Orientation studies of novel xanthine oxidase inhibitors using Glide® docking and spectroscopic kinetic assays

Xanthine oxidase (XO) is an enzyme that converts hypoxanthine to xanthine and xanthine to uric acid, the last two steps in purine metabolism. XO is a molybdenum enzyme, which contains reductive and oxidative half reactions and uses oxygen gas as its final electron acceptor. Because O 2 is the final electron receptor, XO generates reducing equivalents as a product of reaction. During an ischemic condition such as a stroke or a heart attack, the oxidative half reaction does not occur, leading to a buildup of reducing equivalents. Upon reintroduction of oxygen, these reducing equivalents rapidly convert oxygen to H 2 O 2 and O 2 ? at a rate that cannot be broken down by other enzymes, leading to oxidative tissue damage. Therefore, the overall goal of this project is to find an inhibitor which slows release of electrons to oxygen. Using computer modeling and docking programs various inhibitors were docked into XO active site and their docking energies estimated. Glide® was used to dock the inhibitors and investigate their different binding modes. Docking results were compared to enzyme assay results to validate our computational approach. Enzyme assays were performed using XO purified from cow's milk. Enzyme concentration was measured by examining the absorbance at 450 nm and calculated using the extinction coefficient 37.8 mM/cm AU. Enzyme activity was estimated by measuring the uric acid production at 295nm upon addition of xanthine. Binding of inhibitors to two mutants of Xanthine Dehydrogenase was also investigated computationally. The results showed that 1H‐Benzotriazole‐5‐Carboxylic, Benzotriazole‐1‐methanol and Benzotriazole has the most favorable conformation out of all the inhibitors and these results were used to prepare the enzyme assay. Glide docking result matches the experimental data and we can use these data in future for more experiments.