Drug Metabolism and Pharmacokinetics (R.H.T., S.B., D.Z., S.M.) and Discovery Chemistry (J.M.G., Y.L., P.B.), Genentech, Inc., South San Francisco, California.
Drug Metabolism and Pharmacokinetics (R.H.T., S.B., D.Z., S.M.) and Discovery Chemistry (J.M.G., Y.L., P.B.), Genentech, Inc., South San Francisco, California
Drug Metab Dispos. 2020 Jun;48(6):521-527. doi: 10.1124/dmd.119.090019. Epub 2020 Mar 31.
Two novel homodimer metabolites were identified in rat samples collected during the in vivo study of GDC-0994. In this study, we investigated the mechanism of the formation of these metabolites. We generated and isolated the dimer metabolites using a biomimetic oxidation system for NMR structure elucidation to identify a symmetric dimer formed via carbon-carbon bond between two pyrazoles and an asymmetric dimer formed via an aminopyrazole-nitrogen to pyrazole-carbon bond. In vitro experiments demonstrated formation of these dimers was catalyzed by cytochrome P450 enzymes (P450s) with CYP3A4/5 being the most efficient. Using density functional theory, we determined these metabolites share a mechanism of formation, initiated by an N-H hydrogen atom abstraction by the catalytically active iron-oxo of P450s. Molecular modeling studies also show these dimer metabolites fit in the CYP3A4 binding site in low energy conformations with minimal protein rearrangement. Collectively, the results of these experiments suggest that formation of these two homodimer metabolites is mediated by CYP3A, likely involving activation of two GDC-0994 molecules by a single P450 enzyme and proceeding through a radical coupling mechanism. SIGNIFICANCE STATEMENT: These studies identified structures and enzymology for two distinct homodimer metabolites and indicate a novel biotransformation reaction mediated by CYP3A. In it, two molecules may bind within the active site and combine through radical coupling. The mechanism of dimerization was elucidated using density functional theory computations and supported by molecular modeling.
在 GDC-0994 体内研究中收集的大鼠样本中鉴定出两种新型同二聚代谢物。在这项研究中，我们研究了这些代谢物形成的机制。我们使用仿生氧化系统生成并分离出二聚代谢物，用于 NMR 结构阐明，以鉴定通过两个吡唑之间的碳-碳键形成的对称二聚体和通过氨基吡唑-氮与吡唑-碳键形成的不对称二聚体。体外实验表明，这些二聚体是由细胞色素 P450 酶 (P450s) 催化形成的，其中 CYP3A4/5 最为有效。使用密度泛函理论，我们确定这些代谢物具有相似的形成机制，由 P450s 的催化活性铁-氧原子夺取 N-H 氢原子引发。分子建模研究还表明，这些二聚代谢物以低能量构象拟合 CYP3A4 结合位点，蛋白重排最小。总的来说，这些实验结果表明，这两种同二聚代谢物的形成是由 CYP3A 介导的，可能涉及单个 P450 酶激活两个 GDC-0994 分子，并通过自由基偶联机制进行。意义陈述：这些研究鉴定了两种不同的同二聚代谢物的结构和酶学，并表明了一种由 CYP3A 介导的新型生物转化反应。在这种反应中，两个分子可能在活性位点结合，并通过自由基偶联结合。二聚化的机制通过密度泛函理论计算得到阐明，并得到分子建模的支持。
