A-series agent A-234 belongs to a new generation of nerve agents. The poisoning of a former Russian spy Sergei Skripal and his daughter in Salisbury, England, in March 2018 led to the inclusion of A-234 and other A-series agents into the Chemical Weapons Convention. Even though five years have already passed, there is still very little information on its chemical properties, biological activities, and treatment options with established antidotes. In this article, we first assessed A-234 stability in neutral pH for subsequent experiments. Then, we determined its inhibitory potential towards human recombinant acetylcholinesterase (HssAChE; EC 3.1.1.7) and butyrylcholinesterase (HssBChE; EC 3.1.1.8), the ability of HI-6, obidoxime, pralidoxime, methoxime, and trimedoxime to reactivate inhibited cholinesterases (ChEs), its toxicity in rats and therapeutic effects of different antidotal approaches. Finally, we utilized molecular dynamics to explain our findings. The results of spontaneous A-234 hydrolysis showed a slow process with a reaction rate displaying a triphasic course during the first 72 h (the residual concentration 86.2%). A-234 was found to be a potent inhibitor of both human ChEs (HssAChE IC50 = 0.101 ± 0.003 µM and HssBChE IC50 = 0.036 ± 0.002 µM), whereas the five marketed oximes have negligible reactivation ability toward A-234-inhibited HssAChE and HssBChE. The acute toxicity of A-234 is comparable to that of VX and in the context of therapy, atropine and diazepam effectively mitigate A-234 lethality. Even though oxime administration may induce minor improvements, selected oximes (HI-6 and methoxime) do not reactivate ChEs in vivo. Molecular dynamics implies that all marketed oximes are weak nucleophiles, which may explain the failure to reactivate the A-234 phosphorus-serine oxygen bond characterized by low partial charge, in particular, HI-6 and trimedoxime oxime oxygen may not be able to effectively approach the A-234 phosphorus, while pralidoxime displayed low interaction energy. This study is the first to provide essential experimental preclinical data on the A-234 compound.

University Hospital Hradec Kralove Biomedical Research Centre Sokolska 581 500 05 Hradec Králové Czech Republic

University Hospital Hradec Kralove Department of Neurology Sokolska 581 500 05 Hradec Králové Czech Republic

University Hradec Kralove Department of Chemistry Faculty of Science Rokitanskeho 62 50003 Hradec Králové Czech Republic

University of Defence Military Faculty of Medicine Department of Military Medical Service Organization and Management Trebesska 1575 500 01 Hradec Králové Czech Republic

University of Defence Military Faculty of Medicine Department of Toxicology and Military Pharmacy Trebesska 1575 500 01 Hradec Králové Czech Republic

University of Defence Nuclear Biological and Chemical Defence Institute Vita Nejedleho 1 68203 Vyskov Czech Republic

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Aurbek N, Thiermann H, Szinicz L, et al. Analysis of inhibition, reactivation and aging kinetics of highly toxic organophosphorus compounds with human and pig acetylcholinesterase. Toxicology. 2006;224:91–99. doi: 10.1016/j.tox.2006.04.030. PubMed DOI

Bajgar J. Nerve agents poisoning and its treatment in schematic figures and tables. Elsevier; 2012.

Becker G, Kawan A, Gutzeit D, et al. Direct reaction of oximes with crotylsarin, cyclosarin, or VX in vitro. Arch Toxicol. 2007;81:415–420. doi: 10.1007/s00204-006-0168-z. PubMed DOI

Bhakhoa H, Rhyman L, Ramasami P. Theoretical study of the molecular aspect of the suspected novichok agent A234 of the Skripal poisoning. R Soc Open Sci. 2019;6:181831. doi: 10.1098/rsos.181831. PubMed DOI PMC

Bowie JU, Lüthy R, Eisenberg D. A method to identify protein sequences that fold into a known three-dimensional structure. Science. 1991;253:164–170. doi: 10.1126/science.1853201. PubMed DOI

Carlsen L. After salisbury nerve agents revisited. Mol Inform. 2019;38:e1800106. doi: 10.1002/minf.201800106. PubMed DOI

Chai PR, Hayes BD, Erickson TB, Boyer EW. Novichok agents: a historical, current, and toxicological perspective. Toxicol Commun. 2018;2:45–48. doi: 10.1080/24734306.2018.1475151. PubMed DOI PMC

da Silva JAV, Pereira AF, LaPlante SR, et al. Reactivation of VX-inhibited human acetylcholinesterase by deprotonated pralidoxime. Complement Quant Mech Study Biomol. 2020;10:192. doi: 10.3390/biom10020192. PubMed DOI PMC

de Koning MC, Vieira Soares C, van Grol M, et al. Effective degradation of novichok nerve agents by the zirconium metal-organic framework MOF-808. ACS Appl Mater Interfaces. 2022;14:9222–9230. doi: 10.1021/acsami.1c24295. PubMed DOI

Dhuguru J, Zviagin E, Skouta R. FDA-approved oximes and their significance in medicinal chemistry. Pharmaceuticals. 2022;15:66. doi: 10.3390/ph15010066. PubMed DOI PMC

Ellman GL, Courtney KD, Andres V, Feather-Stone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961;7:88–95. doi: 10.1016/0006-2952(61)90145-9. PubMed DOI

De Farias R. The number of conformers explains the high toxicity of novichok agents. Chem J. 2019;6:24–26.

Forsberg Å, Puu G. Kinetics for the inhibition of acetylcholinesterase from the electric eel by some organophosphates and carbamates. Eur J Biochem. 1984;140:153–156. doi: 10.1111/j.1432-1033.1984.tb08079.x. PubMed DOI

Franca TCC, Kitagawa DAS, de Cavalcante SFA, et al. Novichoks: the dangerous fourth generation of chemical weapons. Int J Mol Sci. 2019;20:E1222. doi: 10.3390/ijms20051222. PubMed DOI PMC

Frisch MJ, Trucks GW, Schlegel HB, et al. Gaussian 16 Rev. C.01. CT: Wallingford; 2016.

Gerlits O, Kong X, Cheng X, et al. Productive reorientation of a bound oxime reactivator revealed in room temperature X-ray structures of native and VX-inhibited human acetylcholinesterase. J Biol Chem. 2019;294:10607–10618. doi: 10.1074/jbc.RA119.008725. PubMed DOI PMC

Gertz B (1997) Russia dodges chemical arms ban. Wash. TIMES

Gorecki L, Hepnarova V, Karasova JZ, et al. Development of versatile and potent monoquaternary reactivators of acetylcholinesterase. Arch Toxicol. 2021;95:985–1001. doi: 10.1007/s00204-021-02981-w. PubMed DOI

Hanwell MD, Curtis DE, Lonie DC, et al. Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. J Cheminform. 2012;4:17. doi: 10.1186/1758-2946-4-17. PubMed DOI PMC

Harvey SP, McMahon LR, Berg FJ. Hydrolysis and enzymatic degradation of Novichok nerve agents. Heliyon. 2020;6:e03153. doi: 10.1016/j.heliyon.2019.e03153. PubMed DOI PMC

Horn G, Kranawetvogl T, John H, et al. Human HepaRG liver spheroids: cold storage protocol and study on pyridinium oxime-induced hepatotoxicity in vitro. Chem Biol Interact. 2023;369:110285. doi: 10.1016/j.cbi.2022.110285. PubMed DOI

Hrvat NM, Kovarik Z. Counteracting poisoning with chemical warfare nerve agents. Arch Ind Hyg Toxicol. 2020;71:266–284. doi: 10.2478/aiht-2020-71-3459. PubMed DOI PMC

Hulse EJ, Davies JOJ, Simpson AJ, et al. Respiratory complications of organophosphorus nerve agent and insecticide poisoning. Implications for respiratory and critical care. Am J Respir Crit Care Med. 2014;190:1342–1354. doi: 10.1164/rccm.201406-1150CI. PubMed DOI PMC

Imrit YA, Bhakhoa H, Sergeieva T, et al. A theoretical study of the hydrolysis mechanism of A-234; the suspected novichok agent in the Skripal attack. RSC Adv. 2020;10:27884–27893. doi: 10.1039/D0RA05086E. PubMed DOI PMC

Jeong K, Choi J. Theoretical study on the toxicity of ‘Novichok’ agent candidates. R Soc Open Sci. 2019;6:190414. doi: 10.1098/rsos.190414. PubMed DOI PMC

Jung H, Heo J, Park N, et al. Elimination of A-234 from the environment: effect of different decontaminants. J Hazard Mater. 2023;451:131150. doi: 10.1016/j.jhazmat.2023.131150. PubMed DOI

Kassa J, Karasova JZ, Pavlikova R, et al. The influence of combinations of oximes on the reactivating and therapeutic efficacy of antidotal treatment of tabun poisoning in rats and mice. J Appl Toxicol. 2010;30:120–124. doi: 10.1002/jat.1477. PubMed DOI

Kassa J, Misik J, Hatlapatkova J, et al. The evaluation of the reactivating and neuroprotective efficacy of two newly prepared bispyridinium oximes (K305, K307) in Tabun-poisoned rats—a comparison with trimedoxime and the oxime K203. Molecules. 2017;22:1152. doi: 10.3390/molecules22071152. PubMed DOI PMC

Kuca K, Musilek K, Pohanka M, et al. Reactivation potency of the acetylcholinesterase reactivator obidoxime is limited. Biomed Pap. 2009;153:259–262. doi: 10.5507/bp.2009.044. PubMed DOI

Kuca K, Jun D, Musilek K, et al. Prophylaxis and post-exposure treatment of intoxications caused by nerve agents and organophosphorus pesticides. Mini-Rev Med Chem. 2013;13:2102–2115. doi: 10.2174/13895575113136660108. PubMed DOI

Lee JY, Lim KC, Kim HS. Characterization and study on fragmentation pathways of a novel nerve agent, ‘Novichok (A234)’, in aqueous solution by liquid chromatography–tandem mass spectrometry. Molecules. 2021;26:1059. doi: 10.3390/molecules26041059. PubMed DOI PMC

Lei C, Sun X. Comparing lethal dose ratios using probit regression with arbitrary slopes. BMC Pharmacol Toxicol. 2018;19:61. doi: 10.1186/s40360-018-0250-1. PubMed DOI PMC

Lewis S. Salisbury, novichok and international law on the use of force. RUSI J. 2018;163:10–19. doi: 10.1080/03071847.2018.1529889. DOI

Lindahl A, Hess S van der (2020) GROMACS 2020 manual. 10.5281/zenodo.3562512

Liu J, Uchea C, Wright L, Pope C. Chapter 34 - chemical warfare agents and the nervous system. In: Gupta RC, editor. Handbook of toxicology of chemical warfare agents. 2. Boston: Academic Press; 2015. pp. 463–475.

Lundy PM, Hamilton MG, Sawyer TW, Mikler J. Comparative protective effects of HI-6 and MMB-4 against organophosphorous nerve agent poisoning. Toxicology. 2011;285:90–96. doi: 10.1016/j.tox.2011.04.006. PubMed DOI

Lüthy R, Bowie JU, Eisenberg D. Assessment of protein models with three-dimensional profiles. Nature. 1992;356:83–85. doi: 10.1038/356083a0. PubMed DOI

Mark P, Nilsson L. Structure and dynamics of the TIP3P, SPC, and SPC/E water models at 298 K. J Phys Chem A. 2001;105:9954–9960. doi: 10.1021/jp003020w. DOI

Marrs TC. Organophosphate poisoning. Pharmacol Ther. 1993;58:51–66. doi: 10.1016/0163-7258(93)90066-M. PubMed DOI

Masson P, Carletti E, Nachon F. Structure, activities and biomedical applications of human butyrylcholinesterase. Protein Pept Lett. 2009;16:1215–1224. doi: 10.2174/092986609789071207. PubMed DOI

Mirzaynov VS. State secrets: an insider’s chroniclr of the Russians chemical weapons program. Outskirts Press; 2008.

Morris GM, Huey R, Lindstrom W, et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem. 2009;30:2785–2791. doi: 10.1002/jcc.21256. PubMed DOI PMC

Nachon F, Brazzolotto X, Trovaslet M, Masson P. Progress in the development of enzyme-based nerve agent bioscavengers. Chem Biol Interact. 2013;206:536–544. doi: 10.1016/j.cbi.2013.06.012. PubMed DOI

Nepovimova E, Kuca K. Chemical warfare agent NOVICHOK—mini-review of available data. Food Chem Toxicol Int J Publ Br Ind Biol Res Assoc. 2018;121:343–350. doi: 10.1016/j.fct.2018.09.015. PubMed DOI

O’Boyle NM, Banck M, James CA, et al. Open Babel: an open chemical toolbox. J Cheminform. 2011;3:33. doi: 10.1186/1758-2946-3-33. PubMed DOI PMC

OPCW (2019) Schedule 1. https://www.opcw.org/chemical-weapons-convention/annexes/annex-chemicals/schedule-1. Accessed 25 Nov 2022

OPCW (2020) Case of Mr Alexei Navalny. https://www.opcw.org/media-centre/featured-topics/case-mr-alexei-navalny. Accessed 25 Nov 2022

Opravil J, Pejchal J, Finger V, et al. A-agents, misleadingly known as “Novichoks”: a narrative review. Arch Toxicol. 2023;97:2587–2607. doi: 10.1007/s00204-023-03571-8. PubMed DOI PMC

Pejchal J, Osterreicher J, Kuca K, et al. The influence of acetylcholinesterase reactivators on selected hepatic functions in rats. Basic Clin Pharmacol Toxicol. 2008;103:119–123. doi: 10.1111/j.1742-7843.2008.00249.x. PubMed DOI

Pettersen EF, Goddard TD, Huang CC, et al. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004;25:1605–1612. doi: 10.1002/jcc.20084. PubMed DOI

Pulkrabkova L, Svobodova B, Konecny J, et al. Neurotoxicity evoked by organophosphates and available countermeasures. Arch Toxicol. 2022 doi: 10.1007/s00204-022-03397-w. PubMed DOI

Radić Z. Shifts in backbone conformation of acetylcholinesterases upon binding of covalent inhibitors. Revers Ligands Substr Cryst. 2021;11:1557. doi: 10.3390/cryst11121557. DOI

Saxena A, Redman AMG, Jiang X, et al. Differences in active-site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase. Chem Biol Interact. 1999;119–120:61–69. doi: 10.1016/S0009-2797(99)00014-9. PubMed DOI

Soukup O, Krůšek J, Kaniaková M, et al. Oxime reactivators and their in vivo and in vitro effects on nicotinic receptors. Physiol Res. 2011;60:679–686. doi: 10.33549/physiolres.932105. PubMed DOI

Soukup O, Kumar UK, Proska J, et al. The effect of oxime reactivators on muscarinic receptors: functional and binding examinations. Environ Toxicol Pharmacol. 2011;31:364–370. doi: 10.1016/j.etap.2011.01.003. PubMed DOI

Soukup O, Jun D, Tobin G, Kuca K. The summary on non-reactivation cholinergic properties of oxime reactivators: the interaction with muscarinic and nicotinic receptors. Arch Toxicol. 2013;87:711–719. doi: 10.1007/s00204-012-0977-1. PubMed DOI

Steindl D, Boehmerle W, Körner R, et al. Novichok nerve agent poisoning. Lancet Lond Engl. 2021;397:249–252. doi: 10.1016/S0140-6736(20)32644-1. PubMed DOI

Tallarida RJ, Murray RB. Manual of pharmacologic calculations with computer programs. New York: Springer-Verlag; 1987.

Trott O, Olson AJ. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010;31:455–461. doi: 10.1002/jcc.21334. PubMed DOI PMC

U.S. government (2019) Fourth Generation Agents Hospital - Medical Management Guidelines - CHEMM

van Helden HP, Busker RW, Melchers BP, Bruijnzeel PL. Pharmacological effects of oximes: how relevant are they? Arch Toxicol. 1996;70:779–786. doi: 10.1007/s002040050340. PubMed DOI

Vanquelef E, Simon S, Marquant G, et al. R.E.D. Server: a web service for deriving RESP and ESP charges and building force field libraries for new molecules and molecular fragments. Nucleic Acids Res. 2011;39:W511–W517. doi: 10.1093/nar/gkr288. PubMed DOI PMC

Vogel G. Vergiftungen mit dem insecticid E 605. Arch Tocxicology. 1953;14:381–395. doi: 10.1007/BF00571919. PubMed DOI

Vogel E, Moats D, Woolgar S, Helgesson C-F. Thinking with imposters: the imposter as analytic. In: Helgesson C-F, Moats D, Vogel E, Woolgar S, editors. The imposter as social theory: thinking with gatecrashers, cheats and charlatans. Bristol University Press; 2021. pp. 1–30.

Wang J, Wang W, Kollman PA, Case DA. Automatic atom type and bond type perception in molecular mechanical calculations. J Mol Graph Model. 2006;25:247–260. doi: 10.1016/j.jmgm.2005.12.005. PubMed DOI

Worek F, Reiter G, Eyer P, Szinicz L. Reactivation kinetics of acetylcholinesterase from different species inhibited by highly toxic organophosphates. Arch Toxicol. 2002;76:523–529. doi: 10.1007/s00204-002-0375-1. PubMed DOI

Worek F, Thiermann H, Szinicz L, Eyer P. Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes. Biochem Pharmacol. 2004;68:2237–2248. doi: 10.1016/j.bcp.2004.07.038. PubMed DOI

Worek F, Aurbek N, Thiermann H. Reactivation of organophosphate-inhibited human AChE by combinations of obidoxime and HI 6 in vitro. J Appl Toxicol JAT. 2007;27:582–588. doi: 10.1002/jat.1241. PubMed DOI

Worek F, Wille T, Aurbek N, et al. Reactivation of organophosphate-inhibited human, cynomolgus monkey, swine and guinea pig acetylcholinesterase by MMB-4: a modified kinetic approach. Toxicol Appl Pharmacol. 2010;249:231–237. doi: 10.1016/j.taap.2010.09.021. PubMed DOI

Worek F, Thiermann H, Wille T. Organophosphorus compounds and oximes: a critical review. Arch Toxicol. 2020;94:2275–2292. doi: 10.1007/s00204-020-02797-0. PubMed DOI PMC

Synthesis and in vitro assessment of the reactivation profile of clinically available oximes on the acetylcholinesterase model inhibited by A-230 nerve agent surrogate

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