Molecular insights from multi-spectroscopy and computer simulations investigation on the xanthine oxidase inhibition...

2',4'-dimethoxy-4-hydroxychalcone (15b) has been previously reported as a reversible competitive inhibitor of xanthine oxidase (XOD) with potent activity (> 10-fold vs. allopurinol), but its molecular interactions with the enzyme active site remain uncharacterized. In this research, an integrated approach consisting of multiple spectroscopic methods, isothermal titration calorimetry (ITC), and computer simulations was employed to investigate the binding mode of 15b on XOD. Consequently, ITC data confirmed that the stable 15b-XOD complex was formed spontaneously and driven mainly by hydrophobic interactions with the dissociation constant of the magnitude of 10^-7 mol·L^-1. Three-dimensional fluorescence and circular dichroism spectroscopy indicated that 15b binding induced a conformational transition in the XOD backbone, leading to an increase in global flexibility characterized by enhanced random coil and reduced α-helix/β-sheet. Synchronous fluorescence analysis demonstrated that 15b preferentially perturbed the microenvironment of tyrosine residues over tryptophan residues in XOD. Molecular docking studies (PDB: 1N5X, 2.5 Å) revealed that 15b occupied the xanthine access channel near the molybdenum-pterin (Mo-pt) center, forming critical hydrogen bonds with Arg880 and Thr1010. This binding sterically obstructed substrate xanthine access and subsequent uric acid production. Molecular dynamics simulations indicated that 15b formed a stable binding complex with XOD. Notably, 15b likely bound at the dimer interface and induced inter-subunit proximity, whereas 15b binding preserved the integrity of the surface polarity topology. Furthermore, 15b showed no cytotoxicity in AML-12 hepatocytes at 20 μmol·L^-1(cell viability >90 %), while suppressing uric acid production (> 40 % reduction at 20 μmol·L^-1). These findings may enrich the interaction mechanism of 15b with XOD and also provide a theoretical basis for ligand-guided design of future novel XOD inhibitors.