Oleanolic Acid Inhibits Acetylcholinesterase and Butyrylcholinesterase Through Gorge‐Dependent Allosteric Mechanisms: An Integrated Kinetic and Molecular Dynamics Study

ABSTRACT

Potent cholinesterase inhibitors are needed for their key role in the management of neurodegenerative disorders including Alzheimer disease (AD). Oleanolic acid, a pentacyclic triterpene with known neuroprotective activity, was deeply studied as a dual inhibitor of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) through combined in vitro enzyme kinetics, molecular docking and dynamics simulations. Oleanolic acid inhibited both enzymes through a non‐competitive mode, with IC ₅₀ of 18.54 ± 0.3 μM for AChE and 66.46 ± 0.4 μM for BChE, and an identical Kᵢ of 10 ± 0.1 μM. This AChE selectivity despite equal binding affinity forms an apparent paradox that was resolved by gorge architecture. MD simulations showed that oleanolic acid migrated away from its initial docked position in the narrow AChE gorge to peripheral/PAS‐adjacent residues, consistent with the non‐competitive kinetic profile and blind docking. In contrast, the wider BChE gorge stably accommodated the ligand through the trajectory, with targeted and blind docking converging on the same intra‐gorge site. The greater AChE inhibitory potency is attributable to the disruption of substrate‐guidance and gorge‐breathing dynamics in the architecturally constrained AChE system. These findings establish oleanolic acid as a gorge‐architecture‐dependent allosteric modulator of both cholinesterase isoforms and support its potential as a scaffold for developing dual ChE inhibitors for AD.