Eugenol-based derivatives as potential acetylcholinesterase-targeting biopesticides: an integrated in silico and in vitro study

The extensive use of synthetic insecticides has raised concerns regarding environmental persistence, resistance development, and toxicity toward non-target organisms, necessitating the development of sustainable alternatives. Eugenol, a plant-derived phenylpropanoid, is recognized for its insecticidal properties; however, its efficacy can be further improved through structural modification. In this study, a prediction-driven approach was employed to evaluate eugenol derivatives as acetylcholinesterase (AChE)-targeting biopesticides. Molecular docking revealed strong binding affinities within the AChE catalytic gorge, with eugenol valerate (–7.4 kcal·mol⁻¹) and eugenol isovalerate (–7.0 kcal·mol⁻¹) outperforming the reference insecticide malathion (–6.7 kcal·mol⁻¹). Molecular dynamics simulations confirmed the structural stability of the ligand–enzyme complexes. In vitro leaf exposure studies against Planococcus citri demonstrated rapid insecticidal activity, with eugenol achieving approximately threefold higher mortality than malathion within 24 h and complete mortality by 48 h. Despite exhibiting weaker AChE inhibitory potency than malathion in vitro, the rapid mortality induced by eugenol may be associated with favourable toxicokinetic properties, including efficient cuticular penetration, moderate lipophilicity, rapid membrane diffusion, and possible secondary neurophysiological effects beyond direct AChE inhibition. Additionally, AChE inhibition assays showed concentration-dependent inhibition, with eugenol exhibiting moderate inhibitory activity (IC₅₀ = 12.18 mM) compared to malathion (IC₅₀ = 2.25 mM). In silico ADME analysis indicated favourable physicochemical and environmental behaviour, including optimal lipophilicity, good aqueous solubility, high absorption potential, and compliance with pesticide-relevant property criteria without PAINS alerts, supporting their suitability as biopesticide candidates. Docking analysis indicated that eugenol derivatives interact with AChE mainly through non-covalent interactions within the catalytic gorge, which may differ from the irreversible inhibition mechanism associated with organophosphate insecticides. Structural modification through esterification may also contribute to improved physicochemical stability and residual activity relative to native eugenol. These findings highlight the potential of eugenol-based derivatives as environmentally compatible, AChE-targeting biopesticides for sustainable pest management. Graphical abstract