Design and Elucidation of a Strain-Relief-Driven Ring Opening Mechanism of Inactivation of Ornithine Aminotransferase by (1 S ,3 R ,5 Allison N. Devitt, Abigail L. Vargas, Cathy Kexin Zhang, Benjamin James Des Soye, Maanasa Narayanomoorthy, Inna Sokolenko, Aaron K. Origenes, Tyler B. Alt, Pathum M. Weerawarna, Graham R. Moran, Neil L. Kelleher, Dali Liu, Richard B. Silverman

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.