TY - JOUR T1 - Novel Homodimer Metabolites of GDC-0994 via Cytochrome P450-Catalyzed Radical Coupling JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.119.090019 SP - dmd.119.090019 AU - Ryan H. Takahashi AU - Jessica M. Grandner AU - Sudheer Bobba AU - Yanzhou Liu AU - Paul Beroza AU - Donglu Zhang AU - Shuguang Ma Y1 - 2020/01/01 UR - http://dmd.aspetjournals.org/content/early/2020/03/31/dmd.119.090019.abstract N2 - 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. A symmetric dimer, via formation of a new carbon-carbon bond between two pyrazoles, and an asymmetric dimer, via a new aniline-nitrogen to pyrazole-carbon bond, were identified. In vitro experiments demonstrated formation was catalyzed by cytochrome P450 enzymes, with CYP3A4/5 being the most efficient at forming these dimers. Using Density Functional Theory (DFT), we determined these metabolites share a mechanism of formation, initiated by an N-H hydrogen atom abstraction by the catalytically active iron-oxo of CYPs. 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 two GDC-0994 molecules binding within the active site of the P450 enzyme and proceeding through a diradical 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 diradical coupling. The mechanism of dimerization was elucidated using density functional theory computations and supported by molecular modeling. ER -