Cytochrome P450 (CYP) 3A5 characterized with polymorphic and extensive expression in multiple tissues is the most important P450 enzyme among the minor CYP3A isoforms. However, a selective and sensitive probe for CYP3A5 remains unavailable. In this study, we identified and characterized a naturally occurring lignan 12 (schisantherin E) as an isoform-specific probe for selective detection of CYP3A5 activity in complex biological samples. With thorough characterization including LC-MS and NMR, we found that 12 can be metabolized by CYP3A5 to generate a major metabolite 2-O-demethylated 12. Meanwhile, both reaction phenotyping and chemical inhibition experiments further revealed that CYP3A5 selectively catalyzed the 2-O-demethylation of 12. Specifically, the interactions between the Phe210 residue of CYP3A5 and methyl benzoate of 12 might play key roles in 12-O-demethylation, which was revealed by docking simulation and site-directed mutagenesis studies. These findings are beneficial for exploring the role of CYP3A5 in drug metabolism and pathologic process.

中文翻译：

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天然存在的同工型特异性探针，用于高选择性和灵敏地检测人细胞色素P450 3A5

细胞色素P450（CYP）3A5具有多态性，并在多种组织中广泛表达，是次要CYP3A亚型中最重要的P450酶。然而，CYP3A5的选择性和敏感探针仍然不可用。在这项研究中，我们鉴定并鉴定了天然存在的木脂素12（schisantherin E）作为异构体特异性探针，用于选择性检测复杂生物样品中的CYP3A5活性。通过包括LC-MS和NMR在内的全面表征，我们发现12可以被CYP3A5代谢，生成主要的代谢物2- O-去甲基12。同时，反应表型和化学抑制实验均进一步表明CYP3A5选择性催化了2- O。-脱甲基12。具体地说，CYP3A5的Phe210残基与12的苯甲酸甲酯之间的相互作用可能在12 - O-去甲基化中起关键作用，这通过对接模拟和定点诱变研究得以揭示。这些发现对于探索CYP3A5在药物代谢和病理过程中的作用是有益的。