Refined uncharged bis-oximes for rapid CNS reactivation of OP-inhibited hAChE. Project Summary. Reactivation of organophosphate (OP) inhibited acetylcholinesterase (AChE) by anti-OP antidotes is the only true remedy for OP intoxication in nerve agent or OP pesticide exposure. Most of currently approved and experimental anti-OP antidotes are nucleophilic aldoximes that attack and remove phosphorus atom of OPs covalently attached to the AChE active serine. One significant way of improving current antidotal treatment of OP intoxication is by creation of reactivating antidotes that will reach inhibited AChE in peripheral tissue but also in the CNS, where currently approved antidotes cannot enter. We have designed and characterized in vitro seven improved uncharged LG bis-oxime antidotes of a novel scaffold, with better reactivation of sarin, cyclosarin, VX and paraoxon inhibited hAChE than RS194B. The uniquely novel feature of the LG bis-oxime scaffold is that ionizable cyclic amines and aldoxime groups allow single compound to equilibrate between up to sixteen ionic forms differing in nucleophilic reactivity and in conformation. We now propose to explore and structurally optimize this new antidote scaffold, based on our yet unpublished X-ray structural templates of both LG bis-oximes and of the fastest pyridinium bis-oxime MMB4. Templates will be informed by inelastic neutron scattering (INS) data. We will test refined compounds for reactivation potency against OP-hAChE conjugates in vitro and will evaluate the initial PK profile of most promissing reactivators in mice. Interaction of best refined candidates with AChE will be explored by X-ray and INS.