Molecular modeling of Mannich phenols as reactivators of human acetylcholinesterase inhibited by A-series nerve agents.

The A-series is the most recent generation of chemical warfare nerve agents (CWA) which act directly on the inhibition of the human acetylcholinesterase (HssAChE) enzyme. These compounds lack accurate experimental data on their physicochemical properties, and there is no evidence that traditional antidotes effectively reactivate HssAChE inhibited by them. In the search for potential antidotes, we employed virtual screening, molecular docking, and molecular dynamics (MD) simulations for the theoretical assessment of the performance of a library of Mannich phenols as potential reactivators of HssAChE inhibited by the Novichok agents A-230, A-232, and A-234, in comparison with the commercial oximes pralidoxime (2-PAM), asoxime (HI-6), trimedoxime (TMB-4), and obidoxime. Following the near-attack conformation (NAC) approach, our results suggest that the compounds assessed would face difficulties in triggering the proposed nucleophilic in-line displacement mechanism. Despite this, it was observed that certain Mannich phenols presented similar or superior results to those obtained by reference oximes against A-232 and A-234 model, suggesting that these compounds can adopt more favourable conformations. Additional binding energy calculations confirmed the stability of the model/ligands complexes and the reactivating potential observed in the molecular docking and MD studies. Our findings indicate that the Mannich phenols could be alternative antidotes and that their efficacy should be evaluated experimentally against the A-series CWA.

A comparison of neuroprotective efficacy of two novel reactivators of acetylcholinesterase called K920 and K923 with the oxime K203 and trimedoxime in tabun-poisoned rats.

The ability of two newly developed bispyridinium oximes (K920, K923) to reduce tabun-induced acute neurotoxic signs and symptoms was compared with the oxime K203 and trimedoxime using a functional observational battery (FOB). The neuroprotective effects of the oximes studied combined with atropine on rats poisoned with tabun at a sublethal dose (130 µg/kg i.m.; 80% of LD50 value) were evaluated. Tabun-induced neurotoxicity was monitored by FOB at 2 h after tabun administration. The results indicate that all tested oximes combined with atropine enable tabun-poisoned rats to survive till the end of experiment while one non-treated tabun-poisoned rat died within 2 h. Both newly developed oximes (K920, K923) combined with atropine were able to markedly decrease tabun-induced neurotoxicity in the case of sublethal poisoning although they did not eliminate all tabun-induced acute neurotoxic signs and symptoms. Their ability to decrease tabun-induced acute neurotoxicity did not prevail the neuroprotective efficacy of trimedoxime and the oxime K203. Therefore, the newly developed oximes are not suitable for the replacement of currently available oximes (especially trimedoxime) in the treatment of acute tabun poisonings.

In vitro reactivation kinetics of paraoxon- and DFP-inhibited electric eel AChE using mono- and bis-pyridinium oximes.

Oxime-assisted reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) is a crucial step in the post-inhibitory treatment of OP intoxication. The limited efficacy of oxime reactivators for all OP nerve agents and pesticides led to the development of various novel oximes and their thorough kinetic investigations. Hence, in the present investigation, we have tested 10 structurally different pyridinium oxime-based reactivators for their in vitro potency to reactivate paraoxon- and DFP-inhibited electric eel AChE. From structure activity relationship point of view, various oximes such as mono-quaternary (2-PAM, K100, K024) and bis-quaternary symmetric (obidoxime, TMB-4) and asymmetric (K027, K048, K203, K618, K628) oximes bearing different connecting linkers (oxybismethylene, trimethylene, propane, butane, butene, and xylene) have been studied. The observed kinetic data demonstrate that not only the position of oxime group is decisive for the increased reactivation ability of oximes, but the role of connecting linker is also significant. Oximes with aliphatic linkers are superior reactivators than the oximes with unsaturated and aromatic linkers. The optimal chain length for plausible reactivation ability for paraoxon- and DFP-inhibited AChE is 3 or 4 carbon-carbon connecting linker between prydinium rings.

[Studying kinetics of oxime-induced reactivation of malathion-inhibited cholinesterase].

The kinetics of oxime-induced reactivation of malathion-inhibited cholinesterase has been experimentally studied in vitro. It is shown that oximes do not restore the activity of inhibited butyrylcholinesterase. Acetylcholinesterase reactivation peak (5-mins long) was found to take place upon introduction of dipyroxime (32.5%), pralidoxime (18%), carboxyme (16%) at a concentration of 2.5 x 10(-4) mol/l or toxogonine (26%) at a concentration of 5 x 10(-4) mol/l. Toxogonine demonstrated the maximum affinity to phosphorylated enzyme, while dipyroxime is characterized by a high reactivity with respect to oxime. Significant reactivating ability of these preparations (kR -2300 mol(-1) min(-1) makes them promising solution for the treatment of malathion intoxication.

The benefit of combination of oximes for the neuroprotective efficacy of antidotal treatment of sarin-poisoned rats.

The potency of the oxime HI-6 and two combinations of oximes (HI-6 + trimedoxime, HI-6 + K203) to reduce sarin-induced acute neurotoxic signs and symptoms was evaluated in this study. Sarin-induced neurotoxicity and the neuroprotective effects of atropine alone or in combination with HI-6 alone and HI-6 combined with trimedoxime or K203 in rats poisoned with sarin at a sublethal dose (108 µg/kg i.m.; 90% of LD(50) value) were monitored by a functional observatory battery (FOB) 24 h following sarin administration. The results indicate that both mixtures of oximes combined with atropine were able to survive sarin-poisoned rats 24 h following sarin administration while two non-treated sarin-poisoned rats and one sarin-poisoned rat treated with atropine alone or with atropine in combination with the oxime HI-6 died within 24 h following sarin poisoning. All types of antidotal treatment were able to decrease sarin-induced neurotoxic signs and symptoms but not completely. While atropine alone and atropine in combination with the oxime HI-6 were able to eliminate some sarin-induced neurotoxic signs and symptoms, the neuroprotective efficacy of both combinations of oximes with atropine was slightly higher. Thus, both tested combinations of oximes in combination with atropine bring a small benefit for the neuroprotective efficacy of antidotal treatment of acute sarin poisonings.

A comparison of the neuroprotective efficacy of individual oxime (HI-6) and combinations of oximes (HI-6+trimedoxime, HI-6+K203) in soman-poisoned rats.

The ability of two combinations of oximes (HI-6+trimedoxime, HI-6+K203) to reduce soman-induced acute neurotoxic signs and symptoms was compared with the neuroprotective efficacy of the oxime HI-6 alone, using a functional observational battery. Soman-induced neurotoxicity and the neuroprotective effects of HI-6 alone and HI-6 combined with trimedoxime or K203 in rats poisoned with soman at a sublethal dose (90 µg/kg intramuscularly, i.m.; 80% of LD50 value) were monitored by the functional observational battery at 24 hours following soman administration. The results indicate that both tested oxime mixtures combined with atropine were able to allow soman-poisoned rats to survive 24 hours following soman challenge, while 4 nontreated soman-poisoned rats and 1 soman-poisoned rat treated with oxime HI-6 alone combined with atropine died within 24 hours following soman poisoning. While the oxime HI-6 alone combined with atropine treatment was able to eliminate a few soman-induced neurotoxic signs and symptoms, both oxime mixtures showed higher neuroprotective efficacy in soman-poisoned rats. Especially, the combination of HI-6 with trimedoxime was able to eliminate most soman-induced neurotoxic signs and symptoms and markedly reduce acute neurotoxicity of soman in rats. Thus, both tested mixtures of oximes combined with atropine were able to increase the neuroprotective effectiveness of antidotal treatment of acute soman poisonings, compared to the individual oxime.

The influence of combinations of oximes on the reactivating and therapeutic efficacy of antidotal treatment of soman poisoning in rats and mice.

The influence of the combination of oximes on the reactivating and therapeutic efficacy of antidotal treatment of acute soman poisoning was evaluated. The ability of two combinations of oximes (HI-6 + trimedoxime and HI-6 + K203) to reactivate soman-inhibited acetylcholinesterase and reduce acute toxicity of soman was compared with the reactivating and therapeutic efficacy of antidotal treatment involving single oxime (HI-6, trimedoxime, K203) using in vivo model. Studies determining percent of reactivation of soman-inhibited blood and diaphragm acetylcholinesterase in poisoned rats showed that the reactivating efficacy of both combinations of oximes is slightly greater than the reactivating efficacy of the most effective individual oxime, but the difference among them is not significant. Both combinations of oximes were found to be as effective in the reduction of acute lethal toxic effects in soman-poisoned mice as the antidotal treatment involving the most efficacious individual oxime. Thus, the efficacy of oximes is comparative in rats vs mice. A comparison of reactivating and therapeutic efficacy of individual oximes showed that the newly developed oxime K203 is approximately as effective as commonly used trimedoxime; nevertheless, their reactivating and therapeutic efficacy is markedly lower compared to the oxime HI-6. Based on the obtained data, one can conclude that the antidotal treatment involving chosen combinations of oximes does not significantly influence the potency of the most effective individual oxime (HI-6) to reactivate soman-inhibited rat acetylcholinesterase and to reduce acute toxicity of soman.

Effect of five acetylcholinesterase reactivators on tabun-intoxicated rats: induction of oxidative stress versus reactivation efficacy.

Oxime reactivators HI-6, obidoxime, trimedoxime, K347 and K628 were investigated as drugs designed for treatment of tabun intoxication. The experiments were performed on rats in order to simulate real conditions. Rats were intoxicated with one LD(50 )of tabun and treated with atropine and mentioned reactivators. Activities of erythrocyte acetylcholinesterase (AChE), plasma butyrylcholinesterase (BChE) and brain AChE were measured as markers of reactivation efficacy. An estimation of low molecular weight antioxidant levels using cyclic voltammetry was the second examination parameter. The evaluation of cholinesterases activity showed good reactivation potency of blood AChE and plasma BChE by commercially available obidoxime and newly synthesized K347. The potency of oximes to reactivate brain AChE was lower due to the poor blood-brain barrier penetration of used compounds. Commercially available reactivator HI-6 and newly synthesized K628 caused oxidative stress measured by cyclic voltammetry as antioxidant level. The oxidative stress provoked by HI-6 and K628 was found to be significant on probability level P = 0.05. The others reactivators did not affect antioxidant levels.

An evaluation of therapeutic and reactivating effects of newly developed oximes (K156, K203) and commonly used oximes (obidoxime, trimedoxime, HI-6) in tabun-poisoned rats and mice.

The potency of newly developed monoxime bispyridinium compounds (K156, K203) in reactivating tabun-inhibited acetylcholinesterase and reducing tabun-induced lethal toxic effects was compared with commonly used oximes (obidoxime, trimedoxime, the oxime HI-6) using in vivo methods. Studies determining percentage of reactivation of tabun-inhibited blood and tissue acetylcholinesterase in poisoned rats showed that the reactivating efficacy of newly developed oxime K203 is comparable with obidoxime and trimedoxime in blood and higher than the reactivating potency of trimedoxime and obidoxime in diaphragm and brain, where the difference in reactivating efficacy of obidoxime, trimedoxime and K203 is significant. On the other hand, the potency of newly developed K156 to reactivate tabun-inhibited acetylcholinesterase is comparable with obidoxime or trimedoxime in diaphragm and brain. It is significantly lower than the reactivating efficacy of trimedoxime and obidoxime in blood. Moreover, both newly developed oximes were found to be relatively efficacious in the reduction of lethal toxic effects in tabun-poisoned mice. Especially, the oxime K203 is able to decrease the acute toxicity of tabun nearly two times. The therapeutic efficacy of K156 and K203 corresponds to their potency to reactivate tabun-inhibited acetylcholinesterase, especially in diaphragm and brain. In contrast to obidoxime and trimedoxime, the oxime HI-6 is not effective in reactivation of tabun-inhibited acetycholinesterase and in reducing tabun lethality. While the oxime K156 does not improve the reactivating and therapeutic effectiveness of currently available obidoxime and trimedoxime, the newly developed oxime K203 is markedly more effective in reactivation of tabun-inhibited acetylcholinesterase in rats, especially in brain, and in reducing lethal toxic effects of tabun in mice and, therefore, it is suitable for the replacement of commonly used oximes for the antidotal treatment of acute tabun poisoning.

Unequal efficacy of pyridinium oximes in acute organophosphate poisoning.

The use of organophosphorus pesticides results in toxicity risk to non-target organisms. Organophosphorus compounds share a common mode of action, exerting their toxic effects primarily via acetylcholinesterase (AChE) inhibition. Consequently, acetylcholine accumulates in the synaptic clefts of muscles and nerves, leading to overstimulation of cholinergic receptors. Acute cholinergic crisis immediately follows exposure to organophosphate and includes signs and symptoms resulting from hyperstimulation of central and peripheral muscarinic and nicotinic receptors. The current view of the treatment of organophosphate poisoning includes three strategies, i.e. the use of an anticholinergic drug (e.g., atropine), cholinesterase-reactivating agents (e.g., oximes) and anticonvulsant drugs (e.g., benzodiazepines). Oximes, as a part of antidotal therapy, ensure the recovery of phosphylated enzymes via a process denoted as reactivation of inhibited AChE. However, both experimental results and clinical findings have demonstrated that different oximes are not equally effective against poisonings caused by structurally different organophosphorus compounds. Therefore, antidotal characteristics of conventionally used oximes can be evaluated regarding how close the certain substance is to the theoretical concept of the universal oxime. Pralidoxime (PAM-2), trimedoxime (TMB-4), obidoxime (LüH-6), HI-6 and HLö-7 have all been demonstrated to be very effective in experimental poisonings with sarin and VX. TMB-4 and LüH-6 may reactivate tabun-inhibited AChE, whereas HI-6 possesses the ability to reactivate the soman-inhibited enzyme. An oxime HLö-7 seems to be an efficient reactivator of AChE inhibited by any of the four organophosphorus warfare agents. According to the available literature, the oximes LüH-6 and TMB-4, although relatively toxic, are the most potent to induce reactivation of AChE inhibited by the majority of organophosphorus pesticides. Since there are no reports of controlled clinical trials on the use of TMB-4 in human organophosphate pesticide poisoning, LüH-6 may be a better option.

Currently used cholinesterase reactivators against nerve agent intoxication: comparison of their effectivity in vitro.

In vitro comparison of reactivation efficacy of five currently used oximes - pralidoxime, obidoxime, trimedoxime, methoxime, and HI-6 (at two concentrations: 10-5 and 10-3 M) - against acetylcholinesterase (AChE; E.C. 3.1.1.7) inhibited by six different nerve agents (VX, Russian VX, sarin, cyclosarin, tabun, soman) and organophosphorus insecticide chlorpyrifos was the aim of this study. As a source of AChE in the experiments, rat brain homogenate was used. According to the results obtained, no AChE reactivator was able to reach sufficient potency for AChE inhibited by all nerve agents used. Moreover, oxime HI-6 (the most effective one) was not able to reactivate tabun- and soman-inhibited AChE. Due to this fact, it could be designated as a partially broad-spectrum reactivator.

In vitro evaluation of acetylcholinesterase reactivators as potential antidotes against tabun nerve agent poisonings.

Searching for new potent acetylcholinesterase (AChE; E.C. 3.1.1.7) reactivators (oximes) is a very time-consuming process. At our department, we are able to synthesize more than 50 new AChE reactivators per year. Owing to this fact, we have to select promising reactivators using our in vitro method (potentiometric titration, pH 8 and temperature 25 degrees C; source of cholinesterases, rat brain homogenate; time of inhibition by nerve agents, 30 min; time of reactivation, 10 min) prior to in vivo experiments. For this purpose, we are using two-phase in vitro evaluation of reactivator potency. In the first phase, reactivation potency of all newly synthesized AChE reactivators is tested at two concentrations: 10(-3) M and 10(-5) M. Afterwards, all reactivators achieving reactivation potency over 15% (especially at the concentration 10(-5) M) are tested. The second phase consists of the measurement of the relationship between concentration of the oxime and its reactivation ability. In most cases, the reactivation bell-shaped curve is obtained. The most potent AChE reactivators are selected and provided for further experiments during our development process.

para- and ortho-Pyridinium aldoximes in reaction with acetylthiocholine.

In the oximolysis reaction para-aldoximes K027 and TMB-4 react faster with ATCh than ortho-aldoximes HI-6 and K033. The reaction rate constants at 25 degrees C were 22 M(-1) min(-1) for HI-6 and K033, 230 M(-1) min(-1) for TMB-4 and 306 M(-1) min(-1) for K027. Semi-empirical calculations showed that differences in rates do not origin from different electron density on the oxygen of the oxime group, but can be explained by the steric hindrance of the oxime group within the molecule. Thermodynamic parameters, DeltaG#, DeltaH# and DeltaS#, were also determined for oximolysis reaction.

Reactivation of immobilized acetyl cholinesterase in an amperometric biosensor for organophosphorus pesticide.

Biosensors based on acetyl cholinesterase (AChE) inhibition have been known for monitoring of pesticides in food and water samples. However, strong inhibition of the enzyme is a major drawback in practical application of the biosensor which can be overcome by reactivation of the enzyme for repeated use. In the present study, enzyme reactivation by oximes was explored for this purpose. Two oximes viz., 1,1'-trimethylene bis 4-formylpyridinium bromide dioxime (TMB-4) and pyridine 2-aldoxime methiodide (2-PAM) were compared for the reactivation of the immobilized AChE. TMB-4 was found to be a more efficient reactivator under repeated use, retaining more than 60% of initial activity after 11 reuses, whereas in the case of 2-PAM, the activity retention dropped to less than 50% after only 6 reuses. Investigations also showed that reactivation must be effected within 10 min after each analysis to eliminate the ageing effect, which reduces the efficiency of reactivation.

Influence of trimedoxime bromide on degradation of benactyzine in acidic injectable solutions.

The stability of benactyzine in a multicomponent injectable antidote formulation in deuterium oxide was studied in the presence of various concentrations of trimedoxime bromide. Benactyzine was found to be more stable in the absence of trimedoxime bromide and its degradation rate accelerated linearly with increasing concentrations of trimedoxime bromide. The main reasons for the accelerated decomposition rate of benactyzine were found to be the nucleophilic effect of the bromide ion and the oxime moiety. For each trimedoxime concentration studied, t90 for benactyzine at 25 degrees C was calculated and was found to be 5.3-1.5 years within the concentrations range of 0-120 mg/ml. Rate constants and activation energies for benactyzine degradation were determined for each of the concentrations studied.

Stability of mono- and bisbenzyloxime ethers of the acetylcholinesterase reactivator TMB-4.

Mono- and bisbenzyloxime ethers of the bispyridinium derivative TMB-4 (UNO, DUO) are potent allosteric modulators of the muscarinic receptor attracting clinical interest in case of organophosphate poisoning. In order to work out the stability of these compounds oximes, different oxime ethers and potential degradation products were synthesized and UV- and NMR-spectroscopically characterized. The process of degradation of all compounds was observed under stress conditions at varying pH-values and different temperatures by means of time-dependent NMR- und UV-measurements. The pyridinium aldoxime turned out to be rather stable, whereas the oxime ether and cyano derivatives convert to the pyridone at high pH-values and high temperature. The mechanism of degradation is discussed.

Interactions of oxime reactivators with diethylphosphoryl adducts of human acetylcholinesterase and its mutant derivatives.

Diethylphosphoryl conjugates of human acetylcholinesterase (AChE) and selected mutants, carrying amino acid replacements at the active center and at the peripheral anionic site, were subjected to reactivation with the monopyridinium oxime 2-hydroxy-iminomethyl-1-methylpyridinium chloride and the bispyridinium oximes 1,3-bis(4'-hydroxyiminomethyl-1'-pyridinium),propane dibromide (TMB-4) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4"-carbamoyl-1"-pyridinium)-2 - oxapropane dichloride (HI-6). The kinetic profiles for all of the reactivation reactions indicate single populations of reactivatable species. Replacement of Trp86, the anionic subsite in the active center, lowered the affinity of the free enzyme toward all three reactivators, but in the corresponding diethylphosphoryl conjugate, only affinity toward TMB-4 was affected. Replacement of other constituents of the hydrophobic subsite (Tyr337, Phe338) had no major effect on either affinity to the free enzymes or rates of reactivation. Substitution of residues of the acyl pocket (Phe295, Phe297) lowered the affinities toward reactivators except for the 20-fold increase in affinity of F295A toward HI-6. Replacement of the acidic residues in the active center (Glu202, Glu450) affected mainly the rates of nucleophilic displacement of the phosphoryl moiety. The effect of substituting residues constituting the peripheral anionic site at the rim of the active site gorge (Tyr72, Asp74, Trp286) was particularly puzzling because for 2-hydroxy-iminomethyl-1-methylpyridinium chloride and HI-6, mainly the nucleophilic reaction rate constants were affected, whereas for TMB-4, the affinities of the phosphorylated enzymes were significantly reduced. The fact that perturbations of the functional architecture of HuAChE active center can account for only some of the observed effects on the reactivation rates suggests that the binding modes of oxime to the phosphorylated and nonphosphorylated enzymes are considerably different and/or that interactions of the reactivators with the phosphoryl moieties play a dominant role in the reactivation process.

Conformational analysis, molecular shape comparison, and pharmacophore identification of different allosteric modulators of muscarinic receptors.

Structurally dissimilar compounds such as alcuronium and the newly synthesized substances derived from the bisbenzyl ether TMB4 and from hexamethonium stabilize antagonist binding to M2-cholinoceptors which is indicative of an allosteric action. In order to propose a hypothesis for the common pharmacophore and the corresponding active conformations, seven flexible compounds in a data set were individually aligned onto the most active and, additionally, rigid alcuronium molecule using a torsional angle flexible fit. An S-shape conformation was found to be a plausible general active conformation. In a subsequent molecular shape analysis the overlap and the nonoverlap steric volumes, RMS alignment as well as electrostatic field potentials were employed as possible structure--activity correlation descriptors. The corresponding 3D-QSAR formulation exhibits a correlation between allosteric modulation potency and the nonoverlap steric volume as well as the proton and oxygen anion probe electrostatic field potentials. Because of large structural diversity among the small number of compounds studied, the apparent 3D-QSAR is best thought of as a convenient representation of the common spatial pharmacophore hypothesis.