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

Abstract

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.

Development of a CNS-permeable reactivator for nerve agent exposure: an iterative, multi-disciplinary approach

“…In addition, efficacy needs to be increased and toxicity needs to be reduced by respectively enhancing the antidote-nerve agent complex off rates in adducted AChE and abating off-target binding. Satisfying these requirements is a daunting endeavor and the subject of many efforts; several of which show promise 10 – 28 .…”

Covalent inhibition of hAChE by organophosphates causes homodimer dissociation through long-range allosteric effects

“…They J o u r n a l P r e -p r o o f reveal formation of a 4HB homodimer in the crystal packing. In our 11 recently deposited crystal structures based on the same hAChE construct as the one used in this study, we observe a face-to-face (F-2-F) homodimer in addition to the 4HB dimer, for a total of two simultaneously formed homodimers in the crystal packing (11,21,27).…”

“…S7 ) in complex with wildtype hAChE. High-affinity ligands, (-)huperzine A (K i = 22 nM; ( 19 )) and ambenonium (K i = 250 pM; ( 19 )) at 1.0 mM concentrations, and lower affinity aldoximes, 2PAM (K d = 150 μM; ( 20 )), HI6 (K d = 38 μM; ( 20 )), and RS194B (K i = 157 μM; ( 21 )) at 5 mM concentrations, were used to saturate all hAChE-binding sites in each hAChE–ligand complex. Figure 5 A shows a P(r) profile overlay of wildtype apo hAChE with P(r) profiles recorded for each of the reversible ligand complexes.…”

A Thermophilic Bacterial Esterase for Scavenging Nerve Agents: A Kinetic, Biophysical and Structural Study

“…Despite great efforts for many years, the currently used oximes are not efficient on all OPNA-phosphyl adducts, and do not readily cross the blood–brain barrier, thus preventing an efficient action in the CNS. Recent efforts have focused on uncharged [ 3 ] or zwitterionic [ 4 ] oxime reactivators, in order to favor the CNS tropism of these molecules.…”