De Sousa_2026_Eur.J.Med.Chem_316_119023

Organophosphorus nerve agents exert their acute toxicity by irreversibly inhibiting acetylcholinesterase (AChE), yet currently deployed oxime reactivators exhibit limited efficacy in the central nervous system due to poor blood-brain barrier (BBB) penetration. Addressing this limitation remains a critical challenge in the treatment of nerve agent exposure. We report the design, synthesis, and evaluation of JDS364, an uncharged hybrid oxime reactivator combining a quinoline-based peripheral site ligand with a 3-hydroxypyridinealdoxime nucleophile. In vitro studies using human AChE inhibited by surrogates of G- and V-series nerve agents demonstrated that JDS364 possesses broad-spectrum reactivation activity, notably achieving a 300-fold increase in reactivation efficiency (k(r2)) 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 A 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.