Dosing Inhibition: The Conformational Effect of 1 vs. 2 Dicumarols on NQO1-FAD Inhibition

The overexpression of NAD(P)H oxidoreductase 1 (NQO1) in different tumor types has highlighted this enzyme as a potential therapeutic target. Among the known inhibitors of NQO1 is dicoumarol, a coumarin derivative that binds to the enzyme's active site, competes with the NAD(P)H cofactor, and inhibits its catalytic activity. Although the interaction of dicoumarol with NQO1–FAD is known, conformational changes associated with the number of ligands present in the complex remain poorly explored. Therefore, this study used molecular dynamics (MD) simulations to investigate NQO1–FAD systems containing one and two dicoumarol molecules, aiming to compare the stability and flexibility of the ligand. Molecular dynamics simulations were performed using the crystal structure of the NQO1–FAD–dicoumarol complex (PDB ID: 2F1O) as the starting structure, using GROMACS 2025.4, the AMBER99SB-ILDN force field, and the SPC/E water model. Systems containing one or two dicoumarol molecules were neutralized with Na⁺ and Cl⁻ ions at a concentration of 0.15 mol/L and subjected to energy minimization until the maximum force was below 100.0 kJ/mol. Equilibration was initially performed under the NVT ensemble at 300 K, followed by the NPT ensemble using the Parrinello–Rahman barostat. The resulting trajectories were analyzed using root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (Rg), and principal component analysis (PCA), allowing the influence of the presence of one or two dicoumarol molecules on the conformational behavior of NQO1–FAD to be assessed. The results demonstrated differences in the stability and conformational flexibility of the systems containing one and two dicoumarol molecules. RMSD, RMSF, Rg, and PCA analyses indicated that the number of ligands influences the dynamics of the NQO1–FAD complex, suggesting different conformational patterns as a function of dicoumarol occupancy.