Structural basis of VAChT inhibition by spiroindolines and alkylsulfones.

Cholinergic signaling relies on the vesicular acetylcholine transporter (VAChT) to package acetylcholine (ACh) into synaptic vesicles. VAChT dysfunction is implicated in neurological and neuromuscular disorders, including Alzheimer's disease and myasthenia gravis, yet current inhibitors such as vesamicol suffer from off-target effects. Here, we present cryo-EM structures of human VAChT bound to two inhibitors, spiroindoline and alkylsulfone, revealing their divergent binding modes that converge in occupying the central substrate pocket. Mutagenesis studies identify conserved residues critical for inhibitor binding. These findings elucidate the molecular mechanisms by which VAChT binds structurally distinct inhibitors, providing a blueprint for designing optimized therapeutics. Furthermore, species-specific structural variations identified here suggest opportunities to develop selective insecticides through ortholog-targeted inhibition. Taken together, this work bridges mechanistic insights with therapeutic and agricultural applications, advancing precision tools for modulating the cholinergic system.