Luminescence-based methods and probes for measuring cytochrome P450 activity

摘要

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 luciferase inhibitors.

主权项

1. A method for determining the effect of a compound on cytochrome P450 enzyme activity in an animal comprising:
(a) administering a test compound to an animal; (b) administering a luminogenic molecule to the animal, wherein the luminogenic molecule is a cytochrome P450 substrate and a pro-substrate of bioluminescent enzyme; (c) obtaining a biological sample from said animal; (d) contacting the biological sample with a reaction mixture comprising a bioluminescent enzyme; and (e) determining cytochrome P450 enzyme activity of said animal after exposure of said animal to the test compound by measuring and comparing luminescence from said biological sample taken from a second biological sample taken from a second animal not exposed to said test compound; and wherein the luminogenic molecule is a compound of formula:wherein
R1 represents hydrogen, hydroxyl, amino, C1-20 alkoxy, substituted C1-20 alkoxy, C2-20 alkenyloxy, substituted C2-20 alkenyloxy, halogenated C2-20 alkoxy, substituted halogenated C2-20 alkoxy, C3-20 alkynyloxy, substituted C3-20 alkynyloxy, C3-20 cycloalkoxy, substituted C3-20 cycloalkoxy, C3-20 cycloalkylamino, substituted C3-20 cycloalkylamino, C1-20 alkylamino, substituted C1-20alkylamino, di C1-20alkylamino, substituted diC1-20alkylamino, C2-20 alkenylamino, substituted C2-20 alkenylamino, di C2-20alkenylamino, substituted di C2-20 alkenylamino, C2-20 alkenyl, C1-20alkylamino, substituted C2-20alkenyl C1-20alkylamino C3-20alkynylamino, substituted C3-20alkynylamino, di C3-20alkynylamino, substituted di alkylamino, C3-20alkynyl C2-20alkenylamino, or substituted C3-20alkynyl C2-20 alkenylamino; R2 and R3 independently represents C or N; R4 and R5 independently represents S, O, NR8, wherein R8 represents hydrogen or C1-20 alkyl, CR9R10 wherein R9 and R10 independently represent H, C1-20 alkyl, or fluorine; R6 represents CH2OH; COR11 wherein R11 represents H, OH, C1-20 alkoxide, C2-20 alkenyl, or NR12R13 wherein R12 and R13 are independently H, or C1-20 alkyl; or —OM+ wherein M+ is an alkali metal or a pharmaceutically acceptable salt; and R7 represent H, C1-6 alkyl, C1-20 alkenyl, halogen, or C1-6 alkoxide,with the proviso that R1 is not OH or NH2, R7 is not H, R6 is not COR11 R11 is not OH, R3 and R2 are not both carbon, and R4 and R5 are not both S at the same time (luciferin and aminoluciferin).