Clobetasol Propionate Is a Heme-Mediated Selective Inhibitor of Human Cytochrome P450 3A5

The human cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A5 metabolize most drugs and have high similarities in their structure and substrate preference. Whereas CYP3A4 is predominantly expressed in the liver, CYP3A5 is upregulated in cancer, contributing to drug resistance. Selective inhibitors of CYP3A5 are, therefore, critical to validating it as a therapeutic target. Here we report clobetasol propionate (clobetasol) as a potent and selective CYP3A5 inhibitor identified by high-throughput screening using enzymatic and cell-based assays. Molecular dynamics simulations suggest a close proximity of clobetasol to the heme in CYP3A5 but not in CYP3A4. UV–visible spectroscopy and electron paramagnetic resonance analyses confirmed the formation of an inhibitory type I heme–clobetasol complex in CYP3A5 but not in CYP3A4, thus explaining the CYP3A5 selectivity of clobetasol. Our results provide a structural basis for selective CYP3A5 inhibition, along with mechanistic insights, and highlight clobetasol as an important chemical tool for target validation.人类细胞色素P450（cytochrome P450，CYP）酶中的CYP3A4与CYP3A5可代谢绝大多数临床药物，二者在蛋白质结构与底物偏好性上高度相似。其中CYP3A4主要在肝脏中生理性表达，而CYP3A5则在肿瘤组织中表达上调，进而参与肿瘤多药耐药的发生。因此，选择性CYP3A5抑制剂对于验证该酶作为抗肿瘤治疗靶点具有关键价值。本研究通过酶学测定与基于细胞的高通量筛选策略，鉴定出丙酸氯倍他索（clobetasol propionate，以下简称氯倍他索）为强效且高选择性的CYP3A5抑制剂。分子动力学模拟结果显示，氯倍他索可与CYP3A5中的血红素紧密毗邻，而在CYP3A4中则无此相互作用模式。紫外-可见光谱与电子顺磁共振分析证实，仅在CYP3A5中形成了抑制性I型血红素-氯倍他索复合物，而CYP3A4中并未检测到该复合物的存在，这一结果阐明了氯倍他索对CYP3A5的选择性作用机制。本研究结果为选择性CYP3A5抑制提供了结构基础与机制阐释，并凸显氯倍他索可作为用于靶点验证的重要化学工具。