Mechanism-Based Inactivation of Human Ornithine Aminotransferase by Ethynyl- and Nitrile-Substituted Cyclopentene Analogues of γ-Aminobutyric Acids.

Abstract

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, plays a central role in glutamine, proline, and polyamine metabolism and is increasingly recognized as a metabolic vulnerability in multiple cancers. Previously, we established a second deprotonation strategy to achieve efficient mechanism-based inactivation of hOAT over closely related aminotransferases. Building on this concept, we report the rational design, synthesis, and mechanistic investigation of cyclopentene-based γ-aminobutyric acid analogues bearing alkyne or nitrile warheads as potent hOAT inactivators. These compounds undergo enzyme-catalyzed γ-deprotonation to form ketimine intermediates, priming for a subsequent tautomerization event that leads to irreversible inhibition. Inhibitory activity evaluation revealed pronounced stereochemical effects on binding affinity and partition ratio, with one nitrile analogue (4b) exhibiting an exceptional inactivation efficiency (kinact/KI = 111.8 mM-1·min-1) and ∼400-fold selectivity for hOAT over γ-aminobutyric acid aminotransferase. Intact protein mass spectrometry and X-ray crystallography demonstrated that alkyne-containing analogues form covalent adducts with hOAT, whereas nitrile-containing analogues generate noncovalent but tight-binding species. Kinetic isotope effect studies identified γ-deprotonation as the rate-determining step, and a complementary small-molecule mass and computational study elucidated the inactivation and turnover pathways. Collectively, these results expand the mechanistic repertoire of PLP-dependent enzyme inactivation and provide a generalizable framework for designing highly selective mechanism-based inactivators.