Abstract
NADPH-cytochrome P450 oxidoreductase (CYPOR) variants have been described in patients with perturbed steroidogenesis and sexual differentiation, related to Antley-Bixler syndrome (ABS). It is important to determine the effect of these variants on CYP3A4, the major drug-metabolizing cytochrome P450 (P450) in humans. In this study, 12 CYPOR_ABS variants were separately coexpressed with CYP3A4 in a robust in vitro system to evaluate the effects of these variants on CYP3A4 activity in a milieu that recapitulates the stoichiometry of the mammalian systems. Full-length CYPOR variants were coexpressed with CYP3A4, resulting in relative expression levels comparable to those found in hepatic tissue. Dibenzylfluorescein (DBF), a CYP3A-specific reporter substrate (Biopharm Drug Dispos 24:375–384, 2003), was used to compare the variants and wild-type (WT) CYPOR activities with that of human liver microsomes. CYP3A4, combined with WT CYPOR, demonstrated kinetic parameters (kcat and Km) equal to those for pooled human liver microsomes. CYPOR variants Y181D, Y459H, V492E, L565P, and R616X all demonstrated maximal loss of CYP3A4 catalytic efficiency, whereas R457H and G539R retained ∼10 and 30% activities, respectively. Conversely, variants P228L, M263V, A287P, and G413S each showed WT-like capacity (kcat/Km), with the A287P variant being formerly reported to exhibit substantially lower catalytic efficiency. In addition, Q153R exhibited 60% of WT CYPOR capacity to support the DBF O-debenzylation reaction, contradicting increased catalytic efficiency (kcat/Km) relative to that for the WT, reported previously. Our data indicate the importance of use of simulated, validated in vitro systems, employing full-length proteins with appropriate stoichiometric incorporation of protein partners, when pharmacogenetic predictions are to be made for P450-mediated biotransformation.
Footnotes
This work was supported in part by the National Institutes of Health National Institute of General Medical Sciences [Grant GM081568] (to B.S.M.); the Robert A. Welch Foundation [Endowed Chair AQ-0012] (to B.S.M.); and the Fundação para a Ciência e a Tecnologia (Portugal) [Grant PTDC/SAU-GMG/71911/2006] (to D.M., R.F., and M.K.).
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
ABBREVIATIONS:
- P450
- cytochrome P450
- CYPOR
- NADPH cytochrome P450 oxidoreductase
- FAD
- flavin adenine dinucleotide
- FNM
- flavin mononucleotide
- ABS
- Antley-Bixler syndrome
- DBF
- dibenzylfluorescein
- BOMCC
- benzyl-O-methyl-cyanocoumarin
- DBOMF
- di-[benzyl-O-methyl]-fluorescein
- WT
- wild type
- ACN
- acetonitrile.
- Received September 16, 2011.
- Accepted January 17, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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