Design and Elucidation of a Strain-Relief-Driven Ring Opening Mechanism of Inactivation of Ornithine Aminotransferase by (1S,3R,5R)-3-Amino-6,6-difluorobicyclo[3.1.0]hexane-1-carboxylic Acid.

Abstract

Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that has been implicated in several cancers due to the essential role in the synthesis of glutamine and proline. Inhibition of hOAT may provide a novel method to treat a variety of cancers. In recent years, selective inhibition of hOAT through mechanism-based inactivation has shown promise in the treatment of hepatocellular carcinoma. Inspired by the recently reported hOAT-selective δ-deprotonation and previously proposed ring-strain mediated mechanisms of inactivation, we designed, synthesized, and evaluated (1S,3R,5R)-3-amino-6,6-difluorobicyclo[3.1.0]hexane-1-carboxylic acid (5), a time-dependent inhibitor of hOAT. Structural and mechanistic studies validated the proposed mechanism of 5, which undergoes a ring-opening mechanism to form a PLP-inactivator adduct that is tightly bound in the active site of hOAT. Intact protein mass spectrometry, 19F NMR spectroscopy, transient-state kinetic studies, X-ray crystallography, and QM calculations were used to determine the final adduct and explore the mechanism of inactivation of 5. This is the first report of a ring-opening event in the active site of hOAT.