CYP2D6 Allelic Variants *34, *17-2, *17-3, and *53 and a Thr309Ala Mutant Display Altered Kinetics and NADPH Coupling in Metabolism of Bufuralol and Dextromethorphan and Altered Susceptibility to Inactivation by SCH 66712

Abstract

Metabolic phenotype can be affected by multiple factors, including allelic variation and interactions with inhibitors. Human CYP2D6 is responsible for approximately 20% of cytochrome P450-mediated drug metabolism but consists of more than 100 known variants; several variants are commonly found in the population, whereas others are quite rare. Four CYP2D6 allelic variants-three with a series of mutations distal to the active site (*34, *17-2, *17-3) and one ultra-metabolizer with mutations near the active site (*53), along with reference *1 and an active site mutant of *1 (Thr309Ala)-were expressed, purified, and studied for interactions with the typical substrates dextromethorphan and bufuralol and the inactivator SCH 66712. We found that *34, *17-2, and *17-3 displayed reduced enzyme activity and NADPH coupling while producing the same metabolites as *1, suggesting a possible role for Arg296 in NADPH coupling. A higher-activity variant, *53, displayed similar NADPH coupling to *1 but was less susceptible to inactivation by SCH 66712. The Thr309Ala mutant showed similar activity to that of *1 but with greatly reduced NADPH coupling. Overall, these results suggest that kinetic and metabolic analysis of individual CYP2D6 variants is required to understand their possible contributions to variable drug response and the complexity of personalized medicine.

Spontaneous Ligand Access Events to Membrane-Bound Cytochrome P450 2D6 Sampled at Atomic Resolution

“…Measurements of the enzymatic activity of allelic variant CYP2D6*53 have revealed increased metabolic rates towards bufuralol, dextromethorphan, and N-desmethyltamoxifen 3,6,7 . In contrast, a recent study reported a decrease in the clearance of primaquine 43 .…”

Rolapitant Is a Reversible Inhibitor of CYP2D6

“…Supersomes (Corning Life Sciences, Tewksbury, MA) were used in all assays except spectral binding titrations and stopped-flow spectroscopy. For spectral binding titrations and stopped-flow spectroscopy, purified CYP2D6 and CYP3A4 were used; expression and purification were as described elsewhere (Gillam et al, 1993(Gillam et al, , 1995Hanna et al, 2001;Glass et al, 2018).…”

“…Samples were then centrifuged (16,100g) for 5 minutes, and 10 ml of the supernatant was injected onto a Waters Symmetry C18 column (5 mm, 3.9 Â 150 mm; Waters Corporation, Milford, MA) connected to a Waters e2965 high-performance liquid chromatography system paired with a Waters 2475 fluorescence detector. The instrument method and mobile phase were the same as previously described with bufuralol (Nagy et al, 2011;Glass et al, 2018). The 19-OH-bufuralol peak area was converted to concentration using a standard curve.…”

“…Reactions were quenched with 20 ml ACN and placed on ice after a time determined to be in the linear range for product formation. Samples were then centrifuged (16,100g) for 5 minutes, and 10 ml of the supernatant was analyzed as previously described (Glass et al, 2018). Lineweaver-Burk and Dixon plots were created using KaleidaGraph (Synergy Software).…”

“…Reactions were quenched with 20 ml of cold ACN after 10 minutes. Samples were then centrifuged (16,100g) for 5 minutes and dextrorphan product formation was measured as previously described (Glass et al, 2018).…”