Xanthine oxidase (XO) is a member of the molybdopterin-containing enzyme family. It interconverts xanthine to uric acid as the last step of purine catabolism in the human body. The high uric acid concentration in the blood directly leads to human diseases like gout and hyperuricemia. Therefore, drugs that inhibit the biosynthesis of uric acid by human XO have been clinically used for many years to decrease the concentration of uric acid in the blood. In this study, the inhibition mechanism of XO and a new promising drug, topiroxostat (code: FYX-051), is investigated by employing molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations. This drug has been reported to act as both a noncovalent and covalent inhibitor and undergoes a stepwise inhibition by all its hydroxylated metabolites, which include 2-hydroxy-FYX-051, dihydroxy-FYX-051, and trihydroxy-FYX-051. However, the detailed mechanism of inhibition of each metabolite remains elusive and can be useful for designing more effective drugs with similar inhibition functions. Hence, herein we present the computational investigation of the structural and dynamical effects of FYX-051 and the calculated reaction mechanism for all of the oxidation steps catalyzed by the molybdopterin center in the active site. Calculated results for the proposed reaction mechanisms for each metabolite's inhibition reaction in the enzyme's active site, binding affinities, and the noncovalent interactions with the surrounding amino acid residues are consistent with previously reported experimental findings. Analysis of the noncovalent interactions via energy decomposition analysis (EDA) and noncovalent interaction (NCI) techniques suggests that residues L648, K771, E802, R839, L873, R880, R912, F914, F1009, L1014, and A1079 can be used as key interacting residues for further hybrid-type inhibitor development.
