https://doi.org/10.1016/j.cbi.2019.04.020 Chemico-Biological Interactions Available online 18 April 2019 In Press, Accepted Manuscript

Butyrylcholinesterase inhibited by nerve agents is efficiently reactivated with chlorinated pyridinium oximes

Tamara Zorbaza, David Malinakb,c, Kamil Kuca,b Kamil Musilekb,c*, Zrinka Kovarika* aInstitute for Medical Research and Occupational Health, Ksaverska cesta 2, HR-10000 Zagreb, Croatia bDepartment of Chemistry, Faculty of Science, University of Hradec Kralove, Rokitanskeho 62, 50003 Hradec Kralove, Czech Republic cUniversity Hospital in Hradec Kralove, Biomedical Research Center, Sokolska 581, 50005 Hradec Kralove, Czech Republic

*Corresponding authors: Z. Kovarik, PhD; [email protected] K. Musilek, PhD; [email protected]

Abstract Bispyridinium oximes with one (K865, K866, K867) or two (K868, K869, K870) ortho-positioned chlorine moiety, analogous to previously known K027, K048 and K203 oximes, and potent reactivators of human (AChE) inhibited by nerve agents, were tested in the reactivation of human butyrylcholinesterase (BChE) inhibited by , , VX, and . A previously highlighted AChE reactivator, dichlorinated bispyridinium oxime with propyl linker (K868), was tested in more detail for reactivation of four -BChE conjugates. Its BChE reactivation potency was showed to be promising when compared to the standard oximes used in medical practice, asoxime (HI-6) and (2-PAM), especially in case of sarin and tabun. This finding could be used in the pseudo-catalytic scavenging of the most nerve agents due to its cumulative capacity to reactivate both AChE and BChE.

Keywords organophosphorus compounds; quaternary oxime; reactivators; ; pseudo-catalytic scavenger

1 https://doi.org/10.1016/j.cbi.2019.04.020 Chemico-Biological Interactions Available online 18 April 2019 In Press, Accepted Manuscript Graphical abstract

Highlights  Ortho-positioned chlorine moiety of pyridinium oximes improves reactivation  Dichlorinated oxime K868 is the lead reactivator of BChE inhibited by nerve agents  K868 is superior reactivator than HI-6 and 2-PAM for sarin- and tabun-BChE  K868 could serve as post-exposure therapy for sarin, cyclosarin, and VX

Abbreviations AChE, acetylcholinesterase; ATCh, acetylthiocholine iodide; BBB, blood−brain barrier; BChE, butyrylcholinesterase; BSA, bovine serum albumin; cyclosarin, cyclohexyl methylphosphonofluoridate; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); HI-6, asoxime [1-(((4- (aminocarbonyl)pyridino)methoxy)methyl)-2-((hydroxyimino)methyl)pyridinium dichloride]; K027, 4-carbamoyl-1-(3-(4-((hydroxyimino)methyl)pyridinium-1-yl)propyl)pyridinium dibromide; K048, 4- carbamoyl-1-(3-(4-((hydroxyimino)methyl)pyridinium-1-yl)butyl)pyridinium dibromide; K203, 4- carbamoyl-1-(3-(4-((hydroxyimino)methyl)pyridinium-1-yl)but-2-en-1-yl)pyridinium dibromide; K865, 4-carbamoyl-1-(3-{3-chloro-4-[(hydroxyimino)methyl]pyridinium-1-yl}propyl)pyridinium dibromide; K866, 4-carbamoyl-1-(4-{3-chloro-4-[(hydroxyimino)methyl]pyridinium-1- yl}butyl)pyridinium dibromide; K867, 4-carbamoyl-1-[(2E)-4-{3-chloro-4- [(hydroxyimino)methyl]pyridinium-1-yl}but-2-en-1-yl]pyridinium dibromide; K868, 4-carbamoyl-1- (4-{3,5-dichloro-4-[(hydroxyimino)methyl]pyridinium-1-yl}propyl)pyridinium dibromide; K869, 4- carbamoyl-1-(4-{3,5-dichloro-4-[(hydroxyimino)methyl]pyridinium-1-yl}butyl)pyridinium dibromide; K870, 4-carbamoyl-1-[(2E)-4-{3,5-dichloro-4-[(hydroxyimino)methyl]pyridinium-1- yl}but-2-en-1-yl]pyridinium dibromide; OP, organophosphorus compound; sarin, isopropyl methylphosphonofluoridate; 2-PAM, pralidoxime [2-pyridinealdoxime methiodide]; tabun, N,N- dimethylamido-O-ethyl cyanophosphate; TMB-4, trimedoxime [1,3-bis(4- hydroxyiminomethylpyridinium)propane dichloride]; VX, O-ethyl S-[2- (disopropylamino)ethyl]methylphosphonothioate

2 https://doi.org/10.1016/j.cbi.2019.04.020 Chemico-Biological Interactions Available online 18 April 2019 In Press, Accepted Manuscript 1. Introduction

Nerve agents are organophosphorus compounds (OPs) with very potent toxicity due to their irreversible inhibition of the essential acetylcholinesterase (AChE; EC 3.1.1.7), which is primarily important in the control of neurotransmission in synapses. The related enzyme butyrylcholinesterase (BChE; EC 3.1.1.8) is inhibited by OPs as well. Although its physiological role is not yet fully understood, BChE is located in blood plasma where it participates in the biotransformation of different xenobiotics and is also present in the central and peripheral nervous system where it is probably involved in the regulation of the cholinergic system [1–4]. The main therapy for organophosphorus compound poisoning comprises atropine as an antimuscarinic drug, oxime as a nucleophilic reactivator of inhibited AChE, and benzodiazepine as an anticonvulsive drug. In addition, BChE was investigated as a possible endogenous or exogenous stoichiometric bioscavenger of OPs [5–8]. Currently, it is also being considered as a pseudo-catalytic scavenger of OPs in the bloodstream by which BChE inhibition is paired with an efficient oxime-assisted BChE reactivation, i.e., cycles of OP inhibition of BChE followed by the reactivation with an oxime should help the degradation of OPs [9–11] and consequently, a lower OP concentration is available for AChE inhibition in the synapses. Similar pseudo-catalytic scavenging was demonstrated with a mutant AChE and oxime pair [12–14].

Fig. 1. Standard oximes used in medicinal practice, 2-PAM, and asoxime.

Novel oximes and molecules with nucleophilic potential have been developed for decades with an aim to find a more efficient and universal reactivator of AChE, i.e., a reactivator sufficiently efficient for all OP compounds, and more recently to find a centrally active reactivator. No oxime used for medicinal and military purposes (i.e., standard oxime), such as 2-PAM, obidoxime and asoxime (Fig. 1), is a universal AChE reactivator due to the structural variability of nerve agents and OPs in general and the fact that reactivation depends on a nucleophilic displacement of the OP moiety bound to the catalytic serine of cholinesterase [15]. Moreover, standard oximes are less efficient reactivators of OP-inhibited BChE than they are for OP-inhibited AChE; and still no oxime have shown an exceptional improvement in BChE reactivation [9,12,16,17]. On the other hand, many approaches have been employed to achieve higher blood-brain barrier (BBB) penetration of the reactivators, i.e., different groups synthesised uncharged oximes [10,11,18–23] and molecules [24] or charged pyridinium oximes with substituents that enhance their lipophilicity [25,26]. Most recently, pyridinium oximes with one or two chlorine moiety in ortho-position (Fig. 2) have been synthesized and proven as efficient reactivators of AChE inhibited by different nerve agents [27]. Chlorine atoms enhance lipophilicity, which resulted in an improved ability of chlorinated oximes to cross an artificial barrier modelled after the BBB, and decrease the pKa value of the oxime group that promoted a high reactivation potential of chlorinated oximes [27]. This work was aimed at the evaluation of the OP- inhibited BChE reactivation potential of these novel mono- and dichlorinated pyridinium oximes in case of sarin, cyclosarin, VX and tabun, and therefore, we wanted to evaluate if they could possibly have cumulative antidotal effect through both AChE and BChE reactivation.

3 https://doi.org/10.1016/j.cbi.2019.04.020 Chemico-Biological Interactions Available online 18 April 2019 In Press, Accepted Manuscript

Fig. 2. Pyridinium oximes with ortho-positioned chlorine moiety.

2. Material and methods

2.1. Chemicals

Monochlorinated oximes 4-carbamoyl-1-(3-{3-chloro-4-[(hydroxyimino)methyl]pyridinium-1-yl} propyl)pyridinium dibromide (K865), 4-carbamoyl-1-(4-{3-chloro-4-[(hydroxyimino)methyl] pyridinium-1-yl}butyl)pyridinium dibromide (K866), and 4-carbamoyl-1-[(2E)-4-{3-chloro-4- [(hydroxyimino)methyl]pyridinium-1-yl}but-2-en-1-yl]pyridinium dibromide (K867), and dichlorinated oximes 4-carbamoyl-1-(4-{3,5-dichloro-4-[(hydroxyimino)methyl]pyridinium-1-yl} propyl)pyridinium dibromide (K868), 4-carbamoyl-1-(4-{3,5-dichloro-4-[(hydroxyimino)methyl] pyridinium-1-yl}butyl)pyridinium dibromide (K869), and 4-carbamoyl-1-[(2E)-4-{3,5-dichloro-4- [(hydroxyimino)methyl]pyridinium-1-yl}but-2-en-1-yl]pyridinium dibromide (K870) were synthesized as described before [27]. Oxime stock solutions (10 mM) were prepared in water and stored at 4°C. Oxime 4-carbamoyl-1-(3-(4-((hydroxyimino)methyl)pyridinium-1-yl)propyl)pyridinium dibromide (K027) was synthesized as described before [28]. OP compounds sarin, cyclosarin, VX and tabun were purchased from NC Laboratory (Spiez, Switzerland). OP stock solutions (5000 μg/mL) were made in isopropyl alcohol, and further dilutions were made in water just before use. Acetylthiocholine iodide (ATCh), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Purified human plasma BChE was a generous gift from Dr Florian Nachon, Département de Toxicologie et Risques Chimiques, Institut de Recherche Biomédicale des Armées, Bretigny-sur-Orge, France.

2.2. Reactivation

The reactivation reaction of returned enzyme activity with an oxime was determined using kinetic measurements on a spectrophotometer (CARY 300, Varian Inc., Mulgrave, Australia) and the Ellman method [29] at 25ºC. The reactivation experiment was started with 95−100% inhibition of the enzyme with a 10-fold excess of the nerve agent for 30-60 min. The excess of unconjugated nerve agent was removed using a filtration column MobiSpin G-50 (MoBiTec GmbH, Germany) and the inhibited enzyme was added to the reactivation mixture (oxime in 0.1 M sodium phosphate buffer pH 7.4 with 0.01% BSA) to start the reactivation reaction. At designated time points, the returned enzyme activity was measured by diluting an aliquot of the mixture in phosphate buffer containing DTNB (final 0.3 mM) and after the addition of ATCh (final 1.0 mM). The control uninhibited enzyme was passed through a parallel filtration column, diluted to the same extent as the reactivation mixture, and control activity was measured in the presence of an oxime at the same concentrations used for reactivation. Both the activities of the control and the reactivation mixture were corrected for oximolysis [30]. No significant spontaneous reactivation of the phosphylated enzyme occurred. Screening reactivation was determined with two oxime concentrations (0.1 mM and 1.0 mM) to select the best oximes based on the achieved first-order reactivation rate constant at a given oxime

4 https://doi.org/10.1016/j.cbi.2019.04.020 Chemico-Biological Interactions Available online 18 April 2019 In Press, Accepted Manuscript concentration (kobs), maximal percentage of reactivation (Reactmax) and time needed to achieve it (t). In addition, by determining kobs at a wider oxime concentration range (up to 1 mM) reactivation parameters were also determined, i.e., maximal first-order reactivation rate constant (k+2), overall second-order reactivation rate constant (kr), and phosphylated enzyme-oxime dissociation constant

(KOX), which were determined as described previously [15,30].

3. Results

The screening reactivation of BChE inhibited by nerve agents at 1.0 mM oxime concentration (Fig. 3) showed that the sarin-BChE conjugate was reactivated most efficiently. Moreover, the reactivation rates with 0.1 mM oximes for the sarin-BChE conjugate were comparable to the ones with 1.0 mM oximes in reactivation of cyclosarin- and VX-BChE. The tabun-BChE conjugate was, expectedly, reactivated at the slowest rate because of the hindrance imposed by steric and electronic properties of the phosphoramidate moiety [31,32]. In most cases, dichlorinated pyridinium oximes (K868, K869, K870) were more efficient reactivators than monochlorinated pyridinium oximes (K865, K866, K867). As oxime K868 was recently highlighted as the lead oxime for human AChE reactivation [27], we determined its reactivation rates in wide concentration range and for reactivation of all OP-BChE conjugates. In addition, K869 was also examined in a wider concentration range for sarin-BChE reactivation. Therefore, detailed reactivation experiments enabled determination of the dissociation constant of OP-BChE conjugate complex with oxime (KOX), maximal first-order reactivation rate (k+2) and overall second-order reactivation rate (kr). For sarin-BChE reactivation, K868 and K869 had a comparable reactivation potency (Table 1 and Fig. 4). Moreover, both oximes had higher reactivation rates than standard oximes 2-PAM and HI-6 and achieved complete BChE reactivation within 10-20 minutes. The reactivation parameters of K868 were similar for cyclosarin and VX and achieved maximal reactivation within 1-1.5 hour. In the cyclosarin-BChE reactivation, K868 showed better reactivation potency than 2-PAM but not better than HI-6. In VX-BChE reactivation, K868 had an overall reactivation rate between those of HI-6 and 2-PAM. K868 was effective in the reactivation of tabun-BChE and more potent than 2-PAM or HI-6. The highest concentrations of K868 achieved 70% of enzyme reactivation within two hours. It is interesting to note that, even though it was extremely potent for reactivation of AChE inhibited by most nerve agents, K868 was completely ineffective for tabun-AChE [27]. A plausible explanation might lie in the more spacious BChE active site [33] that allows the productive conformation of reactivating 3,5-dichloropyridinium-oximate moiety.

5 https://doi.org/10.1016/j.cbi.2019.04.020 Chemico-Biological Interactions Available online 18 April 2019 In Press, Accepted Manuscript Fig. 3. Screening reactivation of BChE inhibited by nerve agents with 0.1 mM and 1 mM oximes: first-order reactivation rates, kobs, on the left y-axis (colored columns) and maximal reactivation percentage, Reactmax, on the right y-axis (grey columns).

Table 1 Detailed parameters for reactivation of BChE inhibited by nerve agents with oximes.

-1 -1 -1 OP Oxime k+2 (min ) KOX (μM) kr (M min ) Reactmax (%) t (min) Sarin K868 a / / 360 ± 20 100 10 K869 0.38 ± 0.08 480 ± 250 790 ± 250 100 15 2-PAM 0.18 ± 0.05 2400 ± 995 75 ± 10 100 120 HI-6 b 0.04 ± 0.02 270 ± 200 150 ± 40 100 300 Cyclosarin K868 0.08 ± 0.01 320 ± 130 240 ± 70 90 80 2-PAM 0.08 ± 0.01 1200 ± 290 65 ± 10 80 60 HI-6 b / / 780 ± 30 90 30 VX K868 0.07 ± 0.01 490 ± 200 150 ± 40 80 80 2-PAM 0.09 ± 0.03 2680 ± 1110 35 ± 5 70 120 HI-6 c 0.12 ± 0.03 370 ± 230 330 ± 230 85 120 Tabun K868 0.028 ± 0.005 480 ± 230 60 ± 20 70 150 2-PAM d 0.0011 ± 0.0002 1270 ± 530 0.9 ± 0.4 20-40 12 h HI-6 e 0.0016 ± 0.0004 1800 ± 1000 0.9 ± 0.6 25 18 h a b c Linear dependence of kobs and oxime concentration (0.05−1.0 mM K868); From ref. [23]; From ref. [16]; d From ref. [34]; e From ref. [35].

Fig. 4. Dependence of reactivation rates (kobs) on oxime K868 or K869 concentration for reactivation of sarin-inhibited BChE.

In addition, we wanted to compare the reactivation of sarin-BChE between analogous oximes that differed in the number of chlorine atoms (zero, one or two). Fig. 5 shows the reactivation in time for 0.5 mM oximes K027, K865, and K868. The reactivation rate seems to increase with the number of

6 https://doi.org/10.1016/j.cbi.2019.04.020 Chemico-Biological Interactions Available online 18 April 2019 In Press, Accepted Manuscript chlorine atoms, which could be the result of decreased pKa and thus increased oximate formation [36], together with the stabilization in productive conformation through interactions between chlorine atoms and certain residues in the enzyme’s active site. A similar conclusion about stabilization was demonstrated for this oxime and cyclosarin-AChE using molecular docking and reactivation studies [27]. In addition, oxime K027 and K865 displayed a biphasic curve as shown previously for several combinations of inhibited AChE and oximes [27].

Fig. 5. Reactivation of BChE inhibited by sarin with 0.5 mM oxime K027, K865, and K868.

4. Discussion

In vivo interactions of oximes with endogenous or exogenous bioscavenger BChE can serve to additionally improve applied oxime therapy [35]. This study demonstrated that the previously reported lead chlorinated oxime K868 for the reactivation of human AChE inhibited by sarin, cyclosarin, and VX was also an efficient reactivator of human BChE inhibited by the same nerve agents [27]. For tabun, K868 was surprisingly effective considering its total inertness in tabun-AChE reactivation. Moreover, K868 had a 10-times higher maximal reactivation rate and better reactivation potency for -1 tabun-BChE than its non-chlorinated analogue oxime K027 (k+2= 0.0035 ± 0.0006 min , KOX=1600 ± -1 -1 700 µM, kr=2.2 ± 1.0 M min ) or other oximes such as trimedoxime (TMB-4) or K-oximes K048, K033 [35], K203 [37], K074, K075, and K114 [38]. The highest reactivation potency was observed for sarin-BChE conjugate with two dichlorinated pyridinium oximes, K868 and K869. A comparison of three analogous oximes that differ in the number of chlorine atoms attached to the pyridinium ring in the reactivation of sarin-BChE once again demonstrated the role of chlorine atoms in the stabilization of productive conformation. The affinity of BChE for these six chlorinated oximes was reported to be 7-12 times lower than the affinity of AChE (i.e., BChE-oxime dissociation constants range from 140 to 290 µM) [27]. A comparison of affinities of native and phosphylated AChE and BChE conjugates for K868 (Fig. 6) suggests that K868 was the most promising in therapy as an AChE reactivator for sarin, cyclosarin, and VX exposure due to their KOX values below 160 µM [27]. In addition, the high affinity of native AChE for K868 suggested that the prophylactic application of K868 could protect the AChE active site from OP binding to the catalytic serine [24,39] and this should be evaluated in greater detail in the future. Furthermore, the observed promising broad-spectrum potency of K868 in sarin, cyclosarin, VX and tabun-BChE reactivation could possibly enable their pseudo-catalytic degradation in blood achieving a certain level of protection of AChE from OP inhibition as we have already demonstrated for sarin, cyclosarin, and VX ex vivo [27]. In addition, there are simultaneous reactivation effects of K868 for sarin-, cyclosarin- and VX-AChE and -BChE conjugates that could solve post-intoxication

7 https://doi.org/10.1016/j.cbi.2019.04.020 Chemico-Biological Interactions Available online 18 April 2019 In Press, Accepted Manuscript demands [27]. This could make an important difference in the onset of toxic symptoms and widen the time window for the application of additional therapy.

Fig. 6. A comparison of affinities of native (1/Ki) and phosphylated (1/KOX) AChE and BChE for oxime K868. Data from Table 1 (KOX for sarin-BChE was estimated higher than 1mM) and ref. [27].

5. Conclusion

In addition to previous study that revealed ortho-dichlorinated oxime K868 as very potent reactivator of human AChE, this study on reactivation of human BChE inhibited by different nerve agents confirmed that K868 is a promising novel antidote for nerve agent poisoning that should be investigated further, both as prophylactic and post-exposure treatment in vivo.

Acknowledgement We are grateful to Dr Florian Nachon for the human purified BChE, and Makso Herman for language editing. This work was supported by the Croatian Science Foundation (no. IP-11-2018-01-7683) and Czech Science Foundation (no. 18-01734S).

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