A Remarkable Difference That One Fluorine Atom Confers on the Mechanisms of Inactivation of Human Ornithine Aminotransferase by Two Cyclohexene Analogues of γ-Aminobutyric Acid

Abstract

Human ornithine aminotransferase (<i>h</i>OAT), a pyridoxal 5'-phosphate-dependent enzyme, plays a critical role in the progression of hepatocellular carcinoma (HCC). Pharmacological selective inhibition of <i>h</i>OAT has been shown to be a potential therapeutic approach for HCC. Inspired by the discovery of the nonselective aminotransferase inactivator (1<i>R</i>,3<i>S</i>,4<i>S</i>)-3-amino-4-fluoro cyclopentane-1-carboxylic acid (<b>1</b>), in this work, we rationally designed, synthesized, and evaluated a novel series of fluorine-substituted cyclohexene analogues, thereby identifying <b>8</b> and <b>9</b> as novel selective <i>h</i>OAT time-dependent inhibitors. Intact protein mass spectrometry and protein crystallography demonstrated <b>8</b> and <b>9</b> as covalent inhibitors of <i>h</i>OAT, which exhibit two distinct inactivation mechanisms resulting from the difference of a single fluorine atom. Interestingly, they share a similar turnover mechanism, according to the mass spectrometry-based analysis of metabolites and fluoride ion release experiments. Molecular dynamics (MD) simulations and electrostatic potential (ESP) charge calculations were conducted, which elucidated the significant influence of the one-fluorine difference on the corresponding intermediates, leading to two totally different inactivation pathways. The novel addition-aromatization inactivation mechanism for <b>9</b> contributes to its significantly enhanced potency, along with excellent selectivity over other aminotransferases.