Structure-Based Discovery of Glyoxalase-I Inhibitors: Evaluating the Role of Active Site Water Molecules.

Background: Glyoxalase-I (GLO-I) is a zinc-dependent metalloenzyme and a promising target for anticancer drug discovery. It catalyzes the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis, a metabolic shift commonly observed in cancer cells. GLO-I overexpression in tumor cells promotes multidrug resistance and tumor progression. However, the role of conserved water molecules within the GLO-I active site remains insufficiently explored, and understanding their influence on ligand binding may improve structure-based inhibitor design. Purpose: This study aimed to identify potential GLO-I inhibitors by examining the effect of conserved active site water molecules on ligand binding and activity predictions using a structure-based drug design approach. Materials and Methods: Three human GLO-I crystal structures were used to generate structure-based pharmacophore models under two conditions: with and without crystallographic active site water molecules. The models were applied to virtually screen the OTAVA Lead-Like library (commercial lead-like compound library). Molecular docking of the resulting hits was then performed under both hydration conditions to evaluate effects on ligand binding affinity and pose orientation. Top-ranked compounds were purchased and evaluated in vitro for GLO-I inhibition. The most active hit was further evaluated by 1000 ns molecular dynamics (MD) simulations (± crystallographic waters), including analysis of pose stability and binding-site water behavior. Results: Among the 22 compounds tested in vitro, five showed inhibitory activity, with IC Conclusion: For GLO-I, docking without conserved active site water molecules provided more accurate results and may represent a more reliable approach for studying ligand binding and guiding inhibitor design.