α-Asaronol, a low-toxicity α-asarone metabolite, suppresses seizures in zebrafish Dravet syndrome via positive modulation of GABAA receptors and LDH inhibition.

Dravet syndrome (DS), a rare and severe developmental encephalopathy in children classified as a refractory epilepsy syndrome, presents significant challenges in therapeutic development with urgent unmet needs. α-Asaronol (α-AOL), a terminal hydroxylated metabolite of α-asarone (α-A) and an active constituent of Acorus plants, has demonstrated potent anti-seizure effects in mouse and zebrafish models based on preliminary studies. However, its therapeutic potential for DS remains unexplored. Given the pediatric focus of this research, parallel investigation of its primary metabolites (α-OA and α-TMCA) is imperative to ensure safety. This study aims to (1) validate the general anti-seizure activities of α-OA and α-TMCA, (2) evaluate their comparative efficacy against DS alongside α-AOL, and (3) elucidate underlying mechanisms. We systematically assessed anti-seizure activities using pentylenetetrazol-induced seizure models in zebrafish and mice, followed by rigorous evaluation in a zebrafish DS model (scn1Lab-/- mutants). Mechanistic pathways were investigated through GABAA receptor modulation, energy metabolism regulation, and molecular docking analyses. Results revealed dose-dependent anti-seizure efficacy across all models, with potency ranking α-AOL > α-A > α-OA ≫ α-TMCA. Notably, α-AOL exhibited superior efficacy in scn1Lab-/- mutants compared to α-asarone and reference drugs stiripentol and cannabidiol. Mechanistic studies identified dual pathways for α-AOL: potentiation of GABAA receptor currents (EC50 = 34.0 μmol/L) and inhibition of neuronal lactate dehydrogenase activity. These findings establish terminal oxidation of α-asarone as a critical metabolic pathway enhancing anti-seizure efficacy while reducing toxicity, positioning α-AOL as a promising lead compound for DS therapeutics.