Rational design, synthesis, and evaluation of uncharged, "smart" bis-oxime antidotes of organophosphate-inhibited human acetylcholinesterase

J Biol Chem. 2020 Mar 27;295(13):4079-4092. doi: 10.1074/jbc.RA119.012400. Epub 2020 Feb 4.


Organophosphate (OP) intoxications from nerve agent and OP pesticide exposures are managed with pyridinium aldoxime-based therapies whose success rates are currently limited. The pyridinium cation hampers uptake of OPs into the central nervous system (CNS). Furthermore, it frequently binds to aromatic residues of OP-inhibited acetylcholinesterase (AChE) in orientations that are nonproductive for AChE reactivation, and the structural diversity of OPs impedes efficient reactivation. Improvements of OP antidotes need to include much better access of AChE reactivators to the CNS and optimized orientation of the antidotes' nucleophile within the AChE active-center gorge. On the basis of X-ray structures of a CNS-penetrating reactivator, monoxime RS194B, reversibly bound to native and venomous agent X (VX)-inhibited human AChE, here we created seven uncharged acetamido bis-oximes as candidate antidotes. Both oxime groups in these bis-oximes were attached to the same central, saturated heterocyclic core. Diverse protonation of the heterocyclic amines and oxime groups of the bis-oximes resulted in equilibration among up to 16 distinct ionization forms, including uncharged forms capable of diffusing into the CNS and multiple zwitterionic forms optimal for reactivation reactions. Conformationally diverse zwitterions that could act as structural antidote variants significantly improved in vitro reactivation of diverse OP-human AChE conjugates. Oxime group reorientation of one of the bis-oximes, forcing it to point into the active center for reactivation, was confirmed by X-ray structural analysis. Our findings provide detailed structure-activity properties of several CNS-directed, uncharged aliphatic bis-oximes holding promise for use as protonation-dependent, conformationally adaptive, "smart" accelerated antidotes against OP toxicity.

Keywords: acetylcholinesterase (AChE); aliphatic bis-oxime; crystal structure; drug design; enzyme structure; molecular modeling; organophosphate intoxication; oxime reactivation; structure-function; uncharged oxime antidote.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acetamides / chemistry
  • Acetamides / therapeutic use
  • Acetylcholinesterase / chemistry*
  • Antidotes / chemical synthesis
  • Antidotes / chemistry*
  • Antidotes / therapeutic use
  • Central Nervous System / drug effects*
  • Central Nervous System / enzymology
  • Cholinesterase Inhibitors / chemical synthesis
  • Cholinesterase Inhibitors / chemistry*
  • Cholinesterase Inhibitors / therapeutic use
  • Cholinesterase Reactivators / chemical synthesis
  • Cholinesterase Reactivators / chemistry*
  • Cholinesterase Reactivators / therapeutic use
  • Crystallography, X-Ray
  • Humans
  • Kinetics
  • Organophosphates / chemistry
  • Organophosphates / toxicity
  • Organophosphorus Compounds / chemistry
  • Organophosphorus Compounds / toxicity
  • Oximes / chemical synthesis
  • Oximes / chemistry
  • Oximes / pharmacology
  • Oximes / therapeutic use
  • Protein Conformation / drug effects
  • Structure-Activity Relationship


  • Acetamides
  • Antidotes
  • Cholinesterase Inhibitors
  • Cholinesterase Reactivators
  • Organophosphates
  • Organophosphorus Compounds
  • Oximes
  • RS194B
  • Acetylcholinesterase

Associated data

  • PDB/6U34
  • PDB/6U37
  • PDB/6U3P
  • PDB/4EY4