Benzbromarone (BBR), a potent uricosuric agent for the management of gout, is known to cause fatal fulminant hepatitis. Although the mechanism of BBR-induced idiosyncratic hepatotoxicity remains unelucidated, cytochrome P450 enzyme–mediated bioactivation of BBR to electrophilic reactive metabolites is commonly regarded as a key molecular initiating event. However, apart from causing aberrant toxicities, reactive metabolites may result in mechanism-based inactivation (MBI) of cytochrome P450. Here, we investigated and confirmed that BBR inactivated CYP3A4 in a time-, concentration-, and NADPH-dependent manner with K_I, k_inact, and partition ratio of 11.61 µM, 0.10 minutes⁻¹, and 110, respectively. Coincubation with ketoconazole, a competitive inhibitor of CYP3A4, attenuated the MBI of CYP3A4 by BBR, whereas the presence of glutathione and catalase did not confer such protection. The lack of substantial recovery of enzyme activity postdialysis and after oxidation with potassium ferricyanide, combined with the absence of a Soret peak in spectral difference scans, implied that MBI of CYP3A4 by BBR did not occur through the formation of quasi-irreversible metabolite-intermediate complexes. Analysis of the reduced CO-difference spectrum revealed an ∼44% reduction in ferrous-CO binding and hinted that inactivation is mediated via irreversible covalent adduction to both the prosthetic heme moiety and the apoprotein. Finally, our in silico covalent docking analysis further suggested the modulation of substrate binding to CYP3A4 via the covalent adduction of epoxide-derived reactive intermediates of BBR to two key cysteine residues (Cys239 and Cys58) vicinal to the entrance of the orthosteric binding site.

中文翻译：

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苯溴马隆基于机理的细胞色素P450 3A4灭活

苯溴马隆（BBR）是用于治疗痛风的有效尿酸尿酸药物，已知会导致致命的暴发性肝炎。尽管尚不清楚BBR诱导的特异性肝毒性的机制，但通常将细胞色素P450酶介导的BBR生物活化为亲电反应性代谢物是关键的分子引发事件。但是，除了引起异常的毒性外，反应性代谢物还可能导致细胞色素P450的基于机制的失活（MBI）。在这里，我们研究并证实BBR以时间，浓度和NADPH依赖性方式灭活CYP3A4，K _I，k_无效，分配比为11.61 µM，0.10分钟^-1，和110。与酮康唑（一种CYP3A4的竞争性抑制剂）共孵育可通过BBR减弱CYP3A4的MBI，而谷胱甘肽和过氧化氢酶的存在则不能提供这种保护。透析后和铁氰化钾氧化后酶活性没有实质性恢复，并且在光谱差示扫描中没有Soret峰，这说明CYP3A4的MBI不会通过类不可逆代谢产物-中间体复合物的形成而发生。对降低的CO差异谱的分析表明，亚铁与CO的结合降低了约44％，这表明失活是通过修复血红素部分和脱辅基蛋白的不可逆共价加合来介导的。最后，