Elucidating the Isorhamnetin-3-O-glucoside-iNOS Interaction via Molecular Dynamics and Hirshfeld Surface Analyses

Abstract Inducible nitric oxide synthase (iNOS) overproduction drives chronic inflammation and oncogenic signaling, yet selective small-molecule modulation remains elusive. We interrogated isorhamnetin-3-O-glucoside (I3OG), a dietary flavonol glycoside, against murine (3E6T) and human (3E7G) iNOS oxygenase domains using a validated in silico pipeline spanning redocking, explicit-solvent molecular dynamics (100 ns), and MM/GBSA free-energy analysis. Redocking reproduced co-crystal poses (RMSD 1.21/1.25 Å), and Vina ranked I3OG favorably (-10.1/-9.7 kcal·mol⁻¹). MD revealed confined ligand motions and intact protein compaction; 3E6T displayed tighter RMSD variability and a denser hydrogen-bond network (≈4-6 persistent bonds) than 3E7G (≈1-3), with damped local flexibility around the pocket. MM/GBSA from equilibrated frames yielded ΔGbind = -44.9 ± 3.9 kcal·mol⁻¹ (3E6T) versus -36.1 ± 3.7 kcal·mol⁻¹ (3E7G), driven by favorable gas-phase van der Waals/electrostatics that outweigh polar desolvation. Hot-spot residues (3E6T: Trp188, Cys194, Trp366, Phe363; 3E7G: Trp194, Phe369, Trp372) rationalize species-dependent stabilization. Complementary Hirshfeld analysis of I3OG crystals highlights dominant O···H/H···O contacts and ancillary π-stacking, mirroring solution-phase recognition. Collectively, I3OG emerges as a mechanism-aware, tractable scaffold for iNOS attenuation, with the murine domain offering a more avid binding environment and a quantitative benchmark for future selective inhibitor design.