In Vitro Evaluation of Oxidative Stress Induced by Oxime Reactivators of Acetylcholinesterase in HepG2 Cells.

Oxime reactivators of acetylcholinesterase (AChE) are used as causal antidotes for intended and unintended poisoning by organophosphate nerve agents and pesticides. Despite all efforts to develop new AChE reactivators, none of these drug candidates replaced conventional clinically used oximes. In addition to the therapeutic efficacy, determining the safety profile is crucial in preclinical drug evaluation. The exact mechanism of oxime toxicity and the structure-toxicity relationship are subjects of ongoing research, with oxidative stress proposed as a possible mechanism. In the present study, we investigated four promising bispyridinium oxime AChE reactivators, K048, K074, K075, and K203, and their ability to induce oxidative stress in vitro. Cultured human hepatoma cells were exposed to oximes at concentrations corresponding to their IC50 values determined by the MTT assay after 24 h. Their potency to generate reactive oxygen species, interfere with the thiol antioxidant system, and induce lipid peroxidation was evaluated at 1, 4, and 24 h of exposure. Reactivators without a double bond in the four-carbon linker, K048 and K074, showed a greater potential to induce oxidative stress compared with K075 and K203, which contain a double bond. Unlike oximes with a three-carbon-long linker, the number of aldoxime groups attached to the pyridinium moieties does not determine the oxidative stress induction for K048, K074, K075, and K203 oximes. In conclusion, our results emphasize that the structure of oximes plays a critical role in inducing oxidative stress, and this relationship does not correlate with their cytotoxicity expressed as the IC50 value. However, it is important to note that oxidative stress cannot be disregarded as a potential contributor to the side effects associated with oximes.

Potential of Vitamin B6 Dioxime Analogues to Act as Cholinesterase Ligands.

Seven pyridoxal dioxime quaternary salts (1-7) were synthesized with the aim of studying their interactions with human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The synthesis was achieved by the quaternization of pyridoxal monooxime with substituted 2-bromoacetophenone oximes (phenacyl bromide oximes). All compounds, prepared in good yields (43-76%) and characterized by 1D and 2D NMR spectroscopy, were evaluated as reversible inhibitors of cholinesterase and/or reactivators of enzymes inhibited by toxic organophosphorus compounds. Their potency was compared with that of their monooxime analogues and medically approved oxime HI-6. The obtained pyridoxal dioximes were relatively weak inhibitors for both enzymes (Ki = 100-400 µM). The second oxime group in the structure did not improve the binding compared to the monooxime analogues. The same was observed for reactivation of VX-, tabun-, and paraoxon-inhibited AChE and BChE, where no significant efficiency burst was noted. In silico analysis and molecular docking studies connected the kinetic data to the structural features of the tested compound, showing that the low binding affinity and reactivation efficacy may be a consequence of a bulk structure hindering important reactive groups. The tested dioximes were non-toxic to human neuroblastoma cells (SH-SY5Y) and human embryonal kidney cells (HEK293).

Molecular modeling-guided optimization of acetylcholinesterase reactivators: A proof for reactivation of covalently inhibited targets.

Covalent drugs have been intensively studied in some very important fields such as anti-tumor and anti-virus, including the currently global-spread SARS-CoV-2. However, these drugs may interact with a variety of biological macromolecules and cause serious toxicology, so how to reactivate the inhibited targets seems to be imperative in the near future. Organophosphate was an extreme example, which could form a covalent bound easily with acetylcholinesterase and irreversibly inhibited the enzyme, causing high toxicology. Some nucleophilic oxime reactivators for organophosphate poisoned acetylcholinesterase had been developed, but the reactivation process was still less understanding. Herein, we proposed there should be a pre-reactivated pose during the reactivating process and compounds whose binding pose was easy to transfer to the pre-reactivated pose might be efficient reactivators. Then we refined the previous reactivators based on the molecular dynamic simulation results, the resulting compounds L7R3 and L7R5 were proven as much more efficient reactivators for organophosphate inhibited acetylcholinesterase than currently used oximes. This work might provide some insights for constructing reactivators of covalently inhibited targets by using computational methods.

Glycosylated-imidazole aldoximes as reactivators of pesticides inhibited AChE: Synthesis and in-vitro reactivation study.

The present armamentarium of commercially available antidotes provides limited protection against the neurological effects of organophosphate exposure. Hence, there is an urgent need to design and develop molecules that can protect and reactivate inhibited-AChE in the central nervous system. Some natural compounds like glucose and certain amino acids (glutamate, the anion of glutamic acid) can easily cross the blood brain barrier although they are highly polar. Glucose is mainly transported by systems like glucose transporter protein type 1 (GLUT1). For this reason, a series of non-quaternary and quaternary glycosylated imidazolium oximes with different alkane linkers have been designed and synthesized. These compounds were evaluated for their in-vitro reactivation ability against pesticide (paraoxon-ethyl and paraoxon-methyl) inhibited-AChE and compared with standards antidote AChE reactivators pralidoxime and obidoxime. Several physicochemical properties including acid dissociation constant (pKa), logP, logD, HBD and HBA, have also been assessed for reported compounds. Out of the synthesized compounds, three have exhibited comparable potency with a standard antidote (pralidoxime).

Cytotoxicity of acetylcholinesterase reactivators evaluated in vitro and its relation to their structure.

The development of acetylcholinesterase reactivators, i.e., antidotes against organophosphorus poisoning, is an important goal of defense research. The aim of this study was to compare cytotoxicity and chemical structure of five currently available oximes (pralidoxime, trimedoxime, obidoxime, methoxime, and asoxime) together with four perspective oximes from K-series (K027, K074, K075, and K203). The cytotoxicity of tested substances was measured using two methods - colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and impedance based real-time cytotoxicity assay - in three different cell lines (HepG2, ACHN, and NHLF). Toxicity was subsequently expressed as toxicological index IC50. The tested compounds showed different cytotoxicity ranging from 0.92 to 40.06 mM. In HepG2 cells, K027 was the least and asoxime was the most toxic reactivator. In ACHN and NHLF cell lines, trimedoxime was the compound with the lowest adverse effects, whereas the highest toxicity was found in methoxime-treated cells. The results show that at least five structural features affect the reactivators' toxicity such as the number of oxime groups in the molecule, their position on pyridinium ring, the length of carbon linker, and the oxygen substitution or insertion of the double bond into the connection chain. Newly synthetized oximes with IC50 ≥ 1 mM evaluated in this three cell lines model might appear suitable for further testing.

Pyridinium Oximes with Ortho-Positioned Chlorine Moiety Exhibit Improved Physicochemical Properties and Efficient Reactivation of Human Acetylcholinesterase Inhibited by Several Nerve Agents.

Six chlorinated bispyridinium mono-oximes, analogous to potent charged reactivators K027, K048, and K203, were synthesized with the aim of improving lipophilicity and reducing the p Ka value of the oxime group, thus resulting in a higher oximate concentration at pH 7.4 compared to nonchlorinated analogues. The nucleophilicity was examined and the p Ka was found to be lower than that of analogous nonchlorinated oximes. All the new compounds efficiently reactivated human AChE inhibited by nerve agents cyclosarin, sarin, and VX. The most potent was the dichlorinated analogue of oxime K027 with significantly improved ability to reactivate the conjugated enzyme due to improved binding affinity and molecular recognition. Its overall reactivation of sarin-, VX-, and cyclosarin-inhibited AChE was, respectively, 3-, 7-, and 8-fold higher than by K027. Its universality, PAMPA permeability, favorable acid dissociation constant coupled with its negligible cytotoxic effect, and successful ex vivo scavenging of nerve agents in whole human blood warrant further analysis of this compound as an antidote for organophosphorus poisoning.

SAR study to find optimal cholinesterase reactivator against organophosphorous nerve agents and pesticides.

Irreversible inhibition of acetylcholinesterase (AChE) by organophosphates leads to many failures in living organism and ultimately in death. Organophosphorus compounds developed as nerve agents such as tabun, sarin, soman, VX and others belong to the most toxic chemical warfare agents and are one of the biggest threats to the modern civilization. Moreover, misuse of nerve agents together with organophosphorus pesticides (e.g. malathion, paraoxon, chlorpyrifos, etc.) which are annually implicated in millions of intoxications and hundreds of thousand deaths reminds us of insufficient protection against these compounds. Basic treatments for these intoxications are based on immediate administration of atropine and acetylcholinesterase reactivators which are currently represented by mono- or bis-pyridinium aldoximes. However, these antidotes are not sufficient to ensure 100 % treatment efficacy even they are administered immediately after intoxication, and in general, they possess several drawbacks. Herein, we have reviewed new efforts leading to the development of novel reactivators and proposition of new promising strategies to design novel and effective antidotes. Structure-activity relationships and biological activities of recently proposed acetylcholinesterase reactivators are discussed and summarized. Among further modifications of known oximes, the main attention has been paid to dual binding site ligands of AChE as the current mainstream strategy. We have also discussed new chemical entities as potential replacement of oxime functional group.

Evaluation and computational characterization of the facilitated transport of Glc carbon C-1 oxime reactivators across a blood brain barrier model.

We are evaluating a facilitative transport strategy to move oximes across the blood brain barrier (BBB) to reactivate inhibited brain acetylcholinesterase (AChE). We selected glucose (Glc) transporters (GLUT) for this purpose as these transporters are highly represented in the BBB. Glc conjugates have successfully moved drugs across the BBB and previous work has shown that Glc-oximes (sugar-oximes, SOxs) can reduce the organophosphonate induced hypothermia response. We previously evaluated the reactivation potential of Glc carbon C-1 SOxs. Here we report the reactivation parameters for VX- and GB-inhibited human (Hu) AChE of the best SOx (13c) and our findings that the kinetics are similar to those of the parent oxime. Although crystals of Torpedo californica AChE were produced, neither soaked or co-crystallized experiments were successful at concentrations below 20mM 13c, and higher concentrations cracked the crystals. 13c was non-toxic to neuroblastoma and kidney cell lines at 12-18 mM, allowing high concentrations to be used in a BBB kidney cell model. The transfer of 13c from the donor side was asymmetric with the greatest loss of 13c from the apical- or luminal-treated side. There was no apparent transfer from the basolateral side. The 13cP(app) results indicate a 'low' transport efficiency; however, mass accounting revealed only a 20% recovery from the apical dose in which high concentrations were found in the cell lysate fraction. Molecular modeling of 13c through the GLUT-1 channel demonstrated that transport of 13c was more restricted than Glc. Selected sites were compared and the 13c binding energies were greater than two times those of Glc.

Photostability of antidotal oxime HI-6, impact on drug development.

HI-6 exhibits superior efficacy in the therapy of intoxication by different highly toxic organophosphorus nerve agents. Therefore HI-6 is a promising candidate for the development of new antidotes against nerve agents. For ethical and safety reasons antidotes containing HI-6 should get marketing authorization. Active pharmaceutical ingredients of medicinal products have to fulfil regulatory conditions in terms of purity and stability. Photostability is an essential parameter in this testing strategy. HI-6 was tested under conditions of ICH Q1B 'Photostability testing of new drug substances and products'. The data showed a marked degradation of HI-6 after exposure to daylight. The mechanism of degradation could be detected as photoisomerism. The light burden dependent rate of photoisomerism was followed quantitatively. Based on these quantitative results on the amount of light induced isomeric product a pharmacological qualification was made. A standardized in vitro test showed a decreased ability of light exposed HI-6 to reactivate sarin- and paraoxon-inhibited human acetylcholinesterase. These results have an impact on the further development of antidotes containing HI-6, as light protection will probably be necessary during handling, packaging, storage and application.

Fullerene antioxidants decrease organophosphate-induced acetylcholinesterase inhibition in vitro.

Although organophosphate (OP)-induced acetylcholinesterase (AChE) inhibition is the critical mechanism causing toxicities that follow exposure, other biochemical events, including oxidative stress, have been reported to contribute to OP toxicity. Fullerenes are carbon spheres with antioxidant activity. Thus, we hypothesized that fullerenes could counteract the effects of OP compounds and tested this hypothesis using two in vitro test systems, hen brain and human neuroblastoma SH-SY5Y cells. Cells were incubated with eight different derivatized fullerene compounds before challenge with paraoxon (0=control, 5×10(-8), 10(-7), 2×10(-7) or 5×10(-7) M) or diisopropylphosphorofluoridate (DFP, 0=control, 5×10(-6), 10(-5), 2×10(-5), and 5×10(-5) M) and measurement of AChE activities. Activities of brain and SH-SY5Y AChE with OP compounds alone ranged from 55-83% lower than non-treated controls after paraoxon and from 60-92% lower than non-treated controls after DFP. Most incubations containing 1 and 10 µM fullerene derivatives brought AChE activity closer to untreated controls, with improvements in AChE activity often >20%. Using dissipation of superoxide anion radicals as an indicator (xanthine oxidation as a positive control), all fullerene derivatives demonstrated significant antioxidant capability in neuroblastoma cells at 1 µM concentrations. No fullerene derivative at 1 µM significantly affected neuroblastoma cell viability, when determined using either Alamar Blue dye retention or a luminescent assay for ATP production. These studies suggest that derivatized fullerene nanomaterials have potential capability to ameliorate OP-induced AChE inhibition resulting in toxicities.

Design, evaluation and structure-activity relationship studies of the AChE reactivators against organophosphorus pesticides.

Organophosphate pesticides (OPPs; e.g. chlorpyrifos, diazinon, paraoxon) are a wide and heterogeneous group of organophosphorus compounds. Their biological activity of inhibiting acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) ranks them as life endangering agents. The necessary treatment after OPP exposure involves the use of parasympatolytics (e.g. atropine), oxime reactivators (e.g. obidoxime), and anticonvulsive drugs (e.g. diazepam). Therefore, the reactivators of AChE are essential compounds in the treatment of OPP intoxications. Commercial AChE reactivators (e.g. pralidoxime, HI-6, obidoxime, trimedoxime, methoxime) were originally developed for other members of the organophosphate family, such as nerve agents (e.g. sarin, soman, tabun, VX). Pralidoxime, HI-6, and methoxime were found to be weak reactivators of OPP-inhibited AChE. Obidoxime and trimedoxime showed satisfactory reactivation against various OPPs with minor toxicity issues. During the last two decades, the treatment of OPP exposure has become more widely discussed because of growing agricultural production, industrialization, and harmful social issues (e.g. suicides). In this review is the summarized design, evaluation, and structure-activity relationship studies of recently produced AChE reactivators. Since pralidoxime, over 300 oximes have been produced or tested against OPP poisoning, and several novel compounds show very promising abilities as comparable (or higher) to commercial oximes. Some of these are highlighted for their further testing of OPP exposure and, additionally, the main structure-activity relationship of AChE reactivators against OPP is discussed.

Effects of oximes on mitochondrial oxidase activity.

Oximes, including 2-pyridinealdoxime methiodide (2-PAM), are reactivators of acetylcholinesterase (AChE) inhibited by organophosphate poisoning. Unfortunately, their clinical use has been limited by their toxicity. To investigate the mechanism of this toxicity, the effects of oximes on the enzymes choline oxidase (ChOD) and cytochrome c oxidase (CyCOD) of the respiratory chain in mitochondria were examined. The oximes 2-PAM, obidoxime, and diacetylmonoxime significantly (P<0.01) inhibited ChOD activity, and the extent of inhibition correlated with the ability to reactivate inhibited AChE. When ChOD activity in mitochondrial extracts was tested, 2-PAM inhibited the activity by 75%, obidoxime and diacetylmonoxime did not significantly inhibit it, and 4-[(hydroxy-imino)methyl]-1-decylpyridinium bromide (4-PAD), which has greater toxicity, increased the amount of product generated in the assay to approximately 200% of normal levels. Similarly, 2-PAM inhibited the activity of CyCOD in mitochondrial extracts whereas obidoxime and diacetylmonoxime did not. One explanation for these findings is that, in addition to their inhibition of mitochondrial oxidases, the oximes may produce excessive reactive oxygen species such as H(2)O(2) in the mitochondrial fraction, which may account for some of their toxicity. This is a preliminary report related to the toxicities of oximes that may participate in the inactivation of mitochondrial oxidase enzymes. This hypothesis should be further investigated by in vivo study, including kinetic determination and free radical work.

In vitro reactivation of sarin inhibited electric eel acetylcholinesterase by bis-pyridinium oximes bearing methoxy ether linkages.

Bis-pyridinium oximes connected by methoxy alkane ether linker were synthesized and their in vitro reactivation efficacy was evaluated for sarin inhibited AChE. Reactivation efficacy of synthesized compounds was compared with 2-PAM and obidoxime. Among the synthesized compounds, 1,2-dimethoxy ethylene bis-[3,3'-(hydroxyiminomethyl) pyridinium] dichloride (3P-2) and 1,3-dimethoxy propylene bis-[3,3'-(hydroxyiminomethyl) pyridinium] dichloride (3P-3) were found to be most potent reactivators for AChE inhibited by nerve agent sarin. 3P-2 and 3P-3, respectively exhibited 80% and 69% regeneration of inhibited AChE, whereas 2-PAM (well known antidote for nerve agent poisoning) showed 42% regeneration.

Structural requirements of acetylcholinesterase reactivators.

Nerve agents (sarin, soman, cyclosarin, tabun and VX agent) and pesticides (paraoxon, chlorpyrifos, TEPP) represent extremely toxic group of organophosphorus compounds (OPCs). These compounds inhibit enzyme acetylcholinesterase (AChE, EC 3.1.1.7) via its phosphorylation or phosphonylation at the serine hydroxy group in its active site. Afterwards, AChE is not able to serve its physiological function and intoxicated organism is died due to overstimulation of cholinergic nervous system. The current standard treatment of poisoning with highly toxic OPCs usually consists of the combined administration of anticholinergic drugs (preferably atropine) and AChE reactivators (called "oximes"). Anticholinergic drugs block effects of accumulated neurotransmitter acetylcholine at nicotinic and muscarinic receptor sites, while oximes reactivate AChE inhibited by OPCs. Unfortunately, none from the currently used oximes is sufficiently effective against all known nerve agents and pesticides. Therefore, to find new oximes able to sufficiently reactivate inhibited AChE (regardless of the type of OPCs) is still very important task for medicinal chemistry with the aim to improve the efficacy of antidotal treatment of the acute poisonings mentioned. In this paper, the relationship between chemical structure of AChE reactivators and their ability to reactivate AChE inhibited by several nerve agents and pesticides is summarized. It is shown that there are several structural fragments possibly involving in the structure of proposed AChE reactivators. Finally, an attempt of a future course of new AChE reactivators development is discussed.

Effect of some acetylcholinesterase reactivators on human platelet aggregation in vitro.

Acetylcholinesterase (AChE) reactivators are employed for the prophylaxis and treatment of intoxications with organophosphorus AChE inhibitors, including nerve agents and pesticides. For the recovery of inhibited enzyme, derivatives from the group of pyridinium or bispyridinium aldoximes (called oximes) are used. Adverse effects of these substances are not well elucidated, because of their narrow and one-shot usage. Owing to this fact, the study evaluated the influence of some currently applied oximes on human platelet aggregation in vitro. The antiplatelet activity of pralidoxime, obidoxime, HI-6, methoxime and HLö 7 was assayed in human platelet rich plasma (2.5 x 10(8) platelets.ml(-1)) at a concentration of 1.35 mM. Arachidonic acid (AA), adenosine diphosphate (ADP), collagen (COL) and thrombin (TR) were used as agonists of platelet aggregation. All tested substances, except pralidoxime and methoxime, caused a significant inhibition of the aggregation process induced by AA, ADP and COL. Of the oximes assayed, none was found to influence TR triggered aggregation. Since reduced platelet aggregation can play an important role as an adverse effect in reactivator administration, further evaluation is needed for the estimation of the real impact of active oximes to the aggregation process in humans.

HLö 7 dimethanesulfonate, a potent bispyridinium-dioxime against anticholinesterases.

HLö 7 dimethanesulfonate (1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2,4-bis [(hydroxyimino)methyl]pyridinium dimethanesulfonate) is a broad-spectrum reactivator against highly toxic organophosphorus compounds. The compound was synthesized by a new route with the carcinogenic bis(chloromethyl)ether being substituted by the non-mutagenic bis(methylsulfonoxymethyl)ether. The very soluble dimethanesulfonate of obidoxime was also prepared by this way. HLö 7 dimethanesulfonate is the first water-soluble salt of HLö 7 that should be suitable for the wet/dry autoinjector technology, because aqueous solutions of HLö 7 are not very stable (calculated shelf-life 0.2 years when stored at 8 degrees C, 1 M solution, pH 2.5). The crystalline preparation contains 96% of the syn/syn-isomer, less than 2% of the syn/anti-isomer and some minor identified by-products. HLö 7 was very efficient in reactivating acetylcholinesterase (AChE) blocked by organophosphates as long as ageing did not prevent dephosphylation. HLö 7 was superior to HI 6 (1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2- [(hydroxyimino)methyl]pyridinium dichloride) in reactivating soman and sarin-inhibited AChE from erythrocytes, and literature data indicate that HLö 7 exceeds HI 6 by far in reactivating tabun-inhibited AChE. In atropine-protected, soman-poisoned mice HLö 7 was three times more potent than HI 6 (protective ratio 5 versus 2.5), and in sarin-poisoned mice HLö 7 was 10 times more potent than HI 6 (protective ratio 8 for both oximes). In atropine-protected guinea-pigs HLö 7 was less effective than HI 6 (protective ratio: 2.3 versus 5.2 for soman; 5.2 versus 6.8 for sarin; 4.3 versus 3.8 for tabun). The mean survival time of anaesthetized guinea-pigs exposed to 5 LD50 soman (6.3 min) was increased by atropine (27 min) and atropine + HLö (57 min). HLö 7 alone did not prolong the survival. The most impressive effect of HLö 7 was on respiration: 3 min after i.v. injection of HLö 7 and atropine, the depressed respiration increased rapidly to 60% of control and remained at that level during the observation period (60 min). With atropine alone, respiration recovered only slowly. Behavioural and physiologic parameters were determined in atropine-protected mice exposed to a sublethal soman dose. The running performance was significantly improved by HLö 7. Even central symptoms, e.g. hypothermia and convulsions, were decreased markedly by HLö 7 (evaluation 60 min after poisoning). The pharmacokinetic data for HLö 7 in male beagle dogs are similar to those of HI 6.(ABSTRACT TRUNCATED AT 400 WORDS)