Thermodynamic and structural paradigms of acetylcholinesterase inhibition: Mapping the superior stability of glucosylated eugenol analogues

Eugenol, a phenylpropene found predominantly in clove, is known for its neuroprotective effects, including the inhibition of human acetylcholinesterase (AChE). While early studies identified eugenols as an effective AChE inhibitor, the relative chemical space of its analogues remains largely unexplored in terms of dynamic binding behavior and long-term stability. This study employed a robust, multi-stage computational protocol, combining virtual screening, absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling, and 100 ns molecular dynamics (MD) simulations to identify promising anti-Alzheimer’s leads among natural eugenol congeners. Initial molecular docking against human AChE (PDB ID: 7XN1) revealed that three analogues, eugenol glucoside (-8.6 kcal/mol), ( E )-pseudoisoeugenyl 2-methylbutyrate (-7.1 kcal/mol), and acetyleugenol (-6.5 kcal/mol), exhibited superior predicted binding affinity compared to the reference eugenol (-6.3 kcal/mol). Subsequent pharmacokinetic profiling confirmed all three candidates satisfied key CNS-related criteria (MW < 500 Da, TPSA < 130 Å), justifying further investigation. The subsequent 100 ns MD simulations revealed distinct stabilization mechanisms. Eugenol glucoside, the top-ranked binder, maintained exceptional dynamic stability, employing a dense network of hydrogen bonds to act as a dual anchor across the peripheral anionic site (PAS) and catalytic anionic site (CAS). This stability was quantitatively confirmed by free energy landscape (FEL) analysis, where the eugenol glucoside complex occupied the lowest global energy minimum (ΔG ≈ 4.632 kJ/mol). The results show the critical role of polar functionalization in achieving stable, energetically favored AChE complexes under dynamic conditions. Eugenol glucoside is presented as a high-potential lead for forthcoming experimental validation towards developing safe and chemically versatile therapeutic options for Alzheimer’s disease.