Discovery and Enzyme Kinetic Characterization of Novel CYP2D6 Variants.

Cytochrome P450 2D6 (CYP2D6) exhibits rich genetic polymorphism, and functional changes caused by variations are the key reasons for differences in substrate drug systemic exposure. Discovering novel variants and defining their enzymatic kinetic characteristics can contribute to the personalized application of drugs. In this study, a data chain of variant-function-structure was established through population-based sequencing, baculovirus insect cell expression, in vitro enzymatic incubation, and ultrahigh performance liquid chromatography tandem mass spectrometry. Results revealed nine novel missense mutations in the exonic regions. After the corresponding microsomes were obtained, the kinetics of the variants were investigated using dextromethorphan as a probe substrate. It was found that the activities of CYP2D6.2, 10, 17, 35, 65, R28G, T76M, and E215K were significantly reduced, while D301V almost led to loss of enzyme function. Additionally, the relative clearance rate of R25Q was significantly increased. From the molecular structure perspective, the mutation sites are distributed outside the dextromethorphan binding pocket, suggesting that they primarily influence CYP2D6 activity via allosteric modulation. These research findings provide fundamental data for the precise application of CYP2D6 substrate drugs.