NSAIDs beyond COX: <i>In Silico</i> and <i>In Vitro</i> Insights into Acetylcholinesterase Modulation

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely prescribed for their cyclooxygenase (COX) inhibitory activity. However, mounting evidence suggests that their pharmacology extends beyond this canonical pathway. In this study, the potential of NSAIDs to modulate human acetylcholinesterase (AChE), a key enzyme linking neuroinflammation, metabolic dysfunction and degenerative diseases, was evaluated. An integrative strategy combining silicon and vitro approaches was employed. Structure-based molecular docking using GOLD, molecular dynamics simulations with AMBER21, and MM-GBSA free energy calculations were performed to assess binding stability and energetic favorability. Among the compounds that were tested, nimesulide displayed the highest binding affinity (Δ<i>G</i> = -24.2 kcal/mol), followed by dipyrone (-16.0 kcal/mol), ibuprofen (-11.0 kcal/mol), and paracetamol (-9.0 kcal/mol). The per-residue energy decomposition revealed the involvement of aromatic and polar residues in the catalytic gorge, displaying interaction patterns analogous to those of classical AChE inhibitors. Experimental enzymatic assays corroborated these predictions, demonstrating a dose-dependent inhibition of AChE activity by nimesulide at micromolar concentrations. These findings suggest that NSAIDs, particularly nimesulide, may act as dual modulators with both anti-inflammatory and cholinergic regulatory effects. This work underscores the translational potential of drug repurposing and highlights the importance of combining computational and biochemical methods to uncover novel therapeutic functions of established drugs.