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

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.

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.

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.

Among the 22 compounds tested in vitro, five showed inhibitory activity, with IC₅₀ values ranging from 12.07 to 25.36 μM. The most potent compound (hit 19) demonstrated an IC₅₀ of 12.07 ± 0.31 μM and 85.63% inhibition at 50 μM. Docking analysis indicated that including crystallographic water molecules often increased docking scores but could distort binding orientations, whereas docking without conserved active site water molecules more consistently produced plausible poses and better agreement with experimental activity trends.

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.