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US 2010/0075351 A1Published application
Bioluminescent detection of cyanohydroxy benzothiazole compounds
AssigneePromega Corporation
InventorsJessica Anderson, Poncho Meisenheimer, John Shultz, James J. Cali, Dongping Ma
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Abstract
The invention provides methods that employ derivatives of 2-cyano-6-hydroxy- or 2-cyano-6-amino-benzothiazole, for example, in a bioluminogenic reaction. Also provided are novel compounds that can be used in the methods. 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.
Claims34
A method to detect or determine the presence or activity of a nonluciferase enzyme in a sample comprising
- a) providing a first mixture comprising a sample, a first reaction mixture for a nonluciferase enzyme-mediated reaction, and a derivative of 2-cyano-6-substituted benzothiazole which includes a substrate for the nonluciferase enzyme, wherein the first mixture does not include a cofactor specific for the activity of the nonluciferase enzyme, and wherein the derivative in the presence of D-cysteine yields a product that is a bioluminogenic substrate for a beetle luciferase in the absence of the cofactor;
- b) contacting at least a portion of the first mixture with a second reaction mixture for a beetle luciferase-mediated reaction which includes D-cysteine, so as to yield a second mixture;
- and
- c) comparing luminescence in the second mixture to luminescence in a control mixture in which a third mixture having the sample, a first reaction mixture for the nonluciferase enzyme-mediated reaction, the derivative of 2-cyano-6-substituted benzothiazole, and a cofactor specific for the nonluciferase enzyme, is contacted with a reaction mixture for the beetle luciferase-mediated reaction which includes D-cysteine, thereby detecting or determining the presence or amount of the nonluciferase enzyme in the sample.
1 more element
A method to detect or determine the presence or amount of UGT in a sample comprising
- a) providing a first mixture comprising a sample, a first reaction mixture for a UGT-mediated reaction, and a derivative of 2-cyano-6-substituted benzothiazole which is a substrate for the UGT, wherein the first mixture does not include UDPGA, and wherein the derivative in the presence of D-cysteine and the UGT yields a product that is a bioluminogenic substrate for a beetle luciferase;
- b) contacting at least a portion of the first mixture with a second reaction mixture for a beetle luciferase-mediated reaction which includes D-cysteine, so as to yield a second mixture;
- and
- c) comparing luminescence in the second mixture to luminescence in a control mixture in which a third mixture having the sample, a first reaction mixture for the UGT-mediated reaction, the derivative of 2-cyano-6-substituted benzothiazole, and UDPGA, is contacted with a reaction mixture for the beetle luciferase-mediated reaction which includes D-cysteine, thereby detecting or determining the presence or amount of UGT in the sample.
1 more element
A method to detect or determine activity of a nonluciferase enzyme modulator in a sample comprising
- a) providing a first mixture comprising a sample that includes microsomes expressing a nonluciferase enzyme, a first reaction mixture for the nonluciferase enzyme-mediated reaction, a compound to be evaluated for the modulator activity with respect to the nonluciferase enzyme, and a derivative of 2-cyano-6-substituted benzothiazole which is a substrate for the nonluciferase enzyme, wherein the first mixture does not include a cofactor specific for the activity of the nonluciferase enzyme, and wherein the derivative in the presence of D-cysteine and the nonluciferase enzyme yields a product that is a bioluminogenic substrate for a beetle luciferase;
- b) contacting at least a portion of the first mixture with a second reaction mixture for a beetle luciferase-mediated reaction which includes D-cysteine, so as to yield a second mixture;
- and
- c) comparing luminescence in the second mixture to luminescence in a control mixture in which a third mixture having a sample that includes microsomes expressing the nonluciferase enzyme, a first reaction mixture for the nonluciferase enzyme-mediated reaction, a compound to be evaluated for modulator activity with respect to the nonluciferase enzyme, the derivative of 2-cyano-6-substituted benzothiazole, and a cofactor specific for the nonluciferase enzyme, is contacted with a reaction mixture for the beetle luciferase-mediated reaction which includes D-cysteine, thereby detecting or determining the modulation activity of the compound.
1 more element
A method to detect or determine activity of a UGT modulator in a sample comprising
- a) providing a first mixture comprising a sample that includes microsomes expressing UGT, a first reaction mixture for a UGT-mediated reaction, a compound to be evaluated for UGT modulator activity, and a derivative of 2-cyano-6-substituted benzothiazole which is a substrate for the UGT, wherein the first mixture does not include UDPGA, and wherein the derivative in the presence of D-cysteine and the UGT would yield a product that is a bioluminogenic substrate for a beetle luciferase;
- b) contacting at least a portion of the first mixture with a second reaction mixture for a beetle luciferase-mediated reaction which includes D-cysteine, so as to yield a second mixture;
- and
- c) comparing luminescence in the second mixture to luminescence in a control mixture in which a third mixture having the sample that includes microsomes expressing UGT, a first reaction mixture for a UGT-mediated reaction, a compound to be evaluated for UGT modulator activity, the derivative of 2-cyano-6-substituted benzothiazole, and UDPGA, is contacted with a reaction mixture for the beetle luciferase-mediated reaction which includes D-cysteine, thereby detecting or determining the UGT modulation activity of the compound to be evaluated in the sample.
1 more element
A compound of formula XV:
- wherein
- Z is hydrogen or a protecting group;
- L is an amino acid or a chain of 2-10 amino acids;
- T is O or NH;
- and
- X is hydrogen or fluorine, with the proviso that if X is H then at least one of the amino acids is R, N, D, C, Q, E, H, K, S, T, W, or Y.
3 more elements
Dependencies are inferred from the claim text.