Exploring the Molecular and Biochemical Mechanisms of Aromasin in Modulating COX-2/iNOS for Alzheimer’s Disease-Associated Synaptic Dysfunction: An In Silico Drug Repurposing Study

<title>Abstract</title> <p>Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are central regulators of synaptic homeostasis, plasticity, and dysfunction during Alzheimer’s disease (AD) progression. Dysregulation of this axis promotes oxidative stress, neuronal injury, impaired synaptic signalling, and cognitive decline. This study aimed to identify a single drug capable of simultaneously modulating the COX-2/iNOS using an in silico drug-repurposing strategy. A library of 3,193 clinically approved compounds was screened, 411 candidates predicted to cross the blood-brain barrier. These compounds were evaluated using molecular docking, ADMET profiling, molecular dynamics simulations, and MM-PBSA free-energy calculations. Aromasin emerged as the most promising candidate, showing strong docking affinities for COX-2 (-9.00 kcal/mol) and iNOS (-7.47 kcal/mol), along with favourable pharmacokinetic properties and predicted CNS safety. MD simulations demonstrated stable protein-ligand complexes, with average RMSD values of ~ 0.09 nm (COX-2) and ~ 0.65 nm (iNOS), supported by RMSF, SASA, hydrogen-bonding, and radius of gyration analyses. PCA indicated ligand-induced stabilization, with the first two eigenmodes capturing ~ 40% of conformational variance. FEL analysis revealed deeper, localized minima upon Aromasin binding, consistent with enhanced thermodynamic stability. MM-PBSA calculations confirmed favourable binding free energies for COX-2 (-24.95 kcal/mol) and iNOS (-21.41 kcal/mol), positioning Aromasin as a promising repurposed modulator for AD-associated synaptic dysfunction. Mechanistically, Aromasin occupied the catalytic pockets through hydrophobic and hydrogen-bond interactions, while dynamic analyses indicated coordinated residue motions and restricted conformational sampling, suggesting that dual target engagement may modulate interconnected inflammatory, oxidative, and nitrosative signalling pathways underlying AD-associated synaptic dysfunction.</p>