Rational Design, Synthesis, and Mechanism of (3<i>S</i>,4<i>R</i>)-3-Amino-4-(difluoromethyl)cyclopent-1-ene-1-carboxylic Acid: Employing a Second-Deprotonation Strategy for Selectivity of Human Ornithine Aminotransferase over GABA Aminotransferase

Abstract

Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that contains a similar active site to that of γ-aminobutyric acid aminotransferase (GABA-AT). Recently, pharmacological inhibition of hOAT was recognized as a potential therapeutic approach for hepatocellular carcinoma. In this work, we first studied the inactivation mechanisms of hOAT by two well-known GABA-AT inactivators (<b>CPP-115</b> and <b>OV329</b>). Inspired by the inactivation mechanistic difference between these two aminotransferases, a series of analogues were designed and synthesized, leading to the discovery of analogue <b>10b</b> as a highly selective and potent hOAT inhibitor. Intact protein mass spectrometry, protein crystallography, and dialysis experiments indicated that <b>10b</b> was converted to an irreversible tight-binding adduct (<b>34</b>) in the active site of hOAT, as was the unsaturated analogue (<b>11</b>). The comparison of kinetic studies between <b>10b</b> and <b>11</b> suggested that the active intermediate (<b>17b</b>) was only generated in hOAT and not in GABA-AT. Molecular docking studies and p<i>K</i>_a computational calculations highlighted the importance of chirality and the endocyclic double bond for inhibitory activity. The turnover mechanism of <b>10b</b> was supported by mass spectrometric analysis of dissociable products and fluoride ion release experiments. Notably, the stopped-flow experiments were highly consistent with the proposed mechanism, suggesting a relatively slow hydrolysis rate for hOAT. The novel second-deprotonation mechanism of <b>10b</b> contributes to its high potency and significantly enhanced selectivity for hOAT inhibition.