Probing the activity of a non-oxime reactivator for acetylcholinesterase inhibited by organophosphorus nerve agents

Abstract

Currently fielded treatments for nerve agent intoxication include atropine, an acetylcholine receptor antagonist, and pralidoxime (2PAM), a small molecule reactivator of acetylcholinesterase (AChE). 2PAM reactivates nerve agent-inhibited AChE via direct nucleophilic attack by the oxime moiety on the phosphorus center of the bound nerve agent. Due to a permanently charged pyridinium motif, 2PAM is not thought to cross the blood brain barrier and therefore cannot act directly in the neuronal junctions of the brain. In this study, ADOC, a non-permanently charged, non-oxime molecule initially identified using pesticide-inhibited AChE, was characterized in vitro against nerve agent-inhibited recombinant human AChE. The inhibitory and reactivation potentials of ADOC were determined with native AChE and AChE inhibited with tabun, sarin, soman, cyclosarin, VX, or VR and then compared to those of 2PAM. Several structural analogs of ADOC were used to probe the reactivation mechanism of the molecule. Finally, guinea pigs were used to examine the protective efficacy of the compound after exposure to sarin. The results of both in vitro and in vivo testing will be useful in the design of future small molecule reactivators.

Keywords: Acetylcholinesterase; Guinea pig; Organophosphorus nerve agent; Oxime; Pralidoxime; Reactivator.

Figure 1.

The chemical structures of six organophosphorus nerve agents and two reactivator compounds referenced in this work.

Figure 2.

A) IC₅₀ of ADOC(●) and 2PAM (▲) was determined with human recombinant acetylcholinesterase (RHuAChE). B) Competitive inhibition assay using ADOC in increasing concentrations as the inhibitor and AtCh as the competitive substrate.

Figure 3.

Rates (k_r, min⁻¹) and dissociation constants (K_D, μM) of the reactivation of inhibited RHuAChE by 2PAM (black) and ADOC (grey). Significant differences between 2PAM and ADOC values are indicated by *(t-test, p<0.05).

Figure 4.

Dose-dependence of In Vivo Reactivation by ADOC. A) Reactivation of red blood cell acetylcholinesterase by ADOC administered at doses of 1.8, 10.0, 18.0, 32.0, or 56.0 mg/kg to animals exposed to 1xLD₅₀ GB (▲), GF (▼) or VX (●). Whole blood reactivation displayed similar trends though absolute reactivation values differed (data not shown). B) Reactivation of mid-brain tissue acetylcholinesterase by ADOC administered at the indicated doses to animals exposed to GB (▲), GF (▼) or VX (●). All other tissues tested (brainstem, cerebellum, cortex, hippocampus, striatum, spinal cord, diaphragm, heart and skeletal muscle) displayed similar reactivation trends though absolute reactivation values differed (data not shown).

Scheme 1.

Reactivation from Worek et al. (2004 [19])