Remarkable and Unexpected Mechanism for (<i>S</i>)-3-Amino-4-(difluoromethylenyl)cyclohex-1-ene-1-carboxylic Acid as a Selective Inactivator of Human Ornithine Aminotransferase

Abstract

Human ornithine aminotransferase (<i>h</i>OAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that was recently found to play an important role in the metabolic reprogramming of hepatocellular carcinoma (HCC) via the proline and glutamine metabolic pathways. The selective inhibition of <i>h</i>OAT by compound <b>10</b> exhibited potent <i>in vivo</i> antitumor activity. Inspired by the discovery of the aminotransferase inactivator (1<i>S</i>,3<i>S</i>)-3-amino-4-(difluoromethylene)cyclopentane-1-carboxylic acid (<b>5</b>), we rationally designed, synthesized, and evaluated a series of six-membered-ring analogs. Among them, <b>14</b> was identified as a new selective <i>h</i>OAT inactivator, which demonstrated a potency 22× greater than that of <b>10</b>. Three different types of protein mass spectrometry approaches and two crystallographic approaches were employed to identify the structure of <i>h</i>OAT-<b>14</b> and the formation of a remarkable final adduct (<b>32'</b>) in the active site. These spectral studies reveal an enzyme complex heretofore not observed in a PLP-dependent enzyme, which has covalent bonds to two nearby residues. Crystal soaking experiments and molecular dynamics simulations were carried out to identify the structure of the active-site intermediate <b>27'</b> and elucidate the order of the two covalent bonds that formed, leading to <b>32'</b>. The initial covalent reaction of the activated warhead occurs with *Thr322 from the second subunit, followed by a subsequent nucleophilic attack by the catalytic residue Lys292. The turnover mechanism of <b>14</b> by <i>h</i>OAT was supported by a mass spectrometric analysis of metabolites and fluoride ion release experiments. This novel mechanism for <i>h</i>OAT with <b>14</b> will contribute to the further rational design of selective inactivators and an understanding of potential inactivation mechanisms by aminotransferases.