Promega Corporation

Cytochrome P450 4F2 and 4F3B Enzyme Assays Using a Novel Bioluminescent Probe Substrate

Share

  • Share
  • Print
  • Email
  • Download PDF

Abstract

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

Mary Sobol, Dongping Ma, Carolyn C. Woodroofe and James J. Cali

Promega Corporation
Publication Date: 2008

Introduction

CYP4F2 and CYP4F3B are cytochrome P450 enzymes (CYPs) that catalyze ω-hydroxylation of fatty acids and arachidonic acid and that metabolize certain drugs (1) (2) (3) . Enzyme assays for CYP4F2 and CYP4F3B typically include a chromatographic separation step that limits ease-of-use and throughput. Thus, there is a need for simple, multiwell plate-based CYP4F2 and CYP4F3 assays for rapid enzyme analyses and inhibitor screening applications.

Luminogenic CYP assays use prosubstrates for the light-generating reaction of firefly luciferase. CYPs convert the prosubstrates to luciferin, which makes light in a second reaction with a luciferase reaction mix called Luciferin Detection Reagent (LDR) (4) (5) . 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 (6) . The CYP enzyme selectivity for a given luminogenic substrate depends on the nature of the derivatization on the luciferin structure.

Here we demonstrated that 2-(6-(4-(methylthio)benzyloxy)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid, a luciferin derivative referred to as Luciferin-4F2/3, is converted to luciferin, most prominently by CYP4F3B and has substantial activity with CYP4F2. Luciferin-4F2/3 can be used for luminogenic CYP4F3B and CYP4F2 enzyme assays 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 coexpressed a human CYP cDNA with P450 reductase or P450 reductase plus cytochrome b5 (Gentest™ Supersomes™, BD Biosciences). A 50mM stock solution of Luciferin-4F2/3 (MW = 416.53) was prepared in acidified dimethylsulfoxide (DMSO with 50mM HCl).* The CYP4F2 or CYP4F3B enzyme assays were performed using 25–50µM Luciferin-4F2/3 substrate (Cat.# P1651) or as indicated in figure legends, 50mM KPO4 (pH 7.4), 20nM CYP enzyme coexpressed with P450 reductase and cytochrome b5 (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 or 20 minutes at 37°C, 50µl of Luciferin Detection Reagent (LDR; Cat.# V8920, V8921) was added to each 50µl CYP reaction to stop the reactions 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 (200mM KPO4 [pH 7.4], 200µM Luciferin-4F2/3, 80nM CYP4F2 or CYP4F3B)*, 4X concentrated test compound solution (e.g., for CYP inhibition assays) and 2X concentrated NADPH Regeneration System (7) ,(8) . For a 50µl reaction in a 96-well plate, 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.

*Note: Luciferin-4F2/3 solubility in KPO4 buffer is improved when the 50mM DMSO stock solution is first diluted to an intermediate concentration of 0.2mM in 100mM Tris-HCl (pH 7.5).

Results and Discussion

Substrate Selectivity

The putative luminogenic CYP substrate, Luciferin-4F2/3, 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 prominent activity with CYP4F3B, a relatively smaller activity with CYP4F2, and minor activities with CYP4F3A and CYP4A11.

CYP enzyme selectivity for the Luciferin-4F2/3 substrate.Figure 1. CYP enzyme selectivity for the Luciferin-4F2/3 substrate.

The anticipated reaction scheme with Luciferin-4F2/3 (I) is shown at the top. Luminescence generated by LDR depends on the conversion of I to luciferin (II). For control samples, CYP enzyme was replaced with microsomes devoid of CYP activity from the insect cell CYP expression system. Fifty microliter reactions with 50μM Luciferin-4F2/3 and 20nM recombinant human CYP enzymes in 96-well plates were incubated for 30 minutes at 37°C. Values are mean ± SD, n = 3.

Time Dependence

A time-dependent increase in luminescence reflecting luciferin accumulation was observed at room temperature and 37°C for up to 60 minutes (Figure 2).

Time course of the CYP4F3B reaction with Luciferin-4F2/3.Figure 2. Time course of the CYP4F3B reaction with Luciferin-4F2/3.

Time-dependent changes in net luminescence were monitored at room temperature (20.5°C) and 37°C. Fifty microliter reactions were performed in 96-well plates with 50µM Luciferin-4F2/3 and 20nM CYP4F3B in 50mM KPO4 (pH 7.4), 50mM Tris-HCl (pH 7.5). 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. Background luminescence from control samples with no CYP enzyme (mean background = 1969 RLU at 20.5°C or 2772 RLU at 37°C) was subtracted to give the net luminescence values shown (mean ± SD, n = 3; error bars not visible due small SD).

Substrate Concentration

The CYP4F3B reaction with Luciferin-4F2/3 showed atypical kinetics with sigmoidicity at the low end of the curve, indicating homotropic cooperativity and substrate inhibition at the high end(9) .

Substrate-concentration dependence of CYP4F3B activity with Luciferin-4F2/3.Figure 3. Substrate-concentration dependence of CYP4F3B activity with Luciferin-4F2/3.

Reactions with 20nM CYP4F3B, 50mM KPO4 (pH 7.4), 25mM Tris-HCl (pH 7.5) and 0–60µM Luciferin-4F2/3 were incubated for 10 minutes at 37°C. 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).

CYP4F3B Inhibition

The CYP4F3B assay with Luciferin-4F2/3 was tested as a tool for measuring CYP4F3B inhibition (Figure 4). Dose-dependent inhibition by 17-octadecynoic acid, a known CYP4F3B inhibitor (10) , was observed (IC50 = 18.4µM).

Measuring CYP4F3B inhibition using Luciferin-4F2/3.Figure 4. Measuring CYP4F3B inhibition using Luciferin-4F2/3.

Using 10nM CYP4F3B, enzyme inhibition was assayed using 25µM Luciferin-4F2/3 in the presence of 17-octadecynoic acid at the indicated concentrations. The 17-octadecynoic acid was diluted from a 50mM stock solution in DMF, and the vehicle was kept constant at 0.4% in all reactions. Reactions were incubated at 37°C for 10 minutes before adding LDR. Background luminescence from control samples with no CYP enzyme (mean background = 2169 RLU) was subtracted to give the net luminescence values shown (mean ± SD, n=3).

Conclusion

Luciferin-4F2/3, a novel luciferin derivative, is a luminogenic cytochrome P450 substrate with a high degree of selectivity for CYP4F3B and CYP4F2. Using Luciferin-4F2/3 with a CYP enzyme assay approach harnesses the exquisite sensitivity, selectivity and simplicity of bioluminescence. This provides simple, rapid, multiwell plate-based CYP4F2 and CYP4F3B enzyme assays for analysis and screening.

References

  1. Christmas, P. et al. (2001) Alternative splicing determines the function of CYP4F3 by switching substrate specificity. J. Biol. Chem. 276, 38166–72.
  2. Lasker, J.M. et al. (2000) Formation of 20-hydroxyeicosatetraenoic acid, a vasoactive and natriuretic eicosanoid, in human kidney. J. Biol. Chem. 275, 4118–26.
  3. Wang, M.Z. et al. (2007) Human enteric microsomal CYP4F enzymes O-demethylate the antiparasitic prodrug paruramidine. Drug Metab. Dispos. 35, 2067–75.
  4. Branchini, B.R. et al. (1998) Site-directed mutagenesis of histidine 245 in firefly luciferase: A proposed model of the active site. Biochemistry 37, 15311–9.
  5. Cali, J.J. et al. (2006) Luminogenic cytochrome P450 assays. Exp. Op. Drug Metab. Toxicol. 2, 629–45.
  6. Guengerich, F.P. (2006) Cytochrome P450s and other enzymes in drug metabolism and toxicity. AAPS J. 8, E101–11.
  7. P450-GloAssays Technical Bulletin, TB325, Promega Corporation.
  8. P450-GloScreening Systems Technical Bulletin, TB340, Promega Corporation.
  9. Tracy, T.S. (2006) Atypical cytochrome p450 kinetics: Implications for drug discovery. Drugs R. D. 7, 349–63.
  10. Wang, M.Z. et al. (2006) CYP4F enzymes are the major enzymes in human liver microsomes that catalyze the O-demethylation of the antiparasitic prodrug DB289 [2,5-Bis(4-amidophenyl)furan-bis-O-methylamidoxime]. Drug Metab. Dispos. 34, 1985–94.

How to Cite This Article

Sobol, M., Ma, D., Woodroofe, C. C. and Cali, J. J. Cytochrome P450 4F2 and 4F3B Enzyme Assays Using a Novel Bioluminescent Probe Substrate. [Internet] 2008. [cited: year, month, date]. Available from: http://kr.promega.com/resources/pubhub/enotes/cytochrome-p450-4f2-and-4f3b-enzyme-assays-using-a-novel-bioluminescent-probe-substrate/

Sobol, M., Ma, D., Woodroofe, C. C. and Cali, J. J. Cytochrome P450 4F2 and 4F3B Enzyme Assays Using a Novel Bioluminescent Probe Substrate. Promega Corporation Web site. http://kr.promega.com/resources/pubhub/enotes/cytochrome-p450-4f2-and-4f3b-enzyme-assays-using-a-novel-bioluminescent-probe-substrate/ Updated 2008. Accessed Month Day, Year.

GloMax is a registered trademark of and P450-Glo is a trademark of Promega Corporation.

Gentest and Supersomes are trademarks of Becton, Dickinson and Company.

Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information.

Figures

CYP enzyme selectivity for the Luciferin-4F2/3 substrate.Figure 1. CYP enzyme selectivity for the Luciferin-4F2/3 substrate.

The anticipated reaction scheme with Luciferin-4F2/3 (I) is shown at the top. Luminescence generated by LDR depends on the conversion of I to luciferin (II). For control samples, CYP enzyme was replaced with microsomes devoid of CYP activity from the insect cell CYP expression system. Fifty microliter reactions with 50μM Luciferin-4F2/3 and 20nM recombinant human CYP enzymes in 96-well plates were incubated for 30 minutes at 37°C. Values are mean ± SD, n = 3.

Time course of the CYP4F3B reaction with Luciferin-4F2/3.Figure 2. Time course of the CYP4F3B reaction with Luciferin-4F2/3.

Time-dependent changes in net luminescence were monitored at room temperature (20.5°C) and 37°C. Fifty microliter reactions were performed in 96-well plates with 50µM Luciferin-4F2/3 and 20nM CYP4F3B in 50mM KPO4 (pH 7.4), 50mM Tris-HCl (pH 7.5). 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. Background luminescence from control samples with no CYP enzyme (mean background = 1969 RLU at 20.5°C or 2772 RLU at 37°C) was subtracted to give the net luminescence values shown (mean ± SD, n = 3; error bars not visible due small SD).

Substrate-concentration dependence of CYP4F3B activity with Luciferin-4F2/3.Figure 3. Substrate-concentration dependence of CYP4F3B activity with Luciferin-4F2/3.

Reactions with 20nM CYP4F3B, 50mM KPO4 (pH 7.4), 25mM Tris-HCl (pH 7.5) and 0–60µM Luciferin-4F2/3 were incubated for 10 minutes at 37°C. 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).

Measuring CYP4F3B inhibition using Luciferin-4F2/3.Figure 4. Measuring CYP4F3B inhibition using Luciferin-4F2/3.

Using 10nM CYP4F3B, enzyme inhibition was assayed using 25µM Luciferin-4F2/3 in the presence of 17-octadecynoic acid at the indicated concentrations. The 17-octadecynoic acid was diluted from a 50mM stock solution in DMF, and the vehicle was kept constant at 0.4% in all reactions. Reactions were incubated at 37°C for 10 minutes before adding LDR. Background luminescence from control samples with no CYP enzyme (mean background = 2169 RLU) was subtracted to give the net luminescence values shown (mean ± SD, n=3).

Prefer a different language?

Your country is set to Korea, Republic Of. Your language is set to 한국어. Please select the language that will best suit your needs:

This is correct, continue to site »

I need additional help

It appears that you have Javascript disabled. Our website requires Javascript to function correctly. For the best browsing experience, please enable Javascript.