TY - JOUR
T1 - Altered human CYP3A4 activity caused by antley-bixler syndrome-related variants of NADPH-cytochrome P450 oxidoreductase measured in a robust in vitro system
AU - Moutinho, Daniela
AU - Marohnic, Christopher C.
AU - Panda, Satya P.
AU - Rueff, José
AU - Masters, Bettie Sue
AU - Kranendonk, Michel
PY - 2012/4
Y1 - 2012/4
N2 - 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 (k cat and K m) 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 (k cat/K m), 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 (k cat/K m) 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.
AB - 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 (k cat and K m) 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 (k cat/K m), 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 (k cat/K m) 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.
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U2 - 10.1124/dmd.111.042820
DO - 10.1124/dmd.111.042820
M3 - Article
C2 - 22252407
AN - SCOPUS:84858412533
VL - 40
SP - 754
EP - 760
JO - Drug Metabolism and Disposition
JF - Drug Metabolism and Disposition
SN - 0090-9556
IS - 4
ER -