Abstract
Background: Virtual screening is a widely adopted technique for the discovery of novel pharmacologically active compounds; however, the risk of identifying false positive hits remains a major challenge. Aim: The aim of this study was to perform a validated structure-based drug design screening to discover multitarget pyrrole-based molecules as selective dual-acting monoamine oxidase (MAO) and acetylcholinesterase (AChE) inhibitors. Methods: The study employed validated docking protocols using Glide (Schrödinger) and GOLD (CCDC), integrating ligand enrichment analysis and robust Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring. These methods were applied to a custom-designed database of pyrrole-based compounds. The top-ranked hits were synthesized and validated through in vitro tests, demonstrating significant inhibitory activities against MAO-A, MAO-B, AChE, and Butyrylcholinesterase (BChE). Results: The docking protocols achieved favorable hit rates, with 25.93% for AChE inhibitors and 44.44% for MAO-B inhibitors. Additionally, structure-activity relationship analysis revealed key substituent effects that significantly influence binding affinity and selectivity. Two compounds, EM-DC-19 (2-(2,5-dimethyl-1H-pyrrol-1-yl)-3-(2H-imidazol-4-yl)propanoic acid) and EM-DC-27 ([4-(2,5-dimethyl-1H-pyrrol-1-yl)phenyl]acetic acid), were identified as selective MAO-B inhibitors with additional moderate AChE inhibitory activity, demonstrating IC(50) values of 0.299 ± 0.10 µM and 0.344 ± 0.10 µM against MAO-B, and 76.15 ± 6.12 µM and 375.20 ± 52.99 µM against AChE, respectively. The absence of statistically significant inhibitory effects of these lead compounds on MAO-A and BChE (IC(50) > 100 µM) underscores their selective inhibitory activity towards MAO-B and AChE. Furthermore, both compounds demonstrated low neurotoxicity and significant neuroprotective and antioxidant effects in rat brain synaptosomes, mitochondria, and microsomes. These effects were particularly evident in models of 6-hydroxydopamine-induced neurotoxicity (6-OHDA) and oxidative stress induced by tert-butyl hydroperoxide and Fe(2+)/ascorbic acid. Conclusions: The findings suggest that these multitarget compounds hold promise for further development, with potential for structural modifications to enhance their enzyme inhibitory and neuroprotective properties.