Department of Molecular Biology and Biochemistry , University of California , Irvine , California 92697-3900 , United States.
Biochemistry. 2019 Feb 19;58(7):930-939. doi: 10.1021/acs.biochem.8b01221. Epub 2019 Jan 24.
Binding of small inhibitory compounds to human cytochrome P450 3A4 (CYP3A4) could interfere with drug metabolism and lead to drug-drug interactions, the underlying mechanism of which is not fully understood due to insufficient structural information. This study investigated the interaction of recombinant CYP3A4 with a nonspecific inhibitor metyrapone, antifungal drug fluconazole, and protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Metyrapone and fluconazole are classic type II ligands that inhibit CYP3A4 with medium strength by ligating to the heme iron, whereas PMSF, lacking the heme-ligating moiety, acts as a weak type I ligand and inhibitor of CYP3A4. High-resolution crystal structures revealed that the orientation of metyrapone is similar but not identical to that in the previously reported 1W0G model, whereas the flexible fluconazole adapts a conformer markedly different from that observed in the target CYP51 enzymes, which could explain its high potential for cross-reactivity. Besides hydrophobic and aromatic interactions with the heme and active site residues, both drugs establish water-mediated contacts that stabilize the inhibitory complexes. PMSF also binds near the catalytic center, with the phenyl group parallel to the heme. However, it does not displace the water ligand and is held in place via strong H-bonds formed by the sulfofluoride moiety with Ser119 and Arg212. Collectively, our data suggest that PMSF might have multiple binding sites and likely occupies the high-affinity site in the crystal structure. Moreover, its hydrolysis product, phenylmethanesulfonic acid, can also access and be retained in the CYP3A4 active site. Therefore, to avoid experimental artifacts, PMSF should be excluded from purification and assay solutions.
小分子抑制剂与人类细胞色素 P450 3A4（CYP3A4）的结合可能会干扰药物代谢，导致药物相互作用，由于结构信息不足，其潜在机制尚未完全了解。本研究探讨了重组 CYP3A4 与非特异性抑制剂甲吡酮、抗真菌药物氟康唑和蛋白酶抑制剂苯甲基磺酰氟（PMSF）的相互作用。甲吡酮和氟康唑是经典的 II 型配体，通过与血红素铁结合，以中等强度抑制 CYP3A4，而缺乏血红素结合部分的 PMSF 作为弱 I 型配体和 CYP3A4 的抑制剂。高分辨率晶体结构表明，甲吡酮的取向与之前报道的 1W0G 模型相似但不完全相同，而柔性氟康唑适应的构象与靶标 CYP51 酶中观察到的构象明显不同，这可以解释其高交叉反应性的潜力。除了与血红素和活性位点残基的疏水和芳香相互作用外，两种药物都建立了稳定抑制复合物的水介导接触。PMSF 也结合在催化中心附近，其苯环与血红素平行。然而，它不会取代水分子配体，而是通过磺氟化物部分与 Ser119 和 Arg212 形成的强氢键固定在位。总之，我们的数据表明，PMSF 可能具有多个结合位点，并且在晶体结构中可能占据高亲和力结合位点。此外，其水解产物苯甲基磺酸也可以进入并保留在 CYP3A4 的活性位点中。因此，为了避免实验假象，PMSF 应从纯化和测定溶液中排除。
