US 8,106,052 B2Granted

Luminescence-based methods and probes for measuring cytochrome P450 activity

AssigneePromega Corporation

InventorsJames J. Cali, Dieter Klaubert, William Daily, Samuel Kin Sang Ho, Susan Frackman, Erika Hawkins, Keith V. Wood

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Abstract

The present invention provides methods, compositions, 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. luciferin or coelenterazines, that are cytochrome P450 substrates and that are also bioluminescent enzyme, e.g., luciferase, pro-substrates are provided. Upon addition of the luciferin derivative or other luminogenic molecule into a P450 reaction, the P450 enzyme metabolizes the molecule into a bioluminescent enzyme substrate, e.g., luciferin and/or luciferin derivative metabolite, in a P450 reaction. The resulting metabolite(s) serves as a substrate of the bioluminescent enzyme, e.g., luciferase, in a second light-generating reaction. Luminescent cytochrome P450 assays with low background signals and high sensitivity are disclosed and isoform selectivity is demonstrated. The present invention also provides an improved method for performing luciferase reactions which employs added pyrophosphatase to remove inorganic pyrophosphate, a luciferase inhibitor which may be present in the reaction mixture as a contaminant or may be generated during the reaction. 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.

Claims13

Dependent claims

A method for measuring the activity of a cytochrome P450 enzyme comprising:

(a) contacting a luminogenic molecule with a least one cytochrome P450 enzyme and at least one luciferase enzyme to produce a reaction mixture, wherein the luminogenic molecule is a cytochrome P450 substrate and a pro-substrate of a luciferase enzyme 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 C1-20 alkylamino, di C1-20 alkylamino, substituted diC1-20 alkylamino, C2-20 alkenylamino, substituted C2-20 alkenylamino, di C2-20 alkenylamino, substituted di C2-20 alkenylamino, C2-20 alkenyl C1-20 alkylamino, substituted C2-20 alkenyl C1-20 alkylamino, C3-20 alkylamino, substituted C3-20 alkynylamino, di C3-20 alkynylamino, substituted di alkylamino, C3-20 alkynyl C2-20 alkenylamino, or substituted C3-20 alkynyl 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);
and
(b) determining cytochrome P450 activity by measuring luminescence of the reaction mixture.

10 more elements

A method for measuring cytochrome P450 enzyme activity in an animal comprising:

(a) administering a luminogenic molecule to the animal, wherein the luminogenic molecule is a cytochrome P450 substrate and a pro-substrate of a luciferase enzyme 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 C1-20 alkylamino, di C1-20 alkylamino, substituted diC1-20 alkylamino, C2-20 alkenylamino, substituted C2-20 alkenylamino, di C2-20 alkenylamino, substituted di C2-20 alkenylamino, C2-20 alkenyl C1-20 alkylamino, substituted C2-20 alkenyl C1-20 alkylamino, C3-20 alkynylamino, substituted C3-20 alkynylamino, di C3-20 alkynylamino, substituted di alkylamino, C3-20 alkynyl C2-20 alkenylamino, or substituted C3-20 alkynyl 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);
(b) obtaining a biological sample from the animal;
(c) contacting the biological sample with a luciferase enzyme;
and
(d) determining cytochrome P450 activity of the animal by measuring luminescence.

12 more elements

A method for measuring cytochrome P450 enzyme activity in a transgeneic animal having a bioluminescent enzyme transgene, said method comprising:

(a) administering a luminogenic molecule to a transgeneic animal having a luciferase enzyme transgene, wherein the luminogenic molecule is a cytochrome P450 substrate and a pro-substrate of the luciferase enzyme 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 cycloalkyamino, substituted C3-20 cycloalkylamino, C1-20 alkylamino, substituted C1-20 alkylamino, di C1-20 alkylamino, substituted diC1-20 alkylamino, C2-20 alkenylamino, substituted C2-20 alkenylamino, di C2-20 alkenylamino, substituted di C2-20 alkenylamino, C2-20 alkenyl C1-20 alkylamino, substituted C2-20 alkenyl C1-20 alkylamino, C3-20 alkynylamino, substituted C3-20 alkynylamino, di C3-20 alkynylamino, substituted di alkylamino, C3-20 alkynyl C2-20 alkenylamino, or substituted C3-20 alkynyl 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);
and
(b) determining cytochrome P450 activity of the animal by measuring luminescence of the transgeneic animal.

10 more elements

Dependencies are inferred from the claim text.

Facts

Application: 12/754,164Filed 5 Apr 2010 · division
Priority: 20 Sep 2002
Published: US 2011/0003316 A16 Jan 2011
Granted: 31 Jan 2012
Term ends: Not estimatedThe source records don’t support an estimate.

Classification13 codes

G01N 33/502
For testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics: for testing non-proliferative effects
A61K 49/0008
Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for…:…
C07D 417/04
Containing two hetero rings: directly linked by a ring-member-to-ring-member bond
C12Q 1/66
Measuring or testing processes involving enzymes, nucleic acids or microorganisms ; Compositions therefor; Processes of preparing such…

Family

Application60/412,254Us provisional application · filed 20 Sep 2002
Application60/483,309Us provisional application · filed 27 Jun 2003
Application10/665,314Parent of this division · filed 19 Sep 2003
US 2011/0003316 A1Pre-grant publication · published 6 Jan 2011
US 7,692,022 B2Parent of this division
US 2004/0171099 A1Published application
US 8,106,052 B2This patent

From the US filing history. Foreign family members aren’t listed.