Skip to main content
PMC home page
AAPS PharmSci logoLink to AAPS PharmSci
. 2003 May 7;5(2):88–98. doi: 10.1208/ps050218

High-Throughput screeening assays for CYP2B6 metabolism and inhibition usuing fluorogenic vivid substrates

Bryan D Marks 1,✉, Tony A Goossens 1, Heidi A Braun 1, Mary S Ozers 1, Ronald W Smith 1, Connie Lebakken 1, Olga V Trubetskoy 1,✉
PMCID: PMC2751525  PMID: 12866948

Abstract

CYP2B6 is a highly polymorphic P450 isozyme involved in the metabolism of endo-and xenobiotics with known implications for the activation of many procarcinogens resulting in carcinogenesis. However, lack of validated high-throughput screening (HTS) CYP2B6 assays has limited the current understanding and full characterization of this isozyme’s involvement in human drug metabolism. Here, we have developed and characterized a fluorescence-based HTS assay employing recombinant human CYP2B6 and 2 novel fluorogenic substrates (the Vivid CYP2B6 Blue and Cyan Substrates). Assay validation included testing the inhibitory potency of a panel of drugs and compounds known to be metabolized by this isozyme, including CYP2B6 substrates, inhibitors, and known inducers. Compound rankings based on inhibitory potency in the Vivid CYP2B6 Blue and Cyan Assays matched compound rankings based on relative affinity measurements from previously published data (Ki, Kd, or Km values) for the CYP2B6 isozyme. In conclusion, these assays are proven to be robust and sensitive, with broad dynamic ranges and kinetic parameters allowing screening in HTS mode of a large panel of compounds for CYP2B6 metabolism and inhibition, and are a valuable new tool for CYP2B6 studies.

Keywords: Cytochrome P450, CYP2B6, fluorescent substrate, drug metabolism, high-throughput screening (HTS)

References

  • 1.Guengerich FP. Human cytochrome P450 enzymes. In: Ortiz de Montellano PR, editor. Cytochrome P450: Structure, Mechanism and Biochemistry. 2nd ed. New York, NY: Plenum Press; 1995. pp. 473–535. [Google Scholar]
  • 2.Raucy JL, Allen SW. Recent advances in P450 research. Pharmacogenomics J. 2001;1:178–186. doi: 10.1038/sj.tpj.6500044. [DOI] [PubMed] [Google Scholar]
  • 3.Lang T, Klein K, Fischer J, et al. Extensive genetic polymorphism in the human CYP2B6 gene with impact on expression and function in human liver. Pharmacogenetics. 2001;11:399–415. doi: 10.1097/00008571-200107000-00004. [DOI] [PubMed] [Google Scholar]
  • 4.Nelson DR, Koymans L, Kamataki T, et al. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics. 1996;6:1–42. doi: 10.1097/00008571-199602000-00002. [DOI] [PubMed] [Google Scholar]
  • 5.Coukell AJ, Markham A. Clopidogrel. Drugs. 1997;54:745–750. doi: 10.2165/00003495-199754050-00006. [DOI] [PubMed] [Google Scholar]
  • 6.Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich FP. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther. 1994;270:414–423. [PubMed] [Google Scholar]
  • 7.Ekins S, Branden M, Ring BJ, Wrighton SA. Examination of purported probes of human CYP2B6. Pharmacogenetics. 1997;7:165–179. doi: 10.1097/00008571-199706000-00001. [DOI] [PubMed] [Google Scholar]
  • 8.Ekins S, Ring BJ, Binkley SN, Hall SD, Wrighton SA. Autoactivation and activation of the cytochrome P450s. Int J Clin Pharmacol Ther. 1998;36:642–651. [PubMed] [Google Scholar]
  • 9.Ekins S, Vandenbranden M, Ring BJ, et al. Further characterization of the expression in liver and catalytic activity of CYP2B6. J Pharmacol Exp Ther. 1998;286:1253–1259. [PubMed] [Google Scholar]
  • 10.Gervot L, Rochat B, Gautier JC, et al. Human CYP2B6: expression, inducibility and catalytic activities. Pharmacogenetics. 1999;9:295–306. doi: 10.1097/00008571-199906000-00004. [DOI] [PubMed] [Google Scholar]
  • 11.Granvil CP, Madan A, Sharkawi M, Parkinson A, Wainer IW. Role of CYP2B6 and CYP3A4 in the in vitro N-dechloroethylation of (R)-and (S)-ifosfamide in human liver microsomes. Drug Metab Dispos. 1999;27:533–541. [PubMed] [Google Scholar]
  • 12.Stevens JC, White RB, Hsu SH, Martinet M. Human liver CYP2B6-catalyzed hydroxylation of RP 73401. J Pharmacol Exp Ther. 1997;282:1389–1395. [PubMed] [Google Scholar]
  • 13.Faucette SR, Hawke RL, Lecluyse EL, et al. Validation of bupropion hydroxylation as a selective marker of human cytochrome P450 2B6 catalytic activity. Drug Metab Dispos. 2000;28:1222–1230. [PubMed] [Google Scholar]
  • 14.Kreth K, Kovar K, Schwab M, Zanger UM. Identification of the human cytochromes P450 involved in the oxidative metabolism of “Ecstasy”-related designer drugs. Biochem Pharmacol. 2000;59:1563–1571. doi: 10.1016/S0006-2952(00)00284-7. [DOI] [PubMed] [Google Scholar]
  • 15.Ekins S, Bravi G, Ring BJ, et al. Three-dimensional quantitative structure activity relationship analyses of substrates for CYP2B6. J Pharmacol Exp Ther. 1999;288:21–29. [PubMed] [Google Scholar]
  • 16.Wang Q, Halpert JR. Combined three-dimensional quantitative structure-activity relationship analysis of cytochrome P450 2B6 substrates and protein homology modeling. Drug Metab Dispos. 2002;30:86–95. doi: 10.1124/dmd.30.1.86. [DOI] [PubMed] [Google Scholar]
  • 17.Ekins S, Groot MJ, Jones JP. Pharmacophore and three-dimensional quantitative structure activity relationship methods for modeling cytochrome p450 active sites. Drug Metab Dispos. 2001;29:936–944. [PubMed] [Google Scholar]
  • 18.Kariv I, Fereshteh MP, Oldenburg KR. Development of a miniaturized 384-well high throughput screen for the detection of substrates of cytochrome P450 2D6 and 3A4 metabolism. J Biomol Screen. 2001;6:91–99. doi: 10.1177/108705710100600205. [DOI] [PubMed] [Google Scholar]
  • 19.Miller VP, Stresser DM, Blanchard AP, Turner S, Crespi CL. Fluorometric high-throughput screening for inhibitors of cytochrome P450. Ann N Y Acad Sci. 2000;919:26–32. doi: 10.1111/j.1749-6632.2000.tb06864.x. [DOI] [PubMed] [Google Scholar]
  • 20.Stresser DM, Blanchard AP, Turner SD, et al. Substrate-dependent modulation of CYP3A4 catalytic activity: analysis of 27 test compounds with four fluorometric substrates. Drug Metab Dispos. 2000;28:1440–1448. [PubMed] [Google Scholar]
  • 21.Chauret N, Dobbs B, Lackman RL, et al. The use of 3-[2-(N,N-diethyl-N-methylammonium)ethyl] 7-methoxy-4-methylcoumarin (AMMC) as a specific CYP2D6 probe in human liver microsomes. Drug Metab Dispos. 2001;29:1196–1200. [PubMed] [Google Scholar]
  • 22.Kent UM, Yanev S, Hollenberg PF. Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by n-propylxanthate. Chem Res Toxicol. 1999;12:317–322. doi: 10.1021/tx9802421. [DOI] [PubMed] [Google Scholar]
  • 23.Ariyoshi N, Miyazaki M, Toide K, Sawamura Y, Kamataki T. A single nucleotide polymorphism of CYP2B6 found in Japanese enhances catalytic activity by autoactivation. Biochem. Biophys Res Commun. 2001;281:1256–1260. doi: 10.1006/bbrc.2001.4524. [DOI] [PubMed] [Google Scholar]
  • 24.Zlokarnik G. Cytochrome P450 assays for miniaturized formats and library screening in Optimizing Lead Selection (Vol 1)-High Throughput Screening Assays, Advance Tech Monitor. 2000:37–69.
  • 25.Marks BD, Smith RW, Braun HA, et al. A novel HTS assay to screen for CYP2E1 metabolism and inhibition using a fluorogenic Vivid P450 substrate. ASSAY Drug Devel Technol. 2002;1(1–1):73–81. doi: 10.1089/154065802761001329. [DOI] [PubMed] [Google Scholar]
  • 26.Jinno H, Tanaka-Kagawa T, Ohno A, et al. Functional characterization of cytochrome P450 2B6 allelic variants. Drug Metab Dispos. 2003;31:398–403. doi: 10.1124/dmd.31.4.398. [DOI] [PubMed] [Google Scholar]
  • 27.Hiratsuka M, Takekuma Y, Endo N, et al. Allele and genotype frequencies of CYP2B6 and CYP3A5 in the Japanese population. Eur J Clin Pharmacol. 2002;58:417–421. doi: 10.1007/s00228-002-0499-5. [DOI] [PubMed] [Google Scholar]
  • 28.Yuan R, Madani S, Wei XX, Reynolds K, Huang SM. Evaluation of cytochrome p450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions. Drug Metab Dispos. 2002;30:1311–1319. doi: 10.1124/dmd.30.12.1311. [DOI] [PubMed] [Google Scholar]
  • 29.Hesse LM, Venkatakrishnan K, Court MH, et al. CYP2B6 mediates the in vitro hydroxylation of bupropion: potential drug interactions with other antidepressants. Drug Metab Dispos. 2000;28:1176–1183. [PubMed] [Google Scholar]
  • 30.Zhang W, Ramamoorthy Y, Kilicarslan T, Nolte H, Tyndale RF, Sellers EM. Inhibition of cytochrome P450 by antifungal imidazole derivatives. Drug Metab Dispos. 2002;30:314–318. doi: 10.1124/dmd.30.3.314. [DOI] [PubMed] [Google Scholar]
  • 31.Guo Z, Raeissi S, White RB, Stevens JC. Orphenadrine and methimazole inhibit multiple cytochrome P450 enzymes in human liver microsomes. Drug Metab Dispos. 1997;25:390–393. [PubMed] [Google Scholar]
  • 32.Nakajima M, Yoshida R, Shimada N, Yamazaki H, Yokoi T. Inhibition and inactivation of human cytochrome P450 isoforms by phenethyl isothiocyanate. Drug Metab Dispos. 2001;29:1110–1113. [PubMed] [Google Scholar]
  • 33.Court MH, Duan SX, Hesse LM, Venkatakrishnan K, Greenblatt DJ. Cytochrome P-450 2B6 is responsible for interindividual variability of propofol hydroxylation by human liver microsomes. Anesthesiology. 2001;94:110–119. doi: 10.1097/00000542-200101000-00021. [DOI] [PubMed] [Google Scholar]
  • 34.Rae JM, Soukhova NV, Flockhart DA, Desta Z. Triethylenethiophosphoramide is a specific inhibitor of cytochrome P450 2B6: implications for cyclophosphamide metabolism. Drug Metab Dispos. 2002;30:525–530. doi: 10.1124/dmd.30.5.525. [DOI] [PubMed] [Google Scholar]
  • 35.Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen. 1999;4:67–73. doi: 10.1177/108705719900400206. [DOI] [PubMed] [Google Scholar]
  • 36.Code EL, Crespi CL, Penman BW, Gonzalez FJ, Chang TK, Waxman DJ. Human cytochrome P4502B6: interindividual hepatic expression, substrate specificity, and role in procarcinogen activation. Drug Metab Dispos. 1997;25:985–993. [PubMed] [Google Scholar]
  • 37.DeLuca JG, Dysart GR, Rasnick D, Bradley MO. A direct, highly sensitive assay for cytochrome P-450 catalyzed O-deethylation using a novel coumarin, analog. Biochem Pharmacol. 1988;37:1731–1739. doi: 10.1016/0006-2952(88)90436-4. [DOI] [PubMed] [Google Scholar]
  • 38.Ito K, Iwatsubo T, Kanamitsu S, Nakajima Y, Sugiyama Y. Quantitative prediction of in vivo drug clearance and drug interactions from in vitro data on metabolism, together with binding and transport. Annu Rev Pharmacol Toxicol. 1998;38:461–499. doi: 10.1146/annurev.pharmtox.38.1.461. [DOI] [PubMed] [Google Scholar]
  • 39.Pascussi JM, Gerbal-Chaloin S, Fabre JM, Maurel P, Vilarem MJ. Dexamethasone, enhances constitutive androstane receptor expression in human hepatocytes: consequences on cytochrome P450 gene regulation. Mol Pharmacol. 2000;58:1441–1450. doi: 10.1124/mol.58.6.1441. [DOI] [PubMed] [Google Scholar]
  • 40.Rae JM, Johnson MD, Lippman ME, Flockhart DA. Rifampin is a selective, pleiotropic inducer of drug metabolism genes in human hepatocytes: studies with cDNA and oligonucleotide expression arrays. J Pharmacol Exp Ther. 2001;299:849–857. [PubMed] [Google Scholar]

Articles from AAPS PharmSci are provided here courtesy of American Association of Pharmaceutical Scientists

RESOURCES