Design, synthesis and evaluation of an uncharged broad spectrum quinoline-oxime hybrid for the reactivation of nerve agent-inhibited human acetylcholinesterase

) over the clinical benchmark obidoxime against tabun-like inhibited hAChE. Evaluation in a human in vitro BBB co-culture model revealed that JDS364 exhibits high permeability, significantly outperforming clinically used quaternary oximes and exceeding the flux of earlier-generation uncharged hybrids. Ex vivo functional profiling studies in mice confirmed rapid systemic availability and significant protection against paraoxon challenge (Protective Index = 6.7 when combined with atropine), though a narrower therapeutic window was observed compared to clinical standards. X-ray crystallographic analysis of JDS364 bound to human AChE uncovered ligand-induced conformational plasticity, providing the first structural evidence of an opening of a "backdoor" via Tyr449 rearrangement in the 20 Å deep active site gorge. This confirms the hypothesis of enzyme "breathing" motions and validates alternative diffusion pathways for reactivation. These findings establish JDS364 as a mechanistically significant, CNS-accessible lead that defines a new structural paradigm for the development of next-generation countermeasures.