UNCORRECTED PROOF ARTICLE INFO Article history: Received 1 August 2016 Received in revised form 14 September 2016 Accepted 16 September 2016 Available online xxx Keywords: Nerve agents Antidotes Imidazole oximes Benzimidazole oximes Butyrylcholinesterase Reactivation ABSTRACT A well-considered treatment of acute nerve agents poisoning involves the exogenous administration of butyryl- cholinesterase (BChE, EC 3.1.1.8) as a stoichiometric bioscavenger efficient in preventing cholinergic crises caused by acetylcholinesterase (AChE, EC 3.1.1.7) inhibition. An additional improvement in medical countermeasures would be to use oximes that could reactivate BChE as well to upgrade bioscavenging from stoichiometric to oxime-assisted catalytic. Therefore, in this paper we investigated the potency of 39 imidazolium and benzimidazolium oximes (36 compounds synthesized for the first time) to be considered as the reactivators specifically designed for reactivation of phosphory- lated human BChE. Their efficiency in the reactivation of paraoxon-, VX-, and tabun-inhibited human BChE, as well as human AChE was tested and compared with the efficiencies of HI-6 and obidoxime, used in medical practice today. A comprehensive analysis was performed for the most promising oximes defining kinetic parameters of reactivation as well as interactions with uninhibited BChE. Furthermore, experimental data were compared with computational studies (docking, QSAR analysis) as a starting point in future oxime structure refinement. Considering the strict criteria set for in vivo applications, we determined the cytotoxicity of lead oximes on two cell lines. Among the tested oxime library, one imidazolium compound was selected for preliminary in vivo antidotal study in mice. The obtained protection in VX poisoning outlines its potential in development oxime-assisted OP-bioscavenging with BChE. © 2016 Published by Elsevier Ltd. Toxicology and Applied Pharmacology xxx (2016) xxx-xxx Contents lists available at ScienceDirect Toxicology and Applied Pharmacology journal homepage: www.elsevier.com A comprehensive evaluation of novel oximes in creation of butyrylcholinesterase-based nerve agent bioscavengers Maja Katalinić a , Nikolina Maček Hrvat a , Krešimir Baumann b , Sara Morasi Piperčić b , Sandro Makarić b , Srđanka Tomić b , Ozren Jović b , Tomica Hrenar b , Ante Miličević a , Dubravko Jelić c , Suzana Žunec a , Ines Primožič b, ⁎ , Zrinka Kovarik a, ⁎ a Institute for Medical Research and Occupational Health, POB 291, HR-10001 Zagreb, Croatia b Department of Chemistry, Faculty of Science, University of Zagreb, HR-10001 Zagreb, Croatia c Fidelta Ltd., HR-10001 Zagreb, Croatia 1. Introduction Butyrylcholinesterase (BChE, EC 3.1.1.8) is a serine hydrolase, re- lated to the neurotransmission-essential acetylcholinesterase (AChE, EC 3.1.1.7) (Darvesh et al., 2003). The two enzymes differ genet- ically, structurally and in their substrate specificities and sensitivi- ties to a wide range of inhibitors. Though BChE is not physiologi- cally essential for humans, it has been shown that BChE is widely distributed in the organism (Chatonnet and Lockridge, 1989; Darvesh et al., 2003; Çokuğras, 2003) and can serve as a first line of de- fence in the case of poisoning by highly toxic anti-acetylcholinesterase compounds like organophosphorus (OP) nerve agents and pesticides. Consequently, BChE has been explored as a possible stoichiometric bioscavenger in such a poisoning (Raveh et al., 1997; Lenz et al., 2010; Doctor and Saxena, 2005; Saxena et al., 2011). Nowadays, a newly considered treatment of acute OP poisoning involves the ex- ogenous administration of BChE as a stoichiometric OP bioscavenger efficient in preventing inhibition of native cholinesterases and death (Cerasoli et al., 2005; Huang et al., 2007; Vučinić et al., 2013). Fur ⁎ Corresponding authors. Email addresses: ines.primozic@chem.pmf.hr (I. Primožič); zkovarik@imi.hr (Z. Kovarik) ther, joint administration of an efficient oxime reactivator of the OP–BChE conjugate and purified BChE protein to assist catalysis and turnover of the OP, should, in theory, reduce the amount of BChE needed for efficient protection by establishing a so called “oxime-as- sisted catalytic bioscavenger” system (Kovarik et al., 2010; Sit et al., 2011, 2014). The feasibility of such an approach was demonstrated re- cently by a cationic non-pyridinium aldoxime, TAB2OH and BChE pair as well as by combination of AChE mutants and pyridinium oximes (Radić et al., 2013a; Kovarik et al., 2015; Maček Hrvat et al., 2016). Reactivation of OP-BChE conjugate by imidazole-based oximes has been recently described in the literature as a promising step for- ward (Sit et al., 2011, 2014). The absence of a charge in some of these imidazole oximes bears the promise of reasonable oral bioavail- ability and retention in tissue as well as potential for central nervous system (CNS) penetration despite some agents having limited solu- bility. The quaternary analogues are expected to have smaller vol- umes of distribution and higher initial plasma concentrations. Qua- ternary imidazolium oximes were extensively studied as mono-oxime OP-AChE reactivators (Grifantini et al., 1972, 1973; Franchetti et al., 1974; Bedford et al., 1984, 1989a, 1989b; Goff et al., 1991; Koolpe et al., 1991). The attention was turned to the imidazole heterocyclic system because it was isosteric with the pyridine ring in known pyri http://dx.doi.org/10.1016/j.taap.2016.09.015 0041-008/© 2016 Published by Elsevier Ltd.