Hayek-Orduz_2025_Front.Chem_13_1479763

Therapeutic strategies for Alzheimer's disease (AD) often involve inhibiting acetylcholinesterase (AChE), underscoring the need for novel inhibitors with high selectivity and minimal side effects. A detailed analysis of the protein-ligand pharmacophore dynamics can facilitate this. In this study, we developed and employed dyphAI, an innovative approach integrating machine learning models, ligand-based pharmacophore models, and complex-based pharmacophore models into a pharmacophore model ensemble. This ensemble captures key protein-ligand interactions, including Pi-cation interactions with Trp-86 and several Pi-Pi interactions with residues Tyr-341, Tyr-337, Tyr-124, and Tyr-72. The protocol identified 18 novel molecules from the ZINC database with binding energy values ranging from -62 to -115 kJ/mol, suggesting their strong potential as AChE inhibitors. To further validate the predictions, nine molecules were acquired and tested for their inhibitory activity against human AChE. Experimental results revealed that molecules, 4 (P-1894047), with its complex multi-ring structure and numerous hydrogen bond acceptors, and 7 (P-2652815), characterized by a flexible, polar framework with ten hydrogen bond donors and acceptors, exhibited IC50 values lower than or equal to that of the control (galantamine), indicating potent inhibitory activity. Similarly, molecules 5 (P-1205609), 6 (P-1206762), 8 (P-2026435), and 9 (P-533735) also demonstrated strong inhibition. In contrast, molecule 3 (P-617769798) showed a higher IC(50) value, and molecules 1 (P-14421887) and 2 (P-25746649) yielded inconsistent results, likely due to solubility issues in the experimental setup. These findings underscore the value of integrating computational predictions with experimental validation, enhancing the reliability of virtual screening in the discovery of potent enzyme inhibitors.