Altered human CYP3A4 activity caused by Antley-Bixler Syndrome-related variants of NADPH-cytochrome P450 oxidoreductase measured in a robust in vitro system

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 (CYP) in humans. In this study, 12 CYPOR_ABS variants were separately co-expressed 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 co-expressed with CYP3A4, resulting in relative expression levels comparable to those found in hepatic tissue. Di-benzylfluorescein (DBF), a CYP3A-specific reporter substrate (Ghosal et al., 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 , 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, while 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 A287P variant being formerly reported to exhibit substantially lower catalytic efficiency. Additionally, Q153R exhibited 60% of WT CYPOR capacity to support the DBF O debenzylation reaction, contradicting increased catalytic efficiency (k cat /K m ) relative to WT, reported previously. Our data indicate the importance of using simulated, validated in vitro systems, employing full-length proteins with appropriate stoichiometric incorporation of protein partners, when pharmacogenetic predictions are to be made for 37ºC and rates were derived from the linear part of the kinetic traces. To maintain a constant NADPH concentration, an NADPH-generating system was used, which consisted of 0.5 mM glucose-6-phosphate and 40 mUnits/mL of G-6-P-dehydrogenase. Initial assays with human liver microsomes and BTC3A4_POR WT membranes were performed with DBF gradients up to 1.25 μ M. Subsequent assays with bacterial membranes were performed with a DBF gradient up to 5 µM, except for membranes containing the Q153R variant for which a maximum DBF concentration of 10 µM was applied. Plots of velocity traces versus DBF concentration were fitted according to the Michaelis-Menten equation in order to determine kinetic parameters k cat and K m . BTC3A4 membranes containing CYPOR variants WT, Q153R or A287P were also tested with the Vivid® CYP450 substrates BOMCC and DBOMF with assay conditions as described above, adapted according to indications of manufacturer. The substrate BOMCC was used up to 100 µM, copying conditions described in the study of Flück et al. (2010). Due to low solubility, the concentration gradient of BOMCC could only be performed with final solvent (ACN) concentrations above 2%, thus inhibiting CYP3A4 well above 30% (Busby et al., 1999). Therefore a different Vivid® CYP450 substrate (DBOMF) was used in the present studies, with a concentration gradient up to 10 µM, keeping the final ACN concentration at 0.5%. The final CYP concentration for this assay was 37.5 nM. Velocity traces were plotted and Michaelis-Menten parameters could be derived appropriately.