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 V_max increased and K_m decreased with increasing BBR concentration. Fitting data to the two-site model yielded interaction factors α and β 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.

中文翻译：

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苯溴马隆与人细胞色素 P450s 3A4 和 3A5 之间的差异可逆和不可逆相互作用

越来越多的证据表明，尽管细胞色素 P450 3A 同种型（即 CYP3A4 和 CYP3A5）之间具有高度的序列同源性，但它们倾向于与单一底物表现出截然不同的不可逆和可逆相互作用。我们之前已经确定苯溴马隆 (BBR) 是一种用于治疗痛风的强效促尿酸排泄剂，通过基于机制的失活 (MBI) 不可逆地抑制 CYP3A4。然而，CYP3A5（其高度同源的对应物）是否容易被 BBR 灭活，尚不清楚。使用三种结构不同的探针底物，我们一致证明 MBI 不会通过 BBR 在 CYP3A5 中引起。我们的计算机共价对接模型和分子动力学模拟表明，对 MBI 敏感性的差异可归因于 BBR 共价加合物对 FF' 环的特定影响。偶然地，我们还发现 BBR 可逆地激活 CYP3A5 介导的利伐沙班羟基化，其中明显随着 BBR 浓度的增加，V _max增加，K _m减少。将数据拟合到双位点模型产生的相互作用因子α和β分别为 0.44 和 5.88，从而证实了 BBR 对 CYP3A5 的异向激活。此外，CYP3A 抑制剂酮康唑以浓度依赖性方式抑制异源活化，并随着预孵育时间的增加而降低，这意味着活化是通过酶活性位点内亲本 BBR 分子的结合来激发的。最后，非共价对接显示，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。