A quantitative high-throughput 96-well plate fluorescence assay for mechanism-based inactivators of cytochromes P450 exemplified using CYP2B6

Nature Protocols

Volumn 5, Issue 10, 2010, Pages 1652-1658

  Kenaan, Cesar ^a   Zhang, Haoming ^a   Hollenberg, Paul F ^a  

^a UNIVERSITY OF MICHIGAN MEDICAL SCHOOL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BERGAMOTTIN; CYTOCHROME P450 2B6; ISOPROTEIN;

ARTICLE; CONTROLLED STUDY; ENZYME ACTIVITY; ENZYME INACTIVATION; ENZYME MECHANISM; FLUORESCENCE ANALYSIS; HIGH THROUGHPUT SCREENING; MICROTITER PLATE ASSAY; PRIORITY JOURNAL; QUANTITATIVE ANALYSIS;

AMINO ACIDS; ARYL HYDROCARBON HYDROXYLASES; COUMARINS; CYTOCHROME P-450 ENZYME SYSTEM; ENZYME INHIBITORS; FLUORESCENT DYES; FLUOROMETRY; HIGH-THROUGHPUT SCREENING ASSAYS; HUMANS; OXIDOREDUCTASES, N-DEMETHYLATING; PSORALENS; TIME FACTORS;

EID: 77957579589     PISSN: 17542189     EISSN: 17502799     Source Type: Journal    
DOI: 10.1038/nprot.2010.125     Document Type: Article

References (17)

1. Williams, A.J. et al. Drug-drug interactions for UDP- glucuronosyltransferase substrates: a pharmacokinetic explanation for typically observed low exposure (AUCI/AUC) ratios. Drug Metab. Dispos. 32, 1201-1208 (2004).
2. Porter, T.D. et al. Cytochrome P-450. Multiplicity of isoforms, substrates, and catalytic and regulatory mechanisms. J. Biol. Chem. 266, 13469-13472 (1991).
3. Evans, W.E. et al. Pharmacogenomics: translating functional genomics into rational therapeutics. Science 286, 487-491 (1999).
4. Kent, U.M. et al. Mechanism-based inactivators as probes of cytochrome P450 structure and function. Curr. Drug Metab. 2, 215-243 (2001).
5. Hollenberg, P.F. et al. Mechanism-based inactivation of human cytochrome P450s: experimental characterization, reactive intermediates, and clinical implications. Chem. Res. Toxicol. 21, 189-205 (2008).
6. Silverman, R.B. Mechanism-Based Enzyme Inactivation: Chemistry and Enzymology (CRC Press, Boca Raton, FL, 1988).
7. Mayer, R.T. et al. A real-time fuorescence assay for measuring N-dealkylation. Drug Metab. Dispos. 35, 103-109 (2007).
8. Blobaum, A.L. et al. P450 active site architecture and reversibility: inactivation of cytochromes P450 2B4 and 2B4 T302A by tert-butyl acetylenes. Biochemistry 44, 3831-3844 (2005).
9. Lin, H. et al. The grapefruit juice effect is not limited to cytochrome 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 (2005).
10. Buters, J.T. et al. A highly sensitive tool for the assay of cytochrome P450 enzyme activity in rat, dog and man. Direct fuorescence monitoring of the deethylation of 7-ethoxy-4-trifuoromethylcoumarin. Biochem. Pharmacol. 46, 1577-1584 (1993).
11. DeLuca, J.G. et al. A direct, highly sensitive assay for cytochrome P-450 catalyzed O-deethylation using a novel coumarin analog. Biochem. Pharmacol. 37, 1731-1739 (1988).
12. Scott, E.E. et al. A truncation of 2B subfamily cytochromes P450 yields increased expression levels, increased solubility, and decreased aggregation while retaining function. Arch. Biochem. Biophys. 395, 57-68 (2001).
13. Rendic, S. Summary of information on human CYP enzymes: human P450 metabolism data. Drug Metab. Rev. 34, 83-448 (2002).
14. Xie, HJ. et al. Role of polymorphic human CYP2B6 in cyclophosphamide bioactivation. Pharmacogenomics J. 3, 53-61 (2003).
15. Schmiedlin-Ren, P. et al. Mechanisms of enhanced oral availability of CYP3A4 substrates by grapefruit constituents: decreased enterocyte CYP3A4 concentration and mechanism-based inactivation by furanocoumarins. Drug Metab. Dispos. 25, 1228-1233 (1997).
16. Bailey, D.G. et al. Grapefruit juice-drug interactions. Br. J. Clin. Pharmacol. 46, 101-110 (1998).
17. Bailey, D.G. et al. Grapefruit-felodipine interaction: effect of unprocessed fruit and probable active ingredients. Clin. Pharmacol. Ther. 68, 468-477 (2000)