Substrate-specific modulation of CYP3A4 activity by genetic variants of cytochrome P450 oxidoreductase

Abstract

Objectives: CYP3A4 receives electrons from P450 oxidoreductase (POR) to metabolize about 50% of clinically used drugs. There is substantial inter-individual variation in CYP3A4 catalytic activity that is not explained by CYP3A4 genetic variants. CYP3A4 is flexible and distensible, permitting it to accommodate substrates varying in shape and size. To elucidate the mechanisms of variability in CYP3A4 catalysis, we examined the effects of genetic variants of POR, and explored the possibility that substrate-induced conformational changes in CYP3A4 differentially affect the ability of POR variants to support catalysis.

Methods: We expressed human CYP3A4 and four POR variants (Q153R, A287P, R457H, A503 V) in bacteria, reconstituted them in vitro and measured the Michaelis constant and maximum velocity with testosterone, midazolam, quinidine and erythromycin as substrates.

Results: POR A287P and R457H had low activity with all substrates; Q153R had 76-94% of wild-type (WT) activity with midazolam and erythromycin, but 129-150% activity with testosterone and quinidine. The A503 V polymorphism reduced the CYP3A4 activity to 61-77% of WT with testosterone and midazolam, but had nearly WT activity with quinidine and erythromycin.

Conclusion: POR variants affect CYP3A4 activities. The impact of a POR variant on catalysis by CYP3A4 is substrate-specific, probably because of substrate-induced conformational changes in CYP3A4.

FIGURE 1. Components studied

A. Chemical structures of the CYP3A4 substrates used. B. Difference spectrum (Fe⁺²-CO vs Fe⁺²) of the human CYP3A4 preparation in E.coli membranes.

FIGURE 2. Lineweaver-Burk plots of CYP3A4 catalysis

In all panels, support by WT POR is indicated by circles and solid lines; Q153R with squares and dotted lines; A287P with triangles and dashed lines; and A503V with diamonds and dash-dot lines. Each point represents the mean of three experiments, each performed in duplicate. Catalysis could not be measured with R457H POR. A. 6β-hydroxylation of testosterone. B. 1-hydroxylation of midazolam. C. 4-hydroxylation of midazolam. D. 3-hydroxylation of quinidine. E. N-demethylation of erythromycin.