DecipHering the synergistic inhibition mechanism of xanthine oxidase by Myricetin and allopurinol: Insights from inhibition kinetics, thermodynamics, and molecular dynamic simulations

Xanthine oxidase (XOD) is a critical biomarker associated with various pathological conditions, and recent advances have enhanced the understanding of its inhibition mechanisms. Binary inhibitor combinations, which exhibit synergistic inhibition of XOD, have attracted increasing attention. However, their underlying molecular mechanisms remain poorly elucidated. In this study, the myricetin–allopurinol (MY@ALL, 1:1) combination is identified as a promising binary inhibitor system targeting XOD. At lower concentrations, the interaction between MY@ALL and XOD likely proceeds via a static quenching mechanism, leading to the formation of a stable ground-state MY@ALL@XOD complex with a pronounced quenching effect. At higher concentrations, a mixed (both static and dynamic) quenching mechanism is observed, resulting in a weaker quenching effect. Furthermore, MY@ALL demonstrates lower binding affinity toward XOD than myricetin alone, accompanied by reduced accessibility. The binding of MY@ALL to XOD to form the MY@ALL@XOD complex is spontaneous and endothermic at low concentrations, contrasting with the exothermic nature of myricetin binding. Additionally, MY@ALL induces a smaller increase in XOD protein hydrophobicity, potentially due to more effective modulation of α-helix and β-turn structural elements. This attenuated increase in hydrophobicity may account for the reduced accessibility of the inhibitor to the enzyme. Kinetic analyses reveal that MY@ALL exhibits mixed-type inhibition, with shifting dominance between non-competitive and competitive modes under different conditions. Molecular docking and thermodynamic simulations further confirm and predict that MY@ALL binds to two distinct sites on XOD. Collectively, this work provides novel insights into the molecular basis of synergistic inhibition by efficient binary inhibitor combinations.