Abstract
The mechanism-based inactivation (MBI) of the human cytochrome P450 (P450 or CYP) drug-metabolizing enzymes may lead to adverse drug–drug interactions, especially for drugs with narrow therapeutic windows. Unlike reversible inhibitors of P450, drug–drug interactions originating from MBI may persist in patients for some time after the body eliminates the offending drug because P450 enzymatic activity can be recovered only after de novo synthesis of the P450. In a pharmaceutical setting, a substantial amount of effort is often expended to understand the potential for mechanism-based inactivation and its possible contribution to the drug–drug interaction profile of drug candidates. Therefore, in vitro assays that identify and characterize which drug candidates are P450 MBIs are critically important in preclinical drug metabolism and pharmacokinetic studies. A detailed method is described for the adaptation of a 7-ethoxytrifluoromethyl coumarin O-deethylation fluorescence activity assay to a 96-well plate format to characterize the K I and k inact values for an MBI. The advantages of this microtiter format compared with the conventional method include a significant reduction in the amount of enzyme used, a reduction in assay time, and an increase in experimental throughput.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Evans WE, Relling MV (1999) Pharmacogenomics: translating functional genomics into rational therapeutics. Science 286:487–491
Hollenberg PF, Kent UM, Bumpus NN (2008) Mechanism-based inactivation of human cytochromes p450s: experimental characterization, reactive intermediates, and clinical implications. Chem Res Toxicol 21:189–205
Fontana E, Dansette PM, Poli SM (2005) Cytochrome p450 enzymes mechanism based inhibitors: common sub-structures and reactivity. Curr Drug Metab 6:413–454
Bachmann KA, Ring BJ, Wrighton SA (2003) Drug-drug interactions and cytochrome P450. FontisMedia SA, Switzerland
Walsky RL, Obach RS (2007) A comparison of 2-phenyl-2-(1-piperidinyl)propane (ppp), 1,1′,1″-phosphinothioylidynetrisaziridine (thioTEPA), clopidogrel, and ticlopidine as selective inactivators of human cytochrome P450 2B6. Drug Metab Dispos 35:2053–2059
Grimm SW, Einolf HJ, Hall SD, He K, Lim HK, Ling KH, Lu C, Nomeir AA, Seibert E, Skordos KW, Tonn GR, Van Horn R, Wang RW, Wong YN, Yang TJ, Obach RS (2009) The conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: a perspective of the pharmaceutical research and manufacturers of America. Drug Metab Dispos 37:1355–1370
Hong Y, Yu B, Sherman M, Yuan YC, Zhou D, Chen S (2007) Molecular basis for the aromatization reaction and exemestane-mediated irreversible inhibition of human aromatase. Mol Endocrinol 21:401–414
Vasaitis TS, Bruno RD, Njar VC (2011) CYP17 inhibitors for prostate cancer therapy. J Steroid Biochem Mol Biol 125:23–31
Blobaum AL, Harris DL, Hollenberg PF (2005) P450 active site architecture and reversibility: inactivation of cytochromes P450 2B4 and 2B4 T302A by tert-butyl acetylenes. Biochemistry 44:3831–3844
Lin HL, Kent UM, Hollenberg PF (2005) The grapefruit juice effect is not limited to cytochrome P450 (P450) 3A4: evidence for bergamottin-dependent inactivation, heme destruction, and covalent binding to protein in P450s 2B6 and 3A5. J Pharmacol Exp Ther 313:154–164
Buters JT, Schiller CD, Chou RC (1993) A highly sensitive tool for the assay of cytochrome P450 enzyme activity in rat, dog and man. Direct fluorescence monitoring of the deethylation of 7-ethoxy-4-trifluoromethylcoumarin. Biochem Pharmacol 46:1577–1584
DeLuca JG, Dysart GR, Rasnick D, Bradley MO (1988) A direct, highly sensitive assay for cytochrome P-450 catalyzed O-deethylation using a novel coumarin analog. Biochem Pharmacol 37:1731–1739
Kenaan C, Zhang H, Hollenberg PF (2010) A quantitative high-throughput 96-well plate fluorescence assay for mechanism-based inactivators of cytochromes P450 exemplified using CYP2B6. Nat Protoc 5:1652–1658
Zhang H, Amunugama H, Ney S, Cooper N, Hollenberg PF (2011) Mechanism-based inactivation of human cytochrome P450 2B6 by clopidogrel: involvement of both covalent modification of cysteinyl residue 475 and loss of heme. Mol Pharmacol 80:839–847
Amunugama H, Zhang H, Hollenberg PF (2012) Mechanism-Based Inactivation of Cytochrome P450 2B6 by Methadone through Destruction of Prosthetic Heme. Drug Metab Dispos 40:1765–1770
Foti RS, Rock DA, Pearson JT, Wahlstrom JL, Wienkers LC (2011) Mechanism-based inactivation of cytochrome P450 3A4 by mibefradil through heme destruction. Drug Metab Dispos 39:1188–1195
Ernest CS 2nd, Hall SD, Jones DR (2005) Mechanism-based inactivation of CYP3A by HIV protease inhibitors. J Pharmacol Exp Ther 312:583–591
Yamazaki H, Shimada T (2006) Cytochrome P450 reconstitution systems. Methods Mol Biol 320:61–71
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Kenaan, C., Zhang, H., Hollenberg, P.F. (2013). High-Throughput Fluorescence Assay for Cytochrome P450 Mechanism-Based Inactivators. In: Phillips, I., Shephard, E., Ortiz de Montellano, P. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology, vol 987. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-321-3_5
Download citation
DOI: https://doi.org/10.1007/978-1-62703-321-3_5
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-320-6
Online ISBN: 978-1-62703-321-3
eBook Packages: Springer Protocols