Discovery\nand\nEnzyme Kinetic Characterization of Novel\nCYP2D6 Variants

Cytochrome\nP450 2D6 (CYP2D6) exhibits rich genetic polymorphism,\nand functional changes caused by variations are the key reasons for\ndifferences in substrate drug systemic exposure. Discovering novel\nvariants and defining their enzymatic kinetic characteristics can\ncontribute to the personalized application of drugs. In this study,\na data chain of variant-function-structure was established through\npopulation-based sequencing, baculovirus insect cell expression, <i>in vitro</i> enzymatic incubation, and ultrahigh performance\nliquid chromatography tandem mass spectrometry. Results revealed nine\nnovel missense mutations in the exonic regions. After the corresponding\nmicrosomes were obtained, the kinetics of the variants were investigated\nusing dextromethorphan as a probe substrate. It was found that the\nactivities of CYP2D6.2, 10, 17, 35, 65, R28G, T76M, and E215K were\nsignificantly reduced, while D301V almost led to loss of enzyme function.\nAdditionally, the relative clearance rate of R25Q was significantly\nincreased. From the molecular structure perspective, the mutation\nsites are distributed outside the dextromethorphan binding pocket,\nsuggesting that they primarily influence CYP2D6 activity via allosteric\nmodulation. These research findings provide fundamental data for the\nprecise application of CYP2D6 substrate drugs.