Development of versatile and potent monoquaternary reactivators of acetylcholinesterase

Abstract

To date, the only treatments developed for poisoning by organophosphorus compounds, the most toxic chemical weapons of mass destruction, have exhibited limited efficacy and versatility. The available causal antidotes are based on reactivation of the enzyme acetylcholinesterase (AChE), which is rapidly and pseudo-irreversibly inhibited by these agents. In this study, we developed a novel series of monoquaternary reactivators combining permanently charged moieties tethered to position 6- of 3-hydroxypyridine-2-aldoxime reactivating subunit. Highlighted representatives (21, 24, and 27; also coded as K1371, K1374, and K1375, respectively) that contained 1-phenylisoquinolinium, 7-amino-1-phenylisoquinolinium and 4-carbamoylpyridinium moieties as peripheral anionic site ligands, respectively, showed efficacy superior or comparable to that of the clinically used standards. More importantly, these reactivators exhibited wide-spectrum efficacy and were minutely investigated via determination of their reactivation kinetics in parallel with molecular dynamics simulations to study their mechanisms of reactivation of the tabun-inhibited AChE conjugate. To further confirm the potential applicability of these candidates, a mouse in vivo assay was conducted. While K1375 had the lowest acute toxicity and the most suitable pharmacokinetic profile, the oxime K1374 with delayed elimination half-life was the most effective in ameliorating the signs of tabun toxicity. Moreover, both in vitro and in vivo, the versatility of the agents was substantially superior to that of clinically used standards. Their high efficacy and broad-spectrum capability make K1374 and K1375 promising candidates that should be further investigated for their potential as nerve agents and insecticide antidotes.

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Fig. 1
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Scheme 1
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Fig. 5

taken from trajectory 1 at 8 ns for K1371, trajectory 2 at 24 ns for K1374, and trajectory 1 at 45 ns for K1375. The upper panels display the molecule in monomer A, and the lower panels display the molecule in monomer B. The reactivators are represented with balls and sticks, and the serine-tabun conjugate is represented with sticks, with carbon atoms in cyan. The residues lining the active site gorge are represented with sticks, with carbon atoms in white. Water molecules are displayed as lines. The black dashed lines render H-bonds. The distance d(P-Ox) between the phosphorus atom and the oxygen of the oxime functional group is given in Å

Fig. 6
Fig. 7

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Acknowledgements

The work was supported by a grant from the Ministry of Health of the Czech Republic (no. 17-32801A), by the Long-term development plan (Faculty of Military Health Sciences), University of Hradec Kralove (no. VT2019-2021), and by the French Ministry of Armed Forces (Direction Générale de l'Armement and Service de Santé des Armées) under contract NBC-5-C-4210.

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Development of versatile and highly potent monoquaternary reactivators of acetylcholinesterase. LG—chemical synthesis, design of the compounds, manuscript writing. VH—in vitro determination of cholinesterase reactivation, in vivo experiments. JZK—in vivo experiments, functional observation battery assessment, pharmacokinetic assessment. MH—in vitro determination of cholinesterase reactivation. CC—enzyme production and purification. JD—enzyme production and purification. TK—prediction of the compound's binding pattern into cholinesterases using in silico techniques. TK—prediction of the blood–brain barrier permeation. LM—cytotoxicity evaluation. LP—high-resolution mass spectrometry analysis of the compounds, pharmacokinetic assessment. DM—chemical synthesis, NMR data interpretation. DJ—in vitro determination of cholinesterase reactivation. KM—chemical synthesis, design of the study. FW—kinetics determination of cholinesterase reactivation. FN—in silico analysis—molecular dynamics. OS—manuscript writing, in vivo toxicity determination, design study. JK—data interpretation, chemical synthesis, manuscript writing, design study.

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Correspondence to Ondrej Soukup or Jan Korabecny.

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Gorecki, L., Hepnarova, V., Karasova, J.Z. et al. Development of versatile and potent monoquaternary reactivators of acetylcholinesterase. Arch Toxicol 95, 985–1001 (2021). https://doi.org/10.1007/s00204-021-02981-w

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Keywords

  • Acetylcholinesterase
  • Butyrylcholinesterase
  • Oxime reactivator
  • Organophosphates
  • Organophosphorus compounds
  • Nerve agents
  • Insecticides