Selective Cytochrome P450 4A11 Enzyme Assay Using a Novel Bioluminescent Probe Substrate

Introduction

CYP4A enzymes are cytochromes P450 that catalyze the ω-hydroxylation of fatty acids and the formation of arachidonic acid metabolites⁽¹⁾ . Genes encoding CYP4A enzymes are induced by agonists of the peroxisome proliferator-activated receptor alpha (PPARα) nuclear receptor⁽²⁾ . CYP4A assays, including human CYP4A11 and rat CYP4A1, CYP4A2 and CYP4A3, commonly require chromatographic steps or cell lysate preparations that limit ease-of-use and throughput. There is a need for simple, rapid, multiwell plate-based biochemical CYP4A enzyme assays and cell-based CYP4A induction assays.

Luminogenic CYP assays use prosubstrates for the light-generating reaction of firefly luciferase. CYPs convert the prosubstrates to an active luciferin, which makes light in a second reaction with a luciferase reaction mix called Luciferin Detection Reagent (LDR)⁽³⁾ . 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⁽⁴⁾ . The CYP enzyme selectivity for a given luminogenic substrate depends on the nature of the derived luciferin core structure.

Here we demonstrate that a luciferin derivative, 2-(6-methoxyquinolin-2-yl)-4,5-dihydrothiazole-4-carboxylic acid, referred to as Luciferin-4A, is selectively converted to quinolylluciferin, an active alternative to native beetle luciferin⁽⁵⁾ , by the human CYP4A11 enzyme. We describe how this luciferase prosubstrate was used in a luminogenic CYP4A11 biochemical assay and also in a cell-based assay that measures CYP4A basal and induced activity in intact rat hepatocytes.

Materials and Methods

The CYP4A11 enzyme assay was performed using the instructions in the P450-Glo™ Assay Technical Bulletin TB325 and P450-Glo™ Screening Systems Technical Bulletin TB340. The cell-based CYP4A assay followed the protocol in TB325.

CYP enzymes used were recombinant human forms in microsomes from insect cells that coexpressed a human CYP cDNA with P450 reductase or P450 reductase plus cytochrome b5 (Gentest™ Supersomes™, BD Biosciences).

A 5mM stock solution of Luciferin-4A (molecular weight = 288.3) was prepared in 100mM KPO₄ (pH 7.4). Reaction conditions specific to CYP4A11 enzyme assays included: 80μM Luciferin-4A substrate (Cat.# P1621), 100mM Tris-HCl (pH 7.5), 20nM CYP4A11 enzyme (1pmol/50μl reaction), and 1X NADPH Regeneration System (Cat.# V9510).

Biochemical assays were assembled and performed in opaque white 96-well plates (e.g., white polystyrene, 96-well plates [Costar Cat.# 3912]). Reactions were incubated for 10 minutes at 37°C or room temperature (20–23°C), then 50μl of Luciferin Detection Reagent (LDR; Cat.# V8920, V8921) was added to each 50μl CYP sample to terminate the reaction and initiate luminescence. After 20 minutes, luminescence was detected using the GloMax^® 96 Microplate Luminometer (Cat.# E6501) and reported as relative light units (RLU).

For convenience, you can prepare a 4X enzyme/buffer/substrate mix (400mM Tris-HCl [pH 7.5], 320µM Luciferin-4A, 80nM CYP4A11), 4X concentrated test compound solution (e.g., for CYP inhibition assays) and 2X concentrated NADPH Regeneration System. For a 50μl reaction in a 96-well plate, combine 12.5μl of the 4X enzyme mixture with 12.5µl of the 4X test compound and initiate the reaction by adding 25μl of the NADPH solution ⁽⁶⁾ ,⁽⁷⁾ . The cell-based assay with rat hepatocytes is described in the legend for Figure 6.

Results and Discussion

Substrate Selectivity

The putative luminogenic CYP substrate was screened for activity in the luminescent assay format against 21 recombinant human CYP enzymes (Figure 1). Under the conditions described in the legend for Figure 1, minor activities with CYP1A2, CYP2C8 and CYP2C9 were observed, but the most prominent activity was with CYP4A11.

Figure 1. CYP enzyme selectivity for the Luciferin-4A substrate.

The anticipated reaction scheme with Luciferin-4A (I) is shown at the top. Luminescence generated by LDR depends on the conversion of I to quinolylluciferin (II). Fifty microliter reactions with 50µM Luciferin-4A and 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

At both room temperature and 37°C, a time-dependent increase in luminescence was observed that reflected quinolylluciferin accumulation (Figure 2).

Figure 2. Time course of CYP4A11 reaction with Luciferin-4A.

Time-dependent changes in net luminescence were monitored at room temperature (20.5°C) and 37°C. Fifty-microliter reactions with 50μM Luciferin-4A and 20nM CYP4A11 were performed in 96-well plates. CYP reactions were initiated by staggered addition of the NADPH Regeneration System. All 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. Values are mean ± SD, n = 3.

Substrate Concentration

A saturable hyperbolic Luciferin-4A dose response with CYP4A11 was observed with a K_m of 80μM (Figure 3).

Figure 3. K_m measurement of CYP4A11 using Luciferin-4A.

Reactions contained 20nM CYP4A11 and were incubated for 30 minutes at 37°C. Luciferin-4A caused partial inhibition of luciferase at the upper end of the concentration range tested. After stopping the reaction with LDR, compensation for the luciferase inhibition was made by adjusting the substrate concentration in all reactions to the highest concentration in the range. Values are mean ± SD, n = 3.

Enzyme Concentration

Signals from the reaction at 80μM Luciferin-4A increased in a linear fashion with increasing CYP4A11 enzyme concentration up to 40nM of the enzyme (Figure 4). A significant signal-to-background ratio was measured at the lowest concentration of CYP4A11 tested (20pM). By extrapolation of the linear regression the limit of detection (LOD) is 2pM, when LOD is defined as the background mean plus three times its standard deviation.

Figure 4. Titration of CYP450 enzyme.

Reactions were performed with a range of CYP4A11 concentrations and 80μM Luciferin-4A, and incubated for 10 minutes at 37°C. Values are mean ± SD, n = 3.

CYP4A11 Inhibition

We tested CYP4A11 inhibition using the CYP4A11 assay with 80μM Luciferin-4A, the K_m concentration (Figure 5). Dose-dependent inhibition by the known CYP4A11 inhibitor, 17-octadecynoic acid (17-ODA), was observed⁽⁸⁾ .

Figure 5. Measuring CYP4A11 inhibition.

Using CYP4A11 (10nM), enzyme inhibition was assayed with 80μM Luciferin-4A in the presence of 17-ODA (Cayman Chemicals) at the indicated concentrations. The 17-ODA was diluted from a 50mM stock solution in dimethylformamide, and the concentration of this vehicle was kept constant at 0.2% in all reactions. Reactions were incubated at 37°C for 10 minutes. Values are mean ± SD, n = 3.

CYP4A Cell-Based Induction Assay

Levels of the CYP4A1 and CYP4A3 enzymes, rat orthologues of human CYP4A11, are induced by certain drugs⁽⁹⁾ . An assay was performed to determine if basal and induced CYP4A activity could be measured with Luciferin-4A in intact cells. Luciferin-4A was applied to monolayer cultures of rat hepatocytes that had been exposed to the CYP4A-inducing drug clofibrate and to vehicle controls (Figure 6). Basal luminescence from controls was in substantial excess over the assay background (basal signal:background = 50), activities were increased five-fold by clofibrate, and the basal and induced activities were inhibited by 17-ODA. Induction by a known CYP4A inducer and inhibition by a known CYP4A inhibitor are consistent with selectivity for rat CYP4A activity.

Figure 6. Cell-based CYP4A induction assay.

Rat hepatocytes in 96-well plates were treated for 48 hours in a 37°C cell culture incubator with 100µM clofibrate (Sigma-Aldrich) or vehicle (0.1% ethanol) for basal measurements in 100µl/well of InVitroGRO™ HI medium (Celsis). Medium was replaced with 80µM Luciferin-4A in medium (60µl/well), with or without 100µM 17-ODA (vehicle = 0.2% dimethylformamide/5mM Tris-HCl, [pH 8.5]) and returned to the incubator for 1 hour. Fifty microliters from each well was transferred to an opaque white 96-well plate and combined with 50µl of LDR. Luminescence was read after 20 minutes. Relative cell counts were measured by applying the CellTiter-Glo® Luminescent Cell Viability Assay (Cat.# G7570) to the cells left intact in culture wells. Luminescence (left axis, gold bars) is net RLU: total RLU minus the cell-free background (mean background RLU = 833). Luminescence/Cell Count (right axis, blue bars) is net RLU/CellTiter-Glo® Assay values expressed as percent of basal activity where the basal mean was set arbitrarily to 100%. Values are mean ± SD, n = 6.

Conclusion

Luciferin-4A, a novel luciferin derivative, is a luminogenic CYP substrate with excellent selectivity for the human CYP4A11 enzyme. Results also show selectivity for rat CYP4A enzymes in rat hepatocytes. The application of Luciferin-4A to the luminogenic CYP enzyme assay harnesses the exquisite sensitivity, selectivity and simplicity of bioluminescence. This provides for simple, rapid, multiwell plate-based CYP4A enzyme assays, CYP4A inhibition assays and cell-based CYP4A induction assays.