Novel Reversible Inhibitors of Xanthine Oxidase Targeting the Active Site of the Enzyme

Abstract

Xanthine oxidase (XO) is a flavoprotein catalysing the oxidation of hypoxanthine to xanthine and then to uric acid, while simultaneously producing reactive oxygen species. Altered functions of XO may lead to severe pathological diseases, including gout-causing hyperuricemia and oxidative damage of tissues. These findings prompted research studies aimed at targeting the activity of this crucial enzyme. During the course of a virtual screening study aimed at the discovery of novel inhibitors targeting another oxidoreductase, superoxide dismutase, we identified four compounds with non-purine-like structures, namely <b>ALS-1</b>, <b>-8</b>, <b>-15</b> and <b>-28</b>, that were capable of causing direct inhibition of XO. The kinetic studies of their inhibition mechanism allowed a definition of these compounds as competitive inhibitors of XO. The most potent molecule was <b>ALS-28</b> (<i>K</i>i 2.7 ± 1.5 µM), followed by <b>ALS-8</b> (<i>K</i>i 4.5 ± 1.5 µM) and by the less potent <b>ALS-15</b> (<i>K</i>i 23 ± 9 µM) and <b>ALS-1</b> (<i>K</i>i 41 ± 14 µM). Docking studies shed light on the molecular basis of the inhibitory activity of <b>ALS-28</b>, which hinders the enzyme cavity channel for substrate entry consistently with the competitive mechanism observed in kinetic studies. Moreover, the structural features emerging from the docked poses of <b>ALS-8</b>, <b>-15</b> and <b>-1</b> may explain the lower inhibition power with respect to <b>ALS-28</b>. All these structurally unrelated compounds represent valuable candidates for further elaboration into promising lead compounds.