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.

• MeSH
• acetylcholinesterasa metabolismus   MeSH
• antidota farmakologie   MeSH
• buňky Hep G2 MeSH
• cholinesterasové inhibitory toxicita   MeSH
• lidé MeSH
• organofosfáty toxicita   MeSH
• oxidační stres MeSH
• oximy farmakologie   chemie   MeSH
• pyridinové sloučeniny farmakologie   chemie   MeSH
• reaktivátory cholinesterázy * farmakologie   chemie   MeSH
• uhlík MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The problem of the efficient treatment of acute organophosphorus (OP) poisoning needs more efforts in the development of a versatile antidote, applicable for treatment of the injuries of both peripheral and central nervous systems. A series of N-H, N-methyl, N-butyl, and N-phenyl derivatives of benzhydroxamic (1a-1d), 3-methoxybenzhydroxamic (2a-2d), 4-methoxybenzhydroxamic (3a-3d) acids, and corresponding salycilhydroxamates (4a-4d) was prepared. Their predicted hydrophobicity (log P) was evaluated as regards to ВВВ score by the open access cheminformatics tools; prediction of the passive transport across the BBB was found by means on the parallel artificial membrane permeability assay (PAMPA). The data on reactivation capacity of human acetylcholinesterase (HssAChE) inhibited by GB, VX, and paraoxon was supported by molecular docking study on binding to the active site of the AChE, viability study against mammalian cells (Chinese hamster ovary CHO-K1), and biodegradability (Closed Bottle test OECD 301D). Among the studied compounds, N-butyl derivatives have better balanced combination of properties; among them, N-butylsalicylhydroxamic acid is most promising. The studied compounds demonstrate modest reactivation capacity; change of N-H by N-Me ensures the reactivation capacity in studied concentrations on all studied OP substrates; among N-butyl derivatives, the N-butylsalicylhydroxamic acid demonstrates most promising results within the series. The found regularities may lead to selection of perspective structures to complement current formulations for medical countermeasures against poisoning by organophosphorus toxicants.

• MeSH
• acetylcholinesterasa metabolismus   MeSH
• antidota farmakologie   MeSH
• CHO buňky MeSH
• cholinesterasové inhibitory chemie   farmakologie   MeSH
• Cricetulus MeSH
• křečci praví MeSH
• lidé MeSH
• otrava organofosfáty * MeSH
• oximy chemie   MeSH
• reaktivátory cholinesterázy * chemie   farmakologie   MeSH
• simulace molekulového dockingu MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The fluorinated bis-pyridinium oximes were designed and synthesized with the aim of increasing their nucleophilicity and potential to reactivate phosphorylated human recombinant acetylcholinesterase (AChE) and human purified plasmatic butyrylcholinesterase (BChE) in relation to chlorinated and non-halogenated oxime analogues. Compared to non-halogenated oximes, halogenated oximes showed lower pKa of the oxime group (fluorinated < chlorinated < non-halogenated) along with higher level of oximate anion formation at the physiological pH, and had a higher binding affinity of both AChE and BChE. The stability tests showed that the fluorinated oximes were stable in water, while in buffered environment di-fluorinated oximes were prone to rapid degradation, which was reflected in their lower reactivation ability. Mono-fluorinated oximes showed comparable reactivation to non-halogenated (except asoxime) and mono-chlorinated oximes in case of AChE inhibited by sarin, cyclosarin, VX, and tabun, but were less efficient than di-chlorinated ones. The same trend was observed in the reactivation of inhibited BChE. The advantage of halogen substituents in the stabilization of oxime in a position optimal for in-line nucleophilic attack were confirmed by extensive molecular modelling of pre-reactivation complexes between the analogue oximes and phosphorylated AChE and BChE. Halogen substitution was shown to provide oximes with additional beneficial properties, e.g., fluorinated oximes gained antioxidative capacity, and moreover, halogens themselves did not increase cytotoxicity of oximes. Finally, the in vivo administration of highly efficient reactivator and the most promising analogue, 3,5-di-chloro-bispyridinium oxime with trimethylene linker, provided significant protection of mice exposed to sarin and cyclosarin.

• MeSH
• acetylcholinesterasa metabolismus   MeSH
• butyrylcholinesterasa metabolismus   MeSH
• cholinesterasové inhibitory chemie   MeSH
• halogeny MeSH
• myši MeSH
• nervová bojová látka * farmakologie   MeSH
• organofosforové sloučeniny MeSH
• oximy chemie   MeSH
• reaktivátory cholinesterázy * chemie   MeSH
• sarin chemie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The nerve agents of the A-series are relatively recent chemical weapons with no antidote available yet. Once inside the human body, those chemicals act similarly to the classic nerve agents, by binding to the catalytic residue Serine 203 (Ser203) of human acetylcholinesterase (HssAChE) and thus preventing the proper function of this enzyme. However, there is no experimental evidence yet if the current antidotes for intoxication by nerve agents are also capable of restoring AChE inhibited by the nerve agents of the A-series. In order to launch some light on this issue, we used computational techniques (molecular docking, molecular dynamics and MM-PBSA interaction energy calculations) to assess the performances of the four currently available commercial oximes (2-PAM, HI-6, obidoxime and trimedoxime) when in contact with HssAChE inhibited by the agent A-242. Based on the near-attack conformation (NAC) criterion, our results suggest that the commercial oximes would have limited efficacy to reactivate the enzyme since they are not able to properly approach the adduct Ser203-A-242. Among those oximes, trimedoxime seems to be the most promising, since it showed lower values of energy in the MM-PBSA calculations, a higher stability inside the catalytic anionic center (CAS) of HssAChE, and was able to adopt a position closer to the NAC that could enable the reactivation mechanism.

• MeSH
• acetylcholinesterasa metabolismus   MeSH
• antidota farmakologie   MeSH
• cholinesterasové inhibitory chemie   toxicita   MeSH
• lidé MeSH
• nervová bojová látka * toxicita   MeSH
• organofosfáty MeSH
• oximy chemie   farmakologie   MeSH
• pyridinové sloučeniny farmakologie   MeSH
• reaktivátory cholinesterázy * farmakologie   MeSH
• simulace molekulového dockingu MeSH
• trimedoxim farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The organophosphorus antidotes, so-called oximes, are able to restore the enzymatic function of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) via cleavage of organophosphate from the active site of the phosphylated enzyme. In this work, the charged pyridinium oximes containing thiocarboxamide moiety were designed, prepared and tested. Their stability and pKa properties were found to be analogous to parent carboxamides (K027, K048 and K203). The inhibitory ability of thiocarboxamides was found in low μM levels for AChE and high μM levels for BChE. Their reactivation properties were screened on human recombinant AChE and BChE inhibited by nerve agent surrogates and paraoxon. One thiocarboxamide was able to effectively restore function of NEMP- and NEDPA-AChE, whereas two thiocarboxamides were able to reactivate BChE inhibited by all tested organophosphates. These results were confirmed by reactivation kinetics, where thiocarboxamides were proved to be effective, but less potent reactivators if compared to carboxamides.

• MeSH
• acetylcholinesterasa metabolismus   MeSH
• butyrylcholinesterasa metabolismus   MeSH
• cholinesterasové inhibitory chemická syntéza   chemie   farmakologie   MeSH
• lidé MeSH
• molekulární struktura MeSH
• organofosfáty chemická syntéza   chemie   farmakologie   MeSH
• oximy chemická syntéza   chemie   farmakologie   MeSH
• pyridinové sloučeniny chemická syntéza   chemie   farmakologie   MeSH
• sulfhydrylové sloučeniny chemická syntéza   chemie   farmakologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Oxime cholinesterase reactivators (oximes) are used to counteract organophosphate intoxication. Charged oximes are administered via intramuscular or intravenous injection when the majority of dose is unmetabolized and is excreted as urine. In this study, the effects of selected double charged oximes were determined in the HK-2 cell line as a model for renal toxicity screening. Some effects on dehydrogenase activity were found for obidoxime, asoxime (syn. HI-6), K027, and K203. The effects of K868 and K869 were found to be unreliable due to rapid degradation of both chlorinated oximes in the assay medium, resulting for K868 in an isoxazole-pyridinium product.

• MeSH
• buněčné linie MeSH
• ledviny účinky léků   metabolismus   MeSH
• lidé MeSH
• molekulární struktura MeSH
• oximy aplikace a dávkování   škodlivé účinky   chemie   MeSH
• reaktivátory cholinesterázy aplikace a dávkování   škodlivé účinky   chemie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• acetylcholinesterasa účinky léků   metabolismus   MeSH
• antidota chemická syntéza   chemie   farmakologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• oximy chemická syntéza   chemie   farmakologie   MeSH
• reaktivátory cholinesterázy chemická syntéza   chemie   farmakologie   MeSH
• simulace molekulární dynamiky MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Poisoning with organophosphorus compounds used as pesticides or misused as chemical weapons remains a serious threat to human health and life. Their toxic effects result from irreversible blockade of the enzymes acetylcholinesterase and butyrylcholinesterase, which causes overstimulation of the cholinergic system and often leads to serious injury or death. Treatment of organophosphorus poisoning involves, among other strategies, the administration of oxime compounds. Oximes reactivate cholinesterases by breaking the covalent bond between the serine residue from the enzyme active site and the phosphorus atom of the organophosphorus compound. Although the general mechanism of reactivation has been known for years, the exact molecular aspects determining the efficiency and selectivity of individual oximes are still not clear. This hinders the development of new active compounds. In our research, using relatively simple and widely available molecular docking methods, we investigated the reactivation of acetyl- and butyrylcholinesterase blocked by sarin and tabun. For the selected oximes, their binding modes at each step of the reactivation process were identified. Amino acids essential for effective reactivation and those responsible for the selectivity of individual oximes against inhibited acetyl- and butyrylcholinesterase were identified. This research broadens the knowledge about cholinesterase reactivation and demonstrates the usefulness of molecular docking in the study of this process. The presented observations and methods can be used in the future to support the search for new effective reactivators.

• MeSH
• acetylcholinesterasa metabolismus   MeSH
• aktivace enzymů MeSH
• butyrylcholinesterasa metabolismus   MeSH
• cholinesterasové inhibitory farmakologie   MeSH
• fosfor chemie   MeSH
• katalytická doména MeSH
• konformace proteinů MeSH
• kvantová teorie MeSH
• lidé MeSH
• ligandy MeSH
• molekulární modely MeSH
• myši MeSH
• organofosfáty chemie   MeSH
• oximy chemie   MeSH
• proteosyntéza MeSH
• reaktivátory cholinesterázy farmakologie   MeSH
• sarin chemie   MeSH
• shluková analýza MeSH
• simulace molekulového dockingu * MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Antidotes against organophosphates often possess physicochemical properties that mitigate their passage across the blood-brain barrier. Cucurbit[7]urils may be successfully used as a drug delivery system for bisquaternary oximes and improve central nervous system targeting. The main aim of these studies was to elucidate the relationship between cucurbit[7]uril, oxime K027, atropine, and paraoxon to define potential risks or advantages of this delivery system in a complex in vivo system. For this reason, in silico (molecular docking combined with umbrella sampling simulation) and in vivo (UHPLC-pharmacokinetics, toxicokinetics; acetylcholinesterase reactivation and functional observatory battery) methods were used. Based on our results, cucurbit[7]urils affect multiple factors in organophosphates poisoning and its therapy by (i) scavenging paraoxon and preventing free fraction of this toxin from entering the brain, (ii) enhancing the availability of atropine in the central nervous system and by (iii) increasing oxime passage into the brain. In conclusion, using cucurbit[7]urils with oximes might positively impact the overall treatment effectiveness and the benefits can outweigh the potential risks.

• MeSH
• atropin chemie   MeSH
• hematoencefalická bariéra MeSH
• imidazoly chemie   MeSH
• myši MeSH
• oximy chemie   MeSH
• paraoxon chemie   toxicita   MeSH
• počítačová simulace MeSH
• přemostěné cyklické sloučeniny chemie   MeSH
• pyridinové sloučeniny chemie   MeSH
• reaktivátory cholinesterázy chemie   toxicita   MeSH
• simulace molekulového dockingu MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Organophosphorus (OP) compounds are used as both chemical weapons and pesticides. However, these agents are very dangerous and toxic to humans, animals, and the environment. Thus, investigations with reactivators have been deeply developed in order to design new antidotes with better efficiency, as well as a greater spectrum of action in the acetylcholinesterase (AChE) reactivation process. With that in mind, in this work, we investigated the behavior of trimedoxime toward the Mus musculus acetylcholinesterase (MmAChE) inhibited by a range of nerve agents, such as chemical weapons. From experimental assays, reactivation percentages were obtained for the reactivation of different AChE-OP complexes. On the other hand, theoretical calculations were performed to assess the differences in interaction modes and the reactivity of trimedoxime within the AChE active site. Comparing theoretical and experimental data, it is possible to notice that the oxime, in most cases, showed better reactivation percentages at higher concentrations, with the best result for the reactivation of the AChE-VX adduct. From this work, it was revealed that the mechanistic process contributes most to the oxime efficiency than the interaction in the site. In this way, this study is important to better understand the reactivation process through trimedoxime, contributing to the proposal of novel antidotes.

• MeSH
• acetylcholinesterasa metabolismus   MeSH
• antidota farmakologie   MeSH
• cholinesterasové inhibitory metabolismus   farmakologie   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• myši MeSH
• nervová bojová látka chemie   MeSH
• organofosforové sloučeniny chemie   MeSH
• oximy chemie   MeSH
• reaktivátory cholinesterázy chemie   farmakologie   MeSH
• trimedoxim farmakologie   terapeutické užití   MeSH
• výpočetní biologie metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH