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

Human ornithine aminotransferase ( h OAT), 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 h OAT 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 h OAT 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 ( k inact / K I = 111.8 mM –1 ·min –1 ) and ∼400-fold selectivity for h OAT over γ-aminobutyric acid aminotransferase. Intact protein mass spectrometry and X-ray crystallography demonstrated that alkyne-containing analogues form covalent adducts with h OAT, 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.