Luminescence-based methods and probes for measuring cytochrome p450 activity

Patent metadata

Field	Value
Publication number	US-2017191985-A1
Application number	US-201715408662-A
Country	US
Kind code	A1
Filing date	Jan 18, 2017
Priority date	Sep 20, 2002
Publication date	Jul 6, 2017
Grant date	—

Abstract

The invention provides compounds, compositions, methods, substrates, and kits useful for analyzing the metabolic activity in cells, tissue, and animals and for screening test compounds for their effect on cytochrome P450 activity. In particular, a one-step and two-step methods using luminogenic molecules, e.g. luciferins or coelenterazines, that are cytochrome P450 substrates and that are also bioluminescent enzyme, e.g., luciferase, pro-substrates are provided. The present method further provides a method for stabilizing and prolonging the luminescent signal in a luciferase-based assay using luciferase stabilizing agents such as reversible lucifetrase inhibitors.

First claim

1 . A method for measuring cytochrome P450 enzyme activity in animal tissue comprising:
(a) contacting an animal tissue with the luminogenic molecule and a bioluminescent enzyme, wherein the molecule is a cytochrome P450 substrate and a pro-substrate of bioluminescent enzyme; (b) determining cytochrome P450 activity of the tissue by measuring luminescence of the mixture; and wherein the luminogenic molecule is a compound of formula:

wherein
R 1 represents hydrogen, hydroxyl, amino, C 1-20 alkoxy, substituted C 1-20 alkoxy, C 2-20 alkenyloxy, substituted C 2-20 alkenyloxy, halogenated C 2-20 alkoxy, substituted halogenated C 2-20 alkoxy, C 3-20 alkynyloxy, substituted C 3-20 alkynyloxy, C 3-20 cycloalkoxy, substituted C 3-20 cycloalkoxy, C 3-20 cycloalkylamino, substituted C 3-20 cycloalkylamino, C 1-20 alkylamino, substituted C 1-20 alkylamino, di C 1-20 alkylamino, substituted di C 1-20 alkylamino, C 2-20 alkenylamino, substituted C 2-20 alkenylamino, di C 2-20 alkenylamino, substituted di C 2-20 alkenylamino, C 2-20 alkenyl C 1-20 alkylamino, substituted C 2-20 alkenyl C 1-20 alkylamino, C 3-20 alkynylamino, substituted C 3-20 alkynylamino, di C 3-20 alkynylamino, substituted di alkylamino, C 3-20 alkynyl C 2-20 alkenylamino, or substituted C 3-20 alkynyl C 2-20 alkenylamino;
R 2 and R 3 independently represents C or N;
R 4 and R 5 independently represents S, O, NR 8 , wherein R 8 represents hydrogen or C 1-20 alkyl, CR 9 R 10 wherein R 9 and R 10 independently represent H, C 1-20 alkyl, or fluorine;
R 6 represents CH 2 OH; COR 11 wherein R 11 , represents H, OH, C 1-2 alkoxide, C 2-20 alkenyl, or NR 12 R 13 wherein R 12 and R 13 are independently H, or C 1-20 alkyl; or —OM + wherein M + is an alkali metal or a pharmaceutically acceptable salt; and
R 7 represent H, C 1-6 alkyl, C 1-20 alkenyl, halogen, or C 1-6 alkoxide, with the proviso that R 1 is not OH or NH 2 , R 7 is not H, R 6 is not COR 11 R 11 is not OH, R 3 and R 2 are not both carbon, and R 4 and R 5 are not both S at the same time (luciferin and aminoluciferin).

2 . The method of claim 1 , wherein the tissue is contacted first with the luminogenic molecule for a first predetermined time period prior to contact with the bioluminescent enzyme to provide a mixture.

3 . The method of claim 1 , wherein the mixture further comprises a detergent and/or a pyrophosphatase.

4 . The method of claim 1 , wherein the animal tissue expresses the bioluminescent enzyme.

5 . The method of claim 2 , wherein the bioluminescent enzyme is part of a reaction mixture.

6 . The method of claim 1 , wherein the bioluminescent enzyme is a luciferase.

7 . The method of claim 6 , wherein the luciferase is a beetle luciferase.

8 . The method of claim 1 , wherein the luminogenic molecule is a luciferin or luciferin derivative.

9 . A method for determining the effect of a compound on cytochrome P450 enzyme activity of animal tissue comprising the steps of:
(a) contacting an animal tissue with the test compound, a luminogenic molecule and a bioluminescent enzyme, wherein the luminogenic molecule is a cytochrome P450 substrate and a pro-substrate of bioluminescent enzyme; (b) determining cytochrome P450 enzyme activity of the tissue, if any, resulting from the exposure of the tissue to the test compound by measuring and comparing luminescence from said tissue with a control tissue not exposed to the test compound; and wherein the luminogenic molecule is a compound of formula:

wherein
R 1 represents hydrogen, hydroxyl, amino, C 1-20 alkoxy, substituted C 1-20 alkoxy, C 2-20 alkenyloxy, substituted C 2-20 alkenyloxy, halogenated C 2-20 alkoxy, substituted halogenated C 2-20 alkoxy, C 3-20 alkynyloxy, substituted C 3-20 alkynyloxy, C 3-20 cycloalkoxy, substituted C 3-20 cycloalkoxy, C 3-20 cycloalkylamino, substituted C 3-20 cycloalkylamino, C 1-20 alkylamino, substituted C 1-20 alkylamino, di C 1-20 alkylamino, substituted diC 1-20 alkylamino, C 2-20 alkenylamino, substituted C 2-20 alkenylamino, di C 2-20 alkenylamino, substituted di C 2-20 alkenylamino, C 2-20 alkenyl C 1-20 alkylamino, substituted C 2-20 alkenyl C 1-20 alkylamino, C 3-20 alkynylamino, substituted C 3-20 alkynylamino, di C 3-20 alkynylamino, substituted di alkylamino, C 3-20 alkynyl C 2-20 alkenylamino, or substituted C 3-20 alkynyl C 2-20 alkenylamino;
R 2 and R 3 independently represents C or N;
R 4 and R 5 , independently represents S, O, NR 8 , wherein R 8 represents hydrogen or C 1-20 alkyl, CR 0 R 10 wherein R 9 and R 10 independently represent H, C 1-20 alkyl, or fluorine;

R 6 represents CH 2 OH; COR 11 wherein R 11 represents H, OH, C 1-20 alkoxide, C 2-20 alkenyl, or NR 12 R 13 wherein R 12 and R 13 are independently H, or C 1-20 alkyl; or —OM + wherein M + is an alkali metal or a pharmaceutically acceptable salt; and
R 7 represent H, C 1-6 alkyl, C 1-20 alkenyl, halogen, or C 1-6 alkoxide, with the proviso that R 1 is not OH or NH 2 , R 7 is not H, R 6 is not COR 11 R 11 is not OH, R 3 and R 2 are not both carbon, and R 4 and R 5 are not both S at the same time (luciferin and aminoluciferin).

10 . The method of claim 9 , wherein the animal tissue expresses the bioluminescent enzyme.

11 . The method of claim 9 , wherein the bioluminescent enzyme is part of a reaction mixture.

12 . The method of claim 9 , wherein the tissue is contacted with the test compound to produce a first mixture prior to contact with the luminogenic molecule to produce a second mixture.

13 . The method of claim 12 , wherein the second mixture further comprises a bioluminescent enzyme.

14 . The method of claim 12 , wherein the second reaction mixture further comprises a detergent and/or pyrophosphatase.

15 . The method of claim 9 , wherein the bioluminescent enzyme is a luciferase.

16 . The method of claim 15 , wherein the luciferase is a beetle luciferase.

17 . The method of claim 9 , wherein the luminogenic molecule is a luciferin or luciferin derivative.

18 . The method of claim 9 , wherein a plurality of compounds are screened in a high throughput method.

19 . The method of claim 18 , wherein the tissue is first contacted with the compounds and luminogenic molecule for a first predetermined time period prior to contact with the bioluminescent enzyme.

20 . The method of claim 19 , further comprising adding a detergent after the first predetermined time period.

21 . The method of claim 20 , wherein the detergent and bioluminescent enzyme may be added at the same time.

22 . The method of claim 9 , wherein the tissue is first contacted with the compounds for a first predetermined time period, then contacted with the luminogenic molecule for a second predetermined time period, then contacted with the bioluminescent enzyme for a third predetermined time period.

23 . The method of claim 22 , further comprising adding a detergent after the second predetermined time period.

24 . The method of claim 23 , wherein the detergent and bioluminescent enzyme may be added at the same time.

25 . The method of claim 23 , wherein the detergent is added prior to addition of the bioluminescent enzyme.

26 . The method

Assignees

• Promega Corp

Inventors

• Cali James J
• Klaubert Dieter
• Daily William
• Ho Samuel Kin Sang
• Frackman Susan
• Hawkins Erika
• Wood Keith V

Classifications

• G01N2500/10

  involving cells · CPC title

• G01N33/582 Primary

  with fluorescent label · CPC title

• G01N33/502 Primary

  for testing non-proliferative effects · CPC title

• C07D417/04

  directly linked by a ring-member-to-ring-member bond · CPC title

• G01N2333/90251

  with a definite EC number (1.14.13.-) · CPC title