Discovery of novel xanthine oxidase inhibitors using deep learning-based composite molecular fingerprint: Screening,...

Xanthine oxidase (XO) confirms as a critical target for the treatment of hyperuricemia (HUA). Current Xanthine oxidase inhibitors (XOIs) used in clinical practice is constrained by adverse effects or suboptimal bioavailability, necessitating the discovery of novel inhibitors. In this study, we generated composite molecular fingerprints with deep learning (DL) techniques and trained an XOI classification model for high-throughput virtual screening and combining molecular docking, ADME prediction and manual screening of predicted conformations. Three active XOIs: Hit 3, Hit 5 and Hit 6 were identified (IC₅₀ =2.08 ± 0.19 μmol·L^-1, 0.62 ± 0.02 μmol·L^-1 and 3.05 ± 0.24 μmol·L^-1, respectively). The inhibition kinetic and binding behavior of the most potent candidate (Hit 5) were investigated using kinetic analysis, spectroscopic techniques, and computational simulations, while its uric acid-lowering effect was evaluated in potassium oxonate/hypoxanthine-induced HUA mice. The results demonstrated that Hit 5 is a reversible, mixed-type XOI and exhibited a strong fluorescence quenching effect through a static quenching procedure. It forms a stable complex within the molybdopterin cavity of the catalytic active site of XO, thereby blocking substrate xanthine access and inducing conformational changes. At the same time, it can directly form a stable bond with the molybdopterin cofactor, thereby affecting the catalytic ability of XO. During the binding process, it is able to compete significantly with 8-anilinonaphthalene-1-sulfonic acid (ANS). It is speculated that it has occupied the hydrophobic cavity of XO and has generated sufficient interactions. It also shows a stable binding with XO in molecular dynamics (MD) simulations. Moreover, Hit 5 significantly decreased serum uric acid levels. In the initial biological safety assessment, Hit 5 demonstrated relatively acceptable biological safety, including low cytotoxicity, liver and kidney safety at low doses, mild liver toxicity at high doses, and good kidney safety. Above findings validate the effectiveness of current DL assisted virtual-screening strategy, provided valuable information for further modification for Hit 5 as a promising lead compound for the treatment of hyperuricemia.