Reactivation Of Acetylcholinesterase Research Articles

Drosophila melanogaster as a model organism for screening acetylcholinesterase reactivators

ABSTRACT The widely used insecticide chlorpyrifos (CP) is known to inhibit acetylcholinesterase (AChE) activity attributed to result in various neurological disorders and acetylcholine-dependent organ functions including heart, skeletal muscle, lung, gastrointestinal tract, and central nervous systems. Enzyme reactivators, such as oximes, are known to restore AChE activity and mitigate adverse effects. The identification of compounds that reactivate AChE constitute agents with important therapeutic beneficial effects in cases of pesticide poisoning. However, the screening of novel drugs using traditional models may raise ethical concerns. This study aimed to investigate the potential of Drosophila melanogaster as a model organism for screening AChE reactivators, with a focus on organophosphate poisoning. The efficacy of several oximes, including pralidoxime, trimedoxime, obidoxime, methoxime, HI-6, K027, and K048, against CP-induced AChE activity inhibition in D. melanogaster was determined in silico, in vitro, and in vivo experiments. Molecular docking studies indicated a strong interaction between studied oximes and the active-site gorge of AChE. Data showed that selected oximes (100 μM) are effective in the reactivation of AChE inhibited by CP (10 μM) in vitro. Finally, in vivo investigations demonstrated that selected oximes, pralidoxime and K048 (1.5 ppm), reversed the locomotor deficits, inhibition of AChE activity as well as lowered the mortality rates induced by CP (0.75 ppm). Our findings contribute to utilization of D. melanogaster as a robust model for determination of actions of identified new AChE inhibitory agents with more effective therapeutic properties that those currently in use in the clinical practice in treatment of AChE associated disorders.

HI-6-Loaded Vehicle of Liposomes Mediated by an Amphiphilic Pillar[5]arene against Paraoxon Poisoning.

Organophosphate (OP) intoxication has become a severe common health matter all over the world. For the treatment of acute OP poisoning, the effective intracerebral delivery of acetylcholinesterase reactivators is crucial. Here, an amphiphilic hydrazide-pillar[5]arene (HP5A-6C), which could be readily integrated into liposomal bilayers' zwitterionic disaturated phosphatidylcholine (DSPC), was synthesized. A T7 peptide-containing guest (G) was attached on the surface via a noncovalent interaction to make mixed liposomes a particularly appealing candidate for brain-targeting delivery. Such coassembly could remain stable at room temperature for up to 6 weeks, and safety evaluations initially verified its fine biological compatibility. The hydrophilic interiors of T7/HP5A-6C@DSPC could further load HI-6 with 89.70% encapsulation efficiency. Support for brain-targeting potency came from imaging results. Notably, intravenous injection of HI-6-loaded vesicles exhibited a remarkable therapeutic effect on paraoxon (POX)-poisoned mice, effectively alleviating seizures and brain damage and significantly increasing the improving survival rate to 60% over the course of 7 days.

Continuous Flow Synthesis of the Nerve Agent Antidote Pralidoxime (2‐PAM) Iodide

AbstractPralidoxime salts are important medical countermeasures towards different organophosphorus compounds, either nerve agents or pesticides, as they act as acetylcholinesterase reactivators, avoiding health issues and even death that can result from intoxication with such chemicals. In this work, we present an expeditious study focuses on the optimization of a two‐step continuous‐flow synthesis of 2‐PAM. Initial attempts to replace the solvent composition in the oxime formation reaction with acetonitrile resulted in lower yields, highlighting the significance of a polar protic solvent. Subsequent optimization under continuous‐flow conditions revealed the successful execution of oxime formation at room temperature with a residence time of 20 minutes, achieving an impressive 96 % conversion, surpassing literature‐reported conditions. The methylation step required a high residence time of 60 minutes at 120 °C for a substantial conversion of 78 %. The two‐step continuous‐flow process demonstrated robustness, yielding 75 %, with space‐time yields of 0.32 g/h and 0.18 g/h. Further concentration adjustments increased space‐time yields to 3.2 g/h and 2.2 g/h. Despite solvent incompatibility, a strategic alteration of the reaction sequence allowed for a successful cascade process, integrating the methylation step in acetonitrile and the oxime formation in water. The continuous‐flow system, incorporating a heat exchanger, exhibited stability, maintaining full conversion and a space‐time yield of 0.3 g/h over extended periods. This systematic optimization not only enhances the efficiency of 2‐PAM synthesis but also provides insights into a scalable and practical continuous‐flow cascade process.

An anesthetized rat assay for evaluating the effects of organophosphate-based compounds and countermeasures on intracranial EEG and respiratory rate

The development of medical countermeasures (MCMs) against organophosphate (OP) induced poisoning is of substantial importance. Use of conventional therapeutics is complicated by off-target effects and restricted penetration of the blood-brain barrier (BBB). Therefore, a concerted effort is underway to discover improved acetylcholinesterase (AChE) reactivators, muscarinic acetylcholine receptor (mAChR) antagonists, and other countermeasures with broader spectrum activity and enhanced CNS efficacy. We recently developed a rat brain slice assay to assess the efficacy of AChE reactivators and mAChR antagonists against the acute effects of the organophosphorus AChE inhibitor 4-nitrophenyl isopropyl methylphosphonate (NIMP) in the basolateral amygdala (BLA). Here we introduce a complimentary anesthetized animal model to evaluate the same compounds in vivo with concurrent monitoring of EEG and respiratory rate. We find that intravenous delivery of 0.5 mg/kg NIMP reliably produces seizure like activity in the BLA, with concurrent respiratory depression and eventual respiratory failure. The central effects of AChE reactivators and mAChR antagonists delivered intravenously are consistent with their expected ability to cross the BBB. Combining our previously described in vitro assay with the methods described here provides a relatively comprehensive set of preclinical tools for evaluating the efficacy of novel MCMs. Notably, using these methods is expected to reduce the need to subject conscious animals to cholinergic crises, which aligns with the AAALAC’s 3Rs principle of refinement.

Synthesis and in vitro assessment of the reactivation profile of clinically available oximes on the acetylcholinesterase model inhibited by A-230 nerve agent surrogate.

The risk of the use of toxic chemicals for unlawful acts has been a matter of concern for different governments and multilateral agencies. The Organisation for the Prohibition of Chemical Weapons (OPCW), which oversees the implementation of the Chemical Weapons Convention (CWC), considering recent events employing chemical warfare agents as means of assassination, has recently included in the CWC "Annex on Chemicals" some organophosphorus compounds that are regarded as acting in a similar fashion to the classical G- and V-series of nerve agents, inhibiting the pivotal enzyme acetylcholinesterase. Therefore, knowledge of the activity of the pyridinium oximes, the sole class of clinically available acetylcholinesterase reactivators to date, is plainly justified. In this paper, continuing our research efforts in medicinal chemistry on this class of toxic chemicals, we synthesized an A-230 nerve agent surrogate and applied a modified Ellman's assay to evaluate its ability to inhibit our enzymatic model, acetylcholinesterase from Electrophorus eel, and if the clinically available antidotes are able to rescue the enzyme activity for the purpose of relating the findings to the previously disclosed in silico data for the authentic nerve agent and other studies with similar A-series surrogates. Our experimental data indicates that pralidoxime is the most efficient compound for reactivating acetylcholinesterase inhibited by A-230 surrogate, which is the opposite of the in silico data previously disclosed.

Quantification of oxidative stress markers in the blood sera following subacute administration of different oximes in rats

Oxidative stress status, as a disruption of redox homeostasis, in the blood sera of Wistar rats caused by repeated application of selected acetylcholinesterase reactivators - asoxime, obidoxime, K027, K048, K074, and K075 were evaluated. Throughout this study, each oxime in a dose of 0.1 of LD50/kg im was given 2x/week for 4 weeks. Then, seven days after the last oximes' application, markers of lipid peroxidation (malondialdehyde, MDA), and protein oxidation (advanced oxidation protein products, AOPP), as well as the activity of antioxidant enzymes (catalase, CAT, superoxide dismutase, SOD, reduced glutathione, GSH, and oxidized glutathione, GSSG), were determined. Oxidative stress parameters, MDA and AOPP were significantly highest in the K048-, K074- and K075-treated groups (p < 0.001). The activity of CAT was significantly elevated in the obidoxime-treated group (p < 0.05), while treatment with K027, K048, and K074 induced high elevation in SOD levels (p < 0.01, p < 0.001). Interestingly, the activity of GSH in each oxime-treated group was significantly elevated. Unlike, treatment with obidoxime caused elevation in GSSG levels (p < 0.01). As a continuation of our previously published data, these results assure that applied oximes following subacute treatment ameliorated the oxidative status and further adverse systemic toxic effects in rats.

ScL-2PAM: A Novel Countermeasure That Ameliorates Neuroinflammation and Neuronal Losses in Mice Exposed to an Anticholinesterase Organophosphate.

Due to their inhibition of acetylcholinesterase, organophosphates are among the most toxic of chemicals. Pralidoxime (a.k.a 2-PAM) is the only acetylcholinesterase reactivator approved in the U.S., but 2-PAM only poorly traverses the blood-brain barrier. Previously, we have demonstrated that scL-2PAM, a nanoformulation designed to enter the brain via receptor-mediated transcytosis, is superior to unencapsulated 2-PAM for reactivating brain acetylcholinesterase, ameliorating cholinergic crisis, and improving survival rates for paraoxon-exposed mice. Here, we employ histology and transcriptome analyses to assess the ability of scL-2PAM to prevent neurological sequelae including microglial activation, expression of inflammatory cytokines, and ultimately loss of neurons in mice surviving paraoxon exposures. Levels of the mRNA encoding chemokine ligand 2 (CCL2) were significantly upregulated after paraoxon exposures, with CCL2 mRNA levels in the brain correlating well with the intensity and duration of cholinergic symptoms. Our nanoformulation of 2-PAM was found to be superior to unencapsulated 2-PAM in reducing the levels of the CCL2 transcript. Moreover, brain histology revealed that scL-2PAM was more effective than unencapsulated 2-PAM in preventing microglial activation and the subsequent loss of neurons. Thus, scL-2PAM appears to be a new and improved countermeasure for reducing neuroinflammation and mitigating brain damage in survivors of organophosphate exposures.

Reactivation by novel pyridinium oximes of rat serum and skeletal muscle acetylcholinesterase inhibited by organophosphates.

The treatment of organophosphate (OP) anticholinesterases currently lacks an effective oxime reactivator of OP-inhibited acetylcholinesterase (AChE) which can penetrate the blood-brain barrier (BBB). Our laboratories have synthesized novel substituted phenoxyalkyl pyridinium oximes and tested them for their ability to promote survival of rats challenged with lethal doses of nerve agent surrogates. These previous studies demonstrated the ability of some of these oximes to promote 24-h survival to rats challenged with a lethal level of highly relevant surrogates for sarin and VX. The reactivation of OP-inhibited AChE in peripheral tissues was likely to be a major contributor to their efficacy in survival of lethal OP challenges. In the present study, twenty of these novel oximes were screened in vitro for reactivation ability for AChE in rat skeletal muscle and serum using two nerve agent surrogates: phthalimidyl isopropyl methylphosphonate (PIMP, a sarin surrogate) and 4-nitrophenyl ethyl methylphosphonate (NEMP, a VX surrogate). The oximes demonstrated a range of 23%-102% reactivation of AChE in vitro across both tissue types. Some of the novel oximes tested in the present study demonstrated the ability to more effectively reactivate AChE in serum than the currently approved oxime, 2-PAM. Therefore, some of these novel oximes have the potential to reverse AChE inhibition in peripheral target tissues and contribute to survival efficacy.

New Heterostilbene and Triazole Oximes as Potential CNS-Active and Cholinesterase-Targeted Therapeutics.

New furan, thiophene, and triazole oximes were synthesized through several-step reaction paths to investigate their potential for the development of central nervous systems (CNS)-active and cholinesterase-targeted therapeutics in organophosphorus compound (OP) poisonings. Treating patients with acute OP poisoning is still a challenge despite the development of a large number of oxime compounds that should have the capacity to reactivate acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The activity of these two enzymes, crucial for neurotransmission, is blocked by OP, which has the consequence of disturbing normal cholinergic nerve signal transduction in the peripheral and CNS, leading to a cholinergic crisis. The oximes in use have one or two pyridinium rings and cross the brain-blood barrier poorly due to the quaternary nitrogen. Following our recent study on 2-thienostilbene oximes, in this paper, we described the synthesis of 63 heterostilbene derivatives, of which 26 oximes were tested as inhibitors and reactivators of AChE and BChE inhibited by OP nerve agents-sarin and cyclosarin. While the majority of oximes were potent inhibitors of both enzymes in the micromolar range, we identified several oximes as BChE or AChE selective inhibitors with the potential for drug development. Furthermore, the oximes were poor reactivators of AChE; four heterocyclic derivatives reactivated cyclosarin-inhibited BChE up to 70%, and cis,trans-5 [2-((Z)-2-(5-((E)-(hydroxyimino)methyl)thiophen-2-yl)vinyl)benzonitrile] had a reactivation efficacy comparable to the standard oxime HI-6. In silico analysis and molecular docking studies, including molecular dynamics simulation, connected kinetic data to the structural features of these oximes and confirmed their productive interactions with the active site of cyclosarin-inhibited BChE. Based on inhibition and reactivation and their ADMET properties regarding lipophilicity, CNS activity, and hepatotoxicity, these compounds could be considered for further development of CNS-active reactivators in OP poisoning as well as cholinesterase-targeted therapeutics in neurodegenerative diseases such as Alzheimer's and Parkinson's.

The reactivation kinetic analysis, molecular docking, and dynamics of oximes against three V-type nerve agents inhibited four human cholinesterases

Nerve agents pose significant threats to civilian and military populations. The reactivation of acetylcholinesterase (AChE) is critical in treating acute poisoning, but there is still lacking broad-spectrum reactivators, which presents a big challenge. Therefore, insights gained from the reactivation kinetic analysis and molecular docking are essential for understanding the behavior of reactivators towards intoxicated AChE. In this research, we present a systematic determination of the reactivation kinetics of three V agents-inhibited four human ChEs [(AChE and butyrylcholinesterase (BChE)) from either native or recombinant resources, namely, red blood cell (RBC) AChE, rhAChE, hBChE, rhBChE) reactivated by five standard oximes. We unveiled the effect of native and recombinant ChEs on the reactivation kinetics of V agents ex vitro, where the reactivation kinetics characteristic of Vs-inhibited BChE was reported for the first time.In terms of the inhibition type, all of the five oxime reactivators exhibited noncompetitive inhibition. The inhibition potency of these reactivators would not lead to the difference in the reactivation kinetics between native and recombinant ChE. Despite the significant differences between the native and recombinant ChEs observed in the inhibition, aging, and spontaneous reactivation kinetics, the reactivation kinetics of V agent-inhibited ChEs by oximes were less differentiated, which were supported by the ligand docking results. We also found differences in the reactivation efficiency between five reactivators and the phosphorylated enzyme, and molecular dynamic simulations can further explain from the perspectives of conformational stability, hydrogen bonding, binding free energies, and amino acid contributions. By Poisson-Boltzmann surface area (MM-PBSA) calculations, the total binding free energy trends aligned well with the experimental kr2 values.

4-Amidophenol Quinone Methide Precursors: Effective and Broad-Scope Nonoxime Reactivators of Organophosphorus-Inhibited Cholinesterases and Resurrectors of Organophosphorus-Aged Acetylcholinesterase.

Acetylcholinesterase (AChE) inhibition by organophosphorus (OP) compounds poses a serious health risk to humans. While many therapeutics have been tested for treatment after OP exposure, there is still a need for efficient reactivation against all kinds of OP compounds, and current oxime therapeutics have poor blood-brain barrier penetration into the central nervous system, while offering no recovery in activity from the OP-aged forms of AChE. Herein, we report a novel library of 4-amidophenol quinone methide precursors (QMP) that provide effective reactivation against multiple OP-inhibited forms of AChE in addition to resurrecting the aged form of AChE after exposure to a pesticide or some phosphoramidates. Furthermore, these QMP compounds also reactivate OP-inhibited butyrylcholinesterase (BChE) which is an in vivo, endogenous scavenger of OP compounds. The in vitro efficacies of these QMP compounds were tested for reactivation and resurrection of soluble forms of human AChE and BChE and for reactivation of cholinesterases within human blood as well as blood and brain samples from a humanized mouse model. We identify compound 10c as a lead candidate due to its broad-scope efficacy against multiple OP compounds as well as both cholinesterases. With methylphosphonates, compound 10c (250 μM, 1 h) shows >60% recovered activity from OEt-inhibited AChE in human blood as well as mouse blood and brain, thus highlighting its potential for future in vivo analysis. For 10c, the effective concentration (EC50) is less than 25 μM for reactivation of three different methylphosphonate-inhibited forms of AChE, with a maximum reactivation yield above 80%. Similarly, for OP-inhibited BChE, 10c has EC50 values that are less than 150 μM for two different methylphosphonate compounds. Furthermore, an in vitro kinetic analysis show that 10c has a 2.2- and 92.1-fold superior reactivation efficiency against OEt-inhibited and OiBu-inhibited AChE, respectively, when compared to an oxime control. In addition to 10c being a potent reactivator of AChE and BChE, we also show that 10c is capable of resurrecting (ethyl paraoxon)-aged AChE, which is another current limitation of oximes.

Neuroprotection of Cholinergic Neurons with a Tau Aggregation Inhibitor and Rivastigmine in an Alzheimer's-like Tauopathy Mouse Model.

Basal forebrain cholinergic dysfunction, most likely linked with tau protein aggregation, is a characteristic feature of Alzheimer's disease (AD). Recent evidence suggests that tau protein is a putative target for the treatment of dementia, and the tau aggregation inhibitor, hydromethylthionine mesylate (HMTM), has emerged as a potential disease-modifying treatment. However, its efficacy was diminished in patients already receiving approved acetylcholinesterase inhibitors. In this study, we ask whether this negative interaction can also be mimicked in experimental tau models of AD and whether the underlying mechanism can be understood. From a previous age profiling study, 6-month-old line 1 (L1) tau transgenic mice were characterized by a severe reduction in several cholinergic markers. We therefore assessed whether long-term pre-exposure with the acetylcholinesterase inhibitor rivastigmine alone and in conjunction with the tau aggregation inhibitor HMTM can reverse cholinergic deficits in L1. Rivastigmine and HMTM, and combinations of the two compounds were administered orally for 11 weeks to both L1 and wild-type mice. The brains were sectioned with a focus on the basal forebrain, motor cortex and hippocampus. Immunohistochemical staining and quantification of choline acetyltransferase (ChAT), tyrosine kinase A (TrkA)-positive neurons and relative optical intensity (ROI) for vesicular acetylcholine transporter (VAChT), and acetylcholinesterase (AChE) reactivity confirmed reversal of the diminished cholinergic phenotype of interneurons (nucleus accumbens, striatum) and projection neurons (medial septum, nucleus basalis magnocellularis) by HMTM, to a greater extent than by rivastigmine alone in L1 mice. Combined administration did not yield additivity but, in most proxies, led to antagonistic effects in which rivastigmine decreased the benefits shown with HMTM alone. Local markers (VAChT and AChE) in target structures of the basal forebrain, motor cortex and hippocampal CA3 seemed to be normalized by HMTM, but not by rivastigmine or the combination of both drugs. HMTM, which was developed as a tau aggregation inhibitor, strongly decreased the tau load in L1 mice, however, not in combination with rivastigmine. Taken together, these data confirm a cholinergic phenotype in L1 tau transgenic mice that resembles the deficits observed in AD patients. This phenotype is reversible by HMTM, but at the same time appears to be subject to a homeostatic regulation induced by chronic pre-treatment with an acetylcholinesterase inhibitor, which interferes with the efficacy of HMTM. The strongest phenotypic reversal coincided with a normalization of the tau load in the cortex and hippocampus of L1, suggesting that tau accumulation underpins the loss of cholinergic markers in the basal forebrain and its projection targets.

Organophosphate Detoxification and Acetylcholinesterase Reactivation Triggered by Zeolitic Imidazolate Framework Structural Degradation.

Organophosphate (OP) toxicity is related to inhibition of acetylcholinesterase (AChE) activity, which plays a key role in the neurotransmission process. In this work, we report the ability of different zinc zeolitic imidazolate frameworks (ZIFs) to behave as potential antidotes against OP poisoning. The Zn-L coordination bond (L = purine, benzimidazole, imidazole, or 2-methylimidazole) is sensitive to the G-type nerve agent model compounds diisopropylfluorophosphate (DIFP) and diisopropylchlorophosphate, leading to P-X (X = F or Cl) bond breakdown into nontoxic diisopropylphosphate. P-X hydrolysis is accompanied by ZIF structural degradation (Zn-imidazolate bond hydrolysis), with the concomitant release of the imidazolate linkers and zinc ions representing up to 95% of ZIF particle dissolution. The delivered imidazolate nucleophilic attack on the OP@AChE adduct gives rise to the recovery of AChE enzymatic function. P-X bond breakdown, ZIF structural degradation, and AChE reactivation are dependent on imidazolate linker nucleophilicity, framework topology, and particle size. The best performance is obtained for 20 nm nanoparticles (NPs) of Zn(2-methylimidazolate)2 (sod ZIF-8) exhibiting a DIFP degradation half-life of 2.6 min and full recovery of AChE activity within 1 h. 20 nm sod ZIF-8 NPs are not neurotoxic, as proven by in vitro neuroblastoma cell culture viability tests.

Restoration of nerve agent impaired neuromuscular transmission in rat diaphragm by bispyridinium non-oximes – Structure-activity relationships

Organophosphate (OP) poisoning is currently treated with atropine, oximes and benzodiazepines. The nicotinic signs, i.e., respiratory impairment, can only be targeted indirectly via the use of oximes as reactivators of OP-inhibited acetylcholinesterase. Hence, compounds selectively targeting nicotinic acetylcholine receptors (nAChRs) might fundamentally improve current treatment options. The bispyridinium compound MB327 has previously shown some therapeutic effect against nerve agents in vitro and in vivo. Nevertheless, compound optimization was deemed necessary, due to limitations (e.g., toxicity and efficacy). The current study investigated a series of 4-tert-butyl bispyridinium compounds and of corresponding bispyridinium compounds without substituents in a rat diaphragm model using an indirect field stimulation technique. The length of the respective linker influenced the ability of the bispyridinium compounds to restore muscle function in rat hemidiaphragms. The current data show structure-activity relationships for a series of bispyridinium compounds and provide insight for future structure-based molecular modeling.

A Pralidoxime Nanocomplex Formulation Targeting Transferrin Receptors for Reactivation of Brain Acetylcholinesterase After Exposure of Mice to an Anticholinesterase Organophosphate.

Organophosphates are among the deadliest of known chemicals based on their ability to inactivate acetylcholinesterase in neuromuscular junctions and synapses of the central and peripheral nervous systems. The consequent accumulation of acetylcholine can produce severe acute toxicities and death. Oxime antidotes act by reactivating acetylcholinesterase with the only such reactivator approved for use in the United States being 2-pyridine aldoxime methyl chloride (a.k.a., pralidoxime or 2-PAM). However, this compound does not cross the blood-brain barrier readily and so is limited in its ability to reactivate acetylcholinesterase in the brain. We have developed a novel formulation of 2-PAM by encapsulating it within a nanocomplex designed to cross the blood-brain barrier via transferrin receptor-mediated transcytosis. This nanocomplex (termed scL-2PAM) has been subjected to head-to-head comparisons with unencapsulated 2-PAM in mice exposed to paraoxon, an organophosphate with anticholinesterase activity. In mice exposed to a sublethal dose of paraoxon, scL-2PAM reduced the extent and duration of cholinergic symptoms more effectively than did unencapsulated 2-PAM. The scL-2PAM formulation was also more effective than unencapsulated 2-PAM in rescuing mice from death after exposure to otherwise-lethal levels of paraoxon. Improved survival rates in paraoxon-exposed mice were accompanied by a higher degree of reactivation of brain acetylcholinesterase. Our data indicate that scL-2PAM is superior to the currently used form of 2-PAM in terms of both mitigating paraoxon toxicity in mice and reactivating acetylcholinesterase in their brains.

Screening of efficient salicylaldoxime reactivators for DFP and paraoxon-inhibited acetylcholinesterase.

Previously we reported two salicylaldoxime conjugates (L7R3 and L7R5) showing equal or even higher reactivating efficiency for both organophosphorus nerve agent and pesticide inhibited acetylcholinesterase in comparison to obidoxime and HI-6. In this study, L7R3 and L7R5 were selected as lead compounds and refined by employing a fragment-based drug design strategy, and a total of 32 novel salicylaldoxime conjugates were constructed and screened for DFP and paraoxon inhibited acetylcholinesterase. The findings demonstrate that the conjugate L73R3, which contains a 4-nitrophenyl group, exhibited a higher reactivation efficacy against paraoxon-inhibited acetylcholinesterase compared to obidoxime and HI-6. It was confirmed that the combination of a 4-pyridinyl or 4-nitrophenyl peripheral site ligand, a piperazine linker and a methyl or chloro-substituted salicylaldoxime could construct efficient nonquaternary oxime reactivators. The results hold promise for developing a new generation of highly effective antidotes for organophosphate poisoning.

Theory-guided design of S-doped Fe/Co dual-atom nanozymes for highly efficient oxidase mimics.

The advent of dual-atom nanozymes (DAzymes) featuring distinctive bimetallic active sites garnered significant attention, representing enhanced iterations of conventional single-atom nanozymes. The quest for an effective and universal strategy to modulate the catalytic activity of DAzymes posed a formidable challenge, yet few published reports addressed this. Herein, we designed and synthesized S-doped Fe/Co DAzymes (S-FeCo-NC) under theoretical guidance and revealed their excellent oxidase-like activity. Experimental and theoretical calculations indicated that the superior oxidase-like activity exhibited by S-FeCo-NC was attributed to the S-doping, which modulated the local electronic structure of the dual-atom active site. This modulation of the local electronic structure significantly optimizes oxygen adsorption energy, thereby accelerating the rate of enzyme-catalyzed reactions. As a proof-of-concept, this study integrated S-FeCo-NC with the cascade inhibition reaction of acetylcholinesterase (AChE) to devise a sensitive analytical platform for detecting organophosphorus pesticides. This study paved the way for elucidating the correlation between the local electronic structure of the active site and enzyme activity, offering novel methodologies and insights for the rational design of DAzymes.

AChE reactivation in precision-cut lung slices following organophosphorus compound poisoning

Precision-cut lung slices (PCLS) are a suitable model for analyzing the acetylcholinesterase (AChE) activity and subsequent effects after exposure to organophosphorus (OP) compounds. In this study, the AChE activity was determined in intact PCLS for the first time. Since the current standard therapy for OP poisoning (atropine + oxime + benzodiazepine) lacks efficiency, reliable models to study novel therapeutic substances are needed. Models should depict pathophysiological mechanisms and help to evaluate the beneficial effects of new therapeutics. Here PCLS were exposed to three organophosphorus nerve agents (OPNAs): sarin (GB), cyclosarin (GF), and VX. They were then treated with three reactivators: HI-6, obidoxime (OBI), and a non-oxime (NOX-6). The endpoints investigated in this study were the AChE activity and the airway area (AA) change. OPNA exposure led to very low residual AChE activities. Depending on the reactivator properties different AChE reactivation results were measured. GB-inhibited PCLS-AChE was reactivated best, followed by VX and GF. To substantiate these findings and to understand the connection between the molecular and the functional levels in a more profound way the results were correlated to the AA changes. These investigations underline the importance of reactivator use and point to the possibilities for future improvements in the treatment of OPNA-exposed victims.

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.

Inhibition with simultaneous spontaneous reactivation and aging of acetylcholinesterase by organophosphorus compounds: Demeton-S-methyl as a model

The kinetic analysis of esterase inhibition by acylating compounds (organophosphorus, carbamates and sulfonylfluorides) sometimes cannot yield consistent results by fitting simple inhibition kinetic models to experimental data of complex systems. In this work kinetic data were obtained for demeton-S-methyl (DSM) with human acetylcholinesterase in two kinds of experiments: (a) time progressive inhibition with a range of concentrations, (b) progressive spontaneous reactivation starting with pre-inhibited enzyme. DSM is an organophosphorus compound used as pesticide and considered a model for studying the dermal exposure of nerve agents such as VX gas. A kinetic model equation was deduced with four different molecular phenomena occurring simultaneously: (1) inhibition; (2) spontaneous reactivation; (3) aging; and (4) ongoing inhibition (inhibition during the substrate reaction). A 3D fit of the model was applied to analyze the inhibition experimental data. The best-fitting model is compatible with a sensitive enzymatic entity. The second-order rate constant of inhibition (ki = 0.0422 μM−1 min−1), the spontaneous reactivation constant (ks = 0.0202 min−1) and the aging constant (kg = 0.0043 min−1) were simultaneously estimated. As an example for testing the model and approach, it was tested also in the presence of 5 % ethanol (conditions as previously used in the literature), the best fitting model is compatible with two apparent sensitive enzymatic entities (17 % and 83 %) and only one spontaneously reactivates and ages. The corresponding second-order rate constants of inhibition (ki = 0.0354 and 0.0119 μM−1 min−1) and the spontaneous reactivation and aging constants for the less sensitive component (kr = 0.0203 min−1 and kg = 0.0088 min−1) were estimated. The results were also consistent with a significant ongoing inhibition. These parameters were similar to those deduced in spontaneous reactivation experiments of the pre-inhibited samples with DSM in the absence or presence of ethanol. The two apparent components fit was interpreted by an equilibrium between ethanol-free and ethanol-bound enzyme. The consistency of results in inhibition and in spontaneous reactivation experiments was considered an internal validation of the methodology and the conclusions.