Department of Pharmacy, Faculty of Science (L.W.T.T., R.P.Y., E.C.Y.C.), and Protein and Proteomics Centre (PPC), SingMass (X.L., L.W., Q.L.), National University of Singapore, Singapore; and Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), Singapore (R.K.V., H.F.).
Department of Pharmacy, Faculty of Science (L.W.T.T., R.P.Y., E.C.Y.C.), and Protein and Proteomics Centre (PPC), SingMass (X.L., L.W., Q.L.), National University of Singapore, Singapore; and Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), Singapore (R.K.V., H.F.)
Mol Pharmacol. 2021 Sep;100(3):224-236. doi: 10.1124/molpharm.121.000256. Epub 2021 Jul 1.
Mounting evidence has revealed that despite the high degree of sequence homology between cytochrome P450 3A isoforms (i.e., CYP3A4 and CYP3A5), they have the propensities to exhibit vastly different irreversible and reversible interactions with a single substrate. We have previously established that benzbromarone (BBR), a potent uricosuric agent used in the management of gout, irreversibly inhibits CYP3A4 via mechanism-based inactivation (MBI). However, it remains unelucidated if CYP3A5-its highly homologous counterpart-is susceptible to inactivation by BBR. Using three structurally distinct probe substrates, we consistently demonstrated that MBI was not elicited in CYP3A5 by BBR. Our in silico covalent docking models and molecular dynamics simulations suggested that disparities in the susceptibilities toward MBI could be attributed to the specific effects of BBR covalent adducts on the F-F' loop. Serendipitously, we also discovered that BBR reversibly activated CYP3A5-mediated rivaroxaban hydroxylation wherein apparent increased and decreased with increasing BBR concentration. Fitting data to the two-site model yielded interaction factors and of 0.44 and 5.88, respectively, thereby confirming heterotropic activation of CYP3A5 by BBR. Furthermore, heteroactivation was suppressed by the CYP3A inhibitor ketoconazole in a concentration-dependent manner and decreased with increasing preincubation time, implying that activation was incited via binding of parent BBR molecule within the enzymatic active site. Finally, noncovalent docking revealed that CYP3A5 can more favorably accommodate both BBR and rivaroxaban in concert as compared with CYP3A4, which further substantiated our experimental observations. SIGNIFICANCE STATEMENT: Although it has been previously demonstrated that benzbromarone (BBR) inactivates CYP3A4, it remains uninterrogated whether it also elicits mechanism-based inactivation in CYP3A5, which shares ∼85% sequence similarity with CYP3A4. This study reported that BBR exhibited differential irreversible and reversible interactions with both CYP3A isoforms and further unraveled the molecular determinants underpinning their diverging interactions. These data offer important insight into differential kinetic behavior of CYP3A4 and CYP3A5, which potentially contributes to interindividual variabilities in drug disposition.
越来越多的证据表明，尽管细胞色素 P4503A 同工型（即 CYP3A4 和 CYP3A5）之间具有高度的序列同源性，但它们对单一底物表现出截然不同的不可逆和可逆相互作用的倾向。我们之前已经证实，苯溴马隆（BBR）是一种用于痛风管理的强效尿酸排泄剂，通过机制基础失活（MBI）不可逆地抑制 CYP3A4。然而，CYP3A5-其高度同源的同工型-是否容易被 BBR 失活仍未阐明。使用三种结构上不同的探针底物，我们一致证明 BBR 不会引起 CYP3A5 的 MBI。我们的计算共价对接模型和分子动力学模拟表明，对 MBI 的敏感性差异可能归因于 BBR 共价加合物对 F-F'环的特定影响。偶然的是，我们还发现 BBR 可逆地激活了 CYP3A5 介导的利伐沙班羟化作用，其中表观 和 随着 BBR 浓度的增加而增加和减少。将数据拟合到双位点模型得出相互作用因子 和 分别为 0.44 和 5.88，从而证实了 BBR 对 CYP3A5 的异源激活。此外，酮康唑以浓度依赖的方式抑制异源激活，并随着预孵育时间的增加而降低，这表明激活是通过结合酶活性位点内的母体 BBR 分子引起的。最后，非共价对接表明，与 CYP3A4 相比，CYP3A5 可以更有利地同时容纳 BBR 和利伐沙班，这进一步证实了我们的实验观察结果。意义：虽然先前已经证明苯溴马隆（BBR）失活 CYP3A4，但仍未探讨它是否也会引起 CYP3A5 的机制基础失活，CYP3A5 与 CYP3A4 共享约 85%的序列相似性。本研究表明，BBR 与两种 CYP3A 同工型表现出不同的不可逆和可逆相互作用，并进一步揭示了支持它们不同相互作用的分子决定因素。这些数据为 CYP3A4 和 CYP3A5 的差异动力学行为提供了重要的见解，这可能导致药物处置的个体间变异性。
