esters, or (ii) (C 1 -C 10 )alkyl, wherein the alkyl is optionally substituted with one to five groups selected from the group consisting of alkoxy, hydroxy, halo, and amino; and
R y is hydrogen or (C 1 -C 10 )alkyl.
25. The method of claim 23 , wherein the derivative is selected from the group consisting of:
26. The method of claim 23 , wherein the non-luciferase enzyme comprises UGT, GST, CYP450, FMO, HDAC, or a protease.
27. The method of claim 23 , wherein the luciferase reaction mixture further comprises an esterase.
28. The method of claim 23 , wherein the cells are hepatocytes.
29. The method of claim 23 , wherein the cells are further contacted with a test compound prior to or simultaneously with step (a).
30. The method of claim 23 , wherein the other bioluminogenic substrate is a derivative of D-luciferin or aminoluciferin.