Kynurenine aminotransferase II (KAT-II) serves as a promising therapeutic target for managing various disorders associated with elevated levels of kynurenic acid (KYNA). Existing KAT-II inactivators, while effective, frequently cause unwanted side effects stemming from their irreversible mode of action. The objective of this investigation was to discover effective and safer KAT-II inhibitors through integrated computational modeling and experimental validation in vitro. Top candidate compounds were selected via virtual screening, MM/GBSA free energy calculations, and molecular dynamics simulations. These were subsequently assessed using enzyme kinetic assays and cytotoxicity testing in cell culture. Among the screened compounds, herbacetin and (-)-Epicatechin demonstrated superior performance. Their Glide docking scores were −8.66 kcal/mol and −8.16 kcal/mol, respectively, with MM/GBSA binding free energies of −50.30 kcal/mol and −51.35 kcal/mol. These values outperformed the reference inhibitor PF-04859989, which recorded a docking score of −7.12 kcal/mol and a binding energy of −38.41 kcal/mol. ADMET predictions indicated that the lead compounds possess desirable pharmacokinetic properties, acceptable bioavailability, and low toxicity risk, suggesting their suitability for further development. Kinetic analyses confirmed that both herbacetin and (-)-Epicatechin act as reversible inhibitors with a competitive mechanism. Their IC50 values were determined to be 5.98 ± 0.18 µM and 8.76 ± 0.76 µM, respectively. Additionally, MTT assays showed no impact on HepG2 cell viability at concentrations required for effective KAT-II inhibition. The findings indicate that herbacetin and (-)-Epicatechin are effective inhibitors of KAT-II and represent viable leads for advancing novel inhibitor design.
