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Cytochrome P450 2J2 Enzyme Assay Using a Novel Bioluminescent Probe Substrate

Dongping Ma, Mary Sobol, Carolyn C. Woodroofe and James J. Cali
Promega Corporation
Publication Date: 2008

Abstract

This is part of a series of seven articles describing new bioluminescent substrates to be used for drug screening and assaying enzymatic activity.

Introduction

CYP2J2 is a cytochrome P450 enzyme (CYP) that catalyzes the formation of arachidonic acid metabolites that regulate blood pressure, cell proliferation and inflammation (1) , (2) , (3) . CYP2J2 is expressed most prominently in the human heart, but it also contributes to the intestinal metabolism of certain drugs (4) , (5) . Enzyme assays for CYP2J2 typically include a chromatographic separation step that limits ease-of-use and throughput; consequently, there is a need for a simple, multiwell plate-based CYP2J2 assay that is met by the luminogenic assay described here.

Luminogenic CYP assays use prosubstrates for the light-generating reaction of firefly luciferase. CYPs convert the prosubstrates to luciferin or a luciferin ester, which produces light in a second reaction with a luciferase reaction mix called Luciferin Detection Reagent (LDR) (6) , (7) . The amount of light generated is proportional to the amount of luciferin produced by the CYP and therefore, to CYP enzyme activity. Multiple CYP enzymes are encoded by families of genes in humans and other organisms (8) . The CYP enzyme selectivity for a given luminogenic substrate depends on the nature of the derivatization on the luciferin structure.

Here we demonstrate that 2-hydroxyethyl 2-(6-(3,3-dimethoxypropoxy)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylate, a luciferin derivative referred to as Luciferin-2J2/4F12, is converted to a luciferin ester, most prominently by CYP2J2 and to a lesser extent by CYP4F12, CYP1A1, CYP1B1 and CYP3A4. We describe how Luciferin-2J2/4F12 can be used for luminogenic CYP2J2 enzyme assays for detecting CYP2J2 activity and inhibitors in a multiwell plate format.

Materials and Methods

The CYP assays were performed using instructions in the P450-Glo™ Assay Technical Bulletin #TB325 and P450-Glo™ Screening Systems Technical Bulletin #TB340. The CYP enzymes used were recombinant human forms in microsomes from insect cells that coexpress a human CYP cDNA with P450 reductase or P450 reductase plus cytochrome b5 (Gentest™ Supersomes™, BD Biosciences). A 50mM stock solution of Luciferin-2J2/4F12 (molecular weight = 426.5) was prepared in acetonitrile. The CYP2J2 enzyme assays were performed using 1µM Luciferin-2J2/4F12 substrate or as indicated in figure legends, 100mM KPO4 (pH 7.4), 2nM CYP2J2 enzyme coexpressed with P450 reductase and cytochrome b5 (0.1pmol/50µl reaction), and 1X NADPH Regeneration System (Cat.# V9510).

Assays were assembled and performed in opaque white 96-well plates (e.g., white polystyrene, 96-well plates [Costar Cat.# 3912]). After incubating reactions for 10 minutes at 37°C, 50µl of LDR with esterase (Cat.# V8930, V8931) was added to each 50µl CYP reaction to stop the reaction and initiate luminescence. The luminescence was read after 20 minutes using the GloMax® 96 Microplate Luminometer (Cat.# E6501), and reported in relative light units (RLU).

For convenience, you can prepare a 4X concentrated enzyme/buffer/substrate mix (400mM KPO4 [pH 7.4], 4µM Luciferin-2J2/4F12, 8nM CYP2J2), 4X concentrated test compound solution (e.g., for CYP inhibition assays) and 2X concentrated NADPH Regeneration System (9) , (10) . For a 50µl reaction, combine 12.5µl of the 4X enzyme mixture with 12.5µl of 4X test compound and initiate the reaction by adding 25µl of the NADPH Regeneration System.

CYP4F12 assays with Luciferin-2J2/4F12 have been performed using a 4X enzyme/buffer/substrate mix which contained 400mM KPO4 (pH 7.4), 200µM Luciferin-2J2/4F12 and 80nM CYP4F12. The reactions were incubated at 37°C for 30 minutes.

Results and Discussion

Substrate Selectivity

The putative luminogenic CYP substrate, Luciferin-2J2/4F12, was initially screened for activity in the luminescent assay format against 21 recombinant human CYP enzymes (Figure 1). Under the conditions used in Figure 1, we observed activities with CYP1A1, CYP1B1, CYP2J2, CYP3A4 and CYP4F12, but the most prominent activity detected was with CYP2J2. Optical substrates for CYP1A1, CYP1B1 and CYP3A4 have been described frequently (7) , (11) , and luminogenic substrates previously described for CYP2J2 and CYP4F12 were turned over at relatively slow rates (7) . Therefore, we considered that the present compound would be most useful for CYP2J2 and CYP4F12 assays.

7748MA.epsFigure 1. CYP enzyme selectivity for the Luciferin-2J2/4F12 substrate.

An anticipated reaction scheme with Luciferin-2J2/4F12 (I) is shown at the top. Intensity of luminescence generated by LDR depends on the conversion of I to luciferin (II). Fifty microliter reactions with 50µM Luciferin-2J2/4F12, 20nM recombinant human CYP enzymes and a no-CYP control in 96-well plates were incubated for 30 minutes at 37°C. Values are mean ± SD, n = 3.

Time Dependence

A linear time-dependent increase in luminescence CYP2J2 activity was observed at 37°C for up to 40 minutes and for the full 60 minutes of the experiment at 21°C (Figure 2).

7749MA.epsFigure 2. Time course of the CYP2J2 reaction with Luciferin-2J2/4F12.

Time-dependent changes in net luminescence were monitored at room temperature (21°C) and 37°C. Fifty microliter reactions were performed in 96-well plates with 1µM Luciferin-2J2/4F12 (from a 1mM stock solution in acetonitrile) and 2nM CYP2J2 in 100mM KPO4 (pH 7.4). CYP reactions were initiated by staggered addition of the NADPH Regeneration System. All CYP reactions were simultaneously terminated and luciferase reactions initiated by adding 50µl of LDR. Zero-time values were measured in samples where the NADPH Regeneration System was withheld until after LDR addition. The luminescence shown was total luminescence of samples minus luminescence of control samples with no CYP enzyme (mean background = 670 RLU at 21°C or 776 RLU at 37°C). Values are mean ± SD, n = 3.

Substrate Concentration

A saturable hyperbolic Luciferin-2J2/4F12 dose response with CYP2J2 was observed with a Km of 1.0µM (Figure 3).

7751MA.epsFigure 3. Km measurement of CYP2J2 using Luciferin-2J2/4F12.

Reactions with 2nM CYP2J2 and 100mM KPO4 (pH 7.4) were incubated for 10 minutes at 37°C. The curve fit and Km calculation were performed using GraphPad Prism® software. Background luminescence from control samples with no CYP enzyme at each substrate concentration was subtracted to give the net luminescence values shown (mean ± SD, n = 3).

CYP2J2 Selectivity

The subset of CYP enzymes that showed activity with Luciferin-2J2/4F12 (Figure 1) were assayed under conditions that were optimal for CYP2J2 (Figure 4). Under these conditions, the substrate had enhanced selectivity for CYP2J2.

7752MA.epsFigure 4. Testing the selectivity of Luciferin-2J2/4F12 substrate using the CYP2J2 enzyme.

The subset of enzymes that tested positive with Luciferin-2J2/4F12 (Figure 1) were reassayed under conditions that favor CYP2J2: 2nM each CYP or a no-CYP control, and 1µM Luciferin-2J2/4F12 in 100mM KPO4 (pH 7.4) for 10 minutes at 37°C. Values are mean ± SD, n = 3.

CYP2J2 Inhibition

The CYP2J2 assay with Luciferin2J2/4F12 at the Km concentration of 1.0µM was tested as a tool for measuring CYP2J2 inhibition (Figure 5). Dose dependent CYP2J2 inhibition by terfenadine was observed (IC50 = 0.3µM). Terfenadine is a CYP2J2 substrate acting as a competitive inhibitor. The IC50 correlates closely with the published Km of 0.4µM for the CYP2J2 reaction with terfenadine (12) .

7753MA.epsFigure 5. Measuring CYP2J2 inhibition using Luciferin-2J2/4F12.

Using 2nM CYP2J2, enzyme inhibition was assayed with 1µM Luciferin-2J2/4F12 in the presence of terfenadine (Sigma Aldrich) at the indicated concentrations. The terfenadine was diluted from a 50mM stock solution in DMSO, and this vehicle was kept constant at 0.1% in all reactions. Background luminescence from control samples with no CYP enzyme (mean background = 633 RLU) was subtracted to give the net luminescence values shown (mean ± SD, n = 3).

Conclusion

Luciferin-2J2/4F12 is a luminogenic CYP enzyme substrate with selectivity for human CYP2J2, CYP4F12, CYP3A4, CYP1A1 and CYP1B1, and a strong selective preference for CYP2J2 under conditions that favor this enzyme. The application of Luciferin-2J2/4F12 to the luminogenic CYP enzyme assay approach harnesses the exquisite sensitivity, selectivity and simplicity of bioluminescence. This provides for simple, rapid, multiwell plate-based CYP enzyme assays and enzyme inhibition assays, as demonstrated for the CYP2J2 enzyme in the above experiments.

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Article References

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How to Cite This Article

Scientific Style and Format, 7th edition, 2006

Ma D, Sobol M, Woodroofe, C C and Cali J J Cytochrome P450 2J2 Enzyme Assay Using a Novel Bioluminescent Probe Substrate. [Internet] 2008. [cited: year, month, date]. Available from: https://www.promega.com/resources/pubhub/enotes/cytochrome-p450-2j2-enzyme-assay-using-a-novel-bioluminescent-probe-substrate/

American Medical Association, Manual of Style, 10th edition, 2007

Ma D, Sobol M, Woodroofe, C C and Cali J J Cytochrome P450 2J2 Enzyme Assay Using a Novel Bioluminescent Probe Substrate. Promega Corporation Web site. https://www.promega.com/resources/pubhub/enotes/cytochrome-p450-2j2-enzyme-assay-using-a-novel-bioluminescent-probe-substrate/ Updated 2008. Accessed Month Day, Year.

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