Evaluation of Potential Xanthine Oxidase Inhibitors by Molecular Docking and Bioassays

For decades, the inhibition of the enzyme xanthine oxidase (XO) by compounds such as allopurinol has been used as an effective treatment of gout. More recently, XO has been implicated in ischemia-reperfusion injury and has emerged as a target for drugs against hyperuricemia and cancer. For the structure-based design of novel XO inhibitors, a validated docking protocol and a reliable bioassay are needed. We successfully docked the structure of the XO inhibitors lumazine and febuxostat into the X-ray crystal structure of XO. Furthermore, we established a bioassay that monitors XO’s activity by measuring the rate of urate production at 290 nm. The assay was performed on a 96-well microplate that permitted the simultaneous testing of eight compounds at twelve different inhibitor concentrations. In order to develop structure-activity relationships, analogues of known inhibitors were identified for experimental testing via similarity and substructure searches of several compound libraries