Department of Pharmacy, Faculty of Science (L.W.T.T., J.F., G.W., E.C.Y.C.), and Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore (L.Z.); Singapore Eye Research Institute (SERI), Singapore (S.K.K., L.Z.); and Ophthalmology and Visual Sciences Academia Clinical Program, Duke-National University of Singapore Medical School, Singapore (L.Z.).
Department of Pharmacy, Faculty of Science (L.W.T.T., J.F., G.W., E.C.Y.C.), and Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore (L.Z.); Singapore Eye Research Institute (SERI), Singapore (S.K.K., L.Z.); and Ophthalmology and Visual Sciences Academia Clinical Program, Duke-National University of Singapore Medical School, Singapore (L.Z.)
Drug Metab Dispos. 2022 Jul;50(7):931-941. doi: 10.1124/dmd.122.000895. Epub 2022 May 5.
Futibatinib (FUT) is a potent inhibitor of fibroblast growth factor receptor (FGFR) 1-4 that is currently under clinical investigation for intrahepatic cholangiocarcinoma. Unlike its predecessors, FUT possesses an acrylamide warhead, which enables it to bind covalently to a free cysteine residue in the FGFR kinase domain. However, it remains uninterrogated if this electrophilic α, -unsaturated carbonyl scaffold could also directly or indirectly engender off-target covalent binding to nucleophilic centers on other cellular proteins. Here, we discovered that FUT inactivated both CYP3A isoforms with inactivator concentration at half-maximum inactivation rate constant, maximum inactivation rate constant, and partition ratios of 12.5 and 51.4 M, 0.25 and 0.06 minutes, and ∼52 and ∼58 for CYP3A4 and CYP3A5, respectively. Along with its time-, concentration-, and cofactor-dependent inhibitory profiles, FUT also exhibited several cardinal features that were consistent with mechanism-based inactivation. Moreover, the nature of inactivation was unlikely to be pseudo-irreversible and instead arose from the covalent modification of the cytochrome P450 apoprotein and/or its heme moiety due to the lack of substantial enzyme activity recovery following dialysis and chemical oxidation, as well as the absence of the diagnostic Soret peak in spectral analyses. Finally, utilizing glutathione (GSH) trapping and high-resolution mass spectrometry, we illuminated that while the acrylamide moiety in FUT could nonenzymatically conjugate to GSH via Michael addition, it was not implicated in the covalent inactivation of CYP3A. Rather, we surmised that it likely stemmed from the metabolic activation of its acrylamide covalent warhead to a highly electrophilic epoxide intermediate that could covalently modify CYP3A and culminate in its catalytic inactivation. SIGNIFICANCE STATEMENT: In this study, we reported for the first time the inactivation of CYP3A by futibatinib (FUT). Furthermore, using FUT as an exemplary targeted covalent inhibitor, our study revealed the propensity for its acrylamide Michael acceptor moiety to be metabolically activated to a highly electrophilic epoxide. Due to the growing resurgence of covalent inhibitors and the well-established toxicological ramifications associated with epoxides, we advocate that closer scrutiny be adopted when profiling the reactive metabolites of compounds possessing an α, -unsaturated carbonyl scaffold.
富替比替尼（FUT）是一种强效的成纤维细胞生长因子受体（FGFR）1-4 抑制剂，目前正在进行临床试验，用于治疗肝内胆管癌。与它的前体不同，FUT 具有丙烯酰胺弹头，使其能够与 FGFR 激酶结构域中的游离半胱氨酸残基共价结合。然而，目前还不清楚这个亲电的α，β-不饱和羰基支架是否也可以直接或间接地导致其他细胞蛋白上的亲核中心的非靶标共价结合。在这里，我们发现 FUT 以半最大失活速率常数、最大失活速率常数和分配比为 12.5 和 51.4 μM、0.25 和 0.06 分钟，以及分别为∼52 和∼58 的方式使 CYP3A 同工酶 4 和 CYP3A5 失活。除了其时间、浓度和辅因子依赖的抑制特征外，FUT 还表现出几种与机制基础失活一致的主要特征。此外，由于透析和化学氧化后酶活性没有显著恢复，以及光谱分析中缺乏特征性的 Soret 峰，失活的性质不太可能是假不可逆的，而是由于细胞色素 P450 脱辅基蛋白和/或其血红素部分的共价修饰所致。最后，利用谷胱甘肽（GSH）捕获和高分辨率质谱，我们阐明了 FUT 中的丙烯酰胺部分虽然可以通过迈克尔加成非酶促地与 GSH 结合，但它与 CYP3A 的共价失活无关。相反，我们推测它可能源自其丙烯酰胺共价弹头的代谢激活，形成高亲电性的环氧化物中间体，可共价修饰 CYP3A 并最终导致其催化失活。意义声明：在这项研究中，我们首次报道了富替比替尼（FUT）对 CYP3A 的失活作用。此外，使用 FUT 作为典型的靶向共价抑制剂，我们的研究揭示了其丙烯酰胺迈克尔受体部分被代谢激活为高亲电性环氧化物的倾向。由于共价抑制剂的重新兴起以及与环氧化物相关的既定毒理学后果，我们主张在对具有α，β-不饱和羰基支架的化合物的反应性代谢物进行分析时，应采取更严格的审查。
