Integrated molecular modeling and in vitro validation identify new acetylcholinesterase-inhibitory scaffolds among FDA-approved drugs

Acetylcholinesterase (AChE) inhibition remains a clinically validated strategy for the symptomatic management of Alzheimer’s disease (AD). However, the limited long-term efficacy of currently available inhibitors continues to drive the search for new scaffolds, making drug repurposing an attractive strategy for accelerating drug discovery. Here, an integrated structure-guided workflow combining molecular docking, molecular dynamics (MD) simulations, MMGBSA binding free-energy analysis, dynamic interaction profiling, and in vitro enzyme inhibition assays was employed to investigate the AChE inhibitory potential of FDA-approved drugs. The docking study identified lumacaftor, olaparib, fexofenadine, and vibegron as the highest-ranked compounds, while MMGBSA predicted favourable binding energetics for lumacaftor, pemetrexed, olaparib, and cilostazol. MD simulations showed that most of the compounds remained favourably accommodated within the AChE active-site gorge while maintaining overall structural integrity of the enzyme. In vitro enzyme inhibition assays identified olaparib, cilostazol, and miconazole with the lowest measured IC50 values (2.86, 2.88, and 2.89 μM, respectively), while vibegron, irbesartan, pemetrexed, lumacaftor, and indomethacin also showed measurable inhibition. Fexofenadine showed 51.26 ± 4.81% inhibition at 20 μM, but its IC50 was not determined. Differences between computational predictions and experimental activity highlight the utility of computational approaches for compound prioritization rather than quantitative prediction of biological activity. Taken together, this study identified FDA-approved drugs with, to the best of our knowledge, previously unreported AChE inhibitory activity and provided mechanistic insights into AChE–ligand recognition. These compounds represent preliminary, experimentally supported AChE-interacting scaffolds for further biochemical, pharmacological, and in vivo investigation. A blue 3D protein structure, panels showing molecular interactions, a 'REPURPOSED' label, a lab device, and two chemical structures.The image depicts a 3D blue protein structure with green and red elements, highlighting molecular interactions. To the right, four labeled panels (A-D) illustrate binding sites and interactions, including marked residues. The central text 'REPURPOSED' is in red, indicating the workflow. Below is a white lab device with a microplate input area. At the bottom, two distinct chemical structures are shown, emphasizing their relevance to the repurposing process. Curved arrows illustrate the flow among these elements, representing a continuous drug repurposing cycle.