Organophosphorus compounds (OP), like pesticides and nerve agents, are potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) due to the phosphylation of their catalytic serine. Current treatment is limited, as approved oximes lack broad-spectrum efficacy and are poor reactivators of inhibited BChE. An alternative approach is pseudo-catalytic OP bioscavenging in which BChE and an efficient reactivator rapidly degrade OP in circulation, preventing it from reaching target tissues enriched with AChE. As imidazolium oximes were previously identified as potent BChE reactivators, we prepared eight novel N-substituted imidazolium oximes and tested them as reactivators of both human AChE and BChE inhibited by pesticide derivative paraoxon, and nerve warfare agents (sarin, cyclosarin, tabun VX, and five A-series agents). Oxime 3 (1,3-dibenzyl-2-hydroxy (imino)methylimidazolium bromide) was identified as the most potent reactivator, showing nanomolar affinity with native BChE and reactivation efficacy superior to standard oximes for cyclosarin-, sarin-, and tabun-BChE conjugate that was 1400-fold, 20-fold and 40-fold higher, respectively. In human whole blood, 30 μM cyclosarin was pseudo-catalytically decomposed by supplemented BChE and oxime 3, restoring 65% of total cholinesterase activity within 10 min. Although less potent, we identified two oximes capable of reactivating A-230-BChE conjugate. Furthermore, oxime 3 was not toxic to neural SH-SY5Y and hepatic HepG2 cells in concentrations relevant for biological activity. These findings highlight oxime 3 as well as the N,N'-dibenzyl imidazolium scaffold as the most potent BChE reactivators reported to date, providing a critical foundation for the advancement of bioscavenging-based therapies for OP poisoning.

Background and purpose:The toxicity of organophosphorus compounds (OPs) and related nerve agents (NAs) impairs the cholinergic system via irreversible inhibition of acetylcholinesterase (AChE) activity by phosphylation of the catalytic serine. Reactivation of the enzyme activity largely depends on the structural compatibility between the enzyme, an oxime reactivator, and the specific OP compound. Experimental approach:For this study, we used our recently published data on the reactivation of human butyrylcholinesterase inhibited by the NAs sarin, cyclosarin, tabun and VX, using a library of 115 oximes. We compared these results with oximes' ADME (absorption, distribution, metabolism, and excretion) parameters relevant to central nervous system activity using principal component analysis (PCA). PCA facilitated the examination of these relatively large datasets by increasing interpretability while minimizing information loss. Key results:Three components with eigenvalues above 1 resulted in 72 % of the cumulative proportion of variance and described 27 variables. PC1 created transformed data that had negative values for most oximes with high reactivation potential, while showing large positive values for oximes with moderate and low efficacy. Distribution of 27 loadings, representing 27 variables, produced a set of 9 positive and 18 negative loadings representing negative and positive data correlation. The efficacy of oxime reactivation was highly correlated with the parameters describing its structure: molecular weight, rotational bonds, molecular volume, and molecular surface area. Conclusion:A large dataset was efficiently analysed by maximizing the preservation of variability and generating new, uncorrelated variables. To our knowledge, this study is the first to apply PCA to assess oxime's reactivation efficacy, thus providing insights into the relationships between oxime properties and their efficacy in restoring OP-inhibited cholinesterase activity.

Butyrylcholinesterase (BChE) plays a key role in cholinergic transmission and the metabolism of various drugs, making its regulation a promising therapeutic strategy for several diseases, including Alzheimer’s disease. Selective inhibition of BChE helps regulate brain acetylcholine levels. However, genetic polymorphisms in the BCHE gene, particularly the Asp70Gly mutation in atypical BChE, can impact treatment outcomes. This study compares the inhibitory potency of 13 carbamates against atypical and usual BChE. Using molecular docking, quantum chemical cluster calculations, and crystallization of wild-type BChE with the most potent carbamate, we identified key differences in carbamylation mechanisms. Atypical BChE shows a less favorable enzyme-inhibitor complex orientation, lacking the hydrogen bond stabilization of the reactive carbonyl oxygen. Additionally, Asp70 in usual BChE contributes to stabilizing the non-reactive carbamate group, whereas Gly70 in atypical BChE is too distant to form such interactions.

Current oxime antidotes exhibit poor blood-brain barrier (BBB) penetration due to their permanent positive charge, limiting the reactivation of organophosphorus (OP)-inhibited cholinesterases in the central nervous system. To address this limitation, the present study investigates nine lipophilic cholesterol- and quinoline-based oximes as potential reactivators of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) inhibited by OP compounds. Several oximes exhibited strong reversible inhibition with pronounced potency toward hBChE (Ki as low as 4.6 nM). However, despite the favourable binding affinities, reactivation assays using sarin- and cyclosarin-inhibited enzymes revealed negligible recovery of enzymatic activity compared with the standard antidote pralidoxime (2-PAM). Molecular modelling of near-attack conformations showed that the oxime group of the most potent inhibitor adopted an unproductive orientation relative to the catalytic serine. These results highlighted that binding affinity alone is insufficient for effective reactivation; precise positioning of the oxime moiety to enable nucleophilic access to the phosphorus centre is critical. Therefore, a strategic framework for designing next-generation oxime reactivators based on such structures is needed to improve functional efficacy. Moreover, the moderate cytotoxicity of nitronesteroids in hepatocarcinoma (HepG2) and neuroblastoma (SH-SY5Y) cell lines warrants further studies to assess their implications for the compounds' therapeutic potential and safety profile.

A library of 100 click-chemistry-derived oximes was evaluated as reactivators of butyrylcholinesterase (BChE) inhibited by the nerve agents (NAs) sarin, cyclosarin, VX, and tabun. While reactivation efficiency was highly dependent on the structure of both the NA and the oxime, for each NA-BChE conjugate, we identified reactivators more effective than currently approved oximes for NA poisoning. Detailed kinetic analysis indicated that this enhancement results from both improved molecular recognition—specifically, enhanced binding affinity of the phosphylated conjugates for the oximes—and increased maximal reactivation rates. Molecular modeling of oximes in a near-attack conformation within inhibited BChE revealed critical interactions for productive reactivation. Among all tested oximes, 5B [1-hexyl-2-((hydroxyimino)methyl)pyridinium chloride] emerged as a particularly efficient reactivator for BChE phosphorylated with cyclosarin, with the highest observed overall reactivation rate of 34,120 M −1 min −1 , which is 525-fold and 44-fold higher than the reference oximes 2-PAM and HI-6, respectively. In general, three mono-pyridinium mono-oximes demonstrated more efficient recovery of BChE activity than bis-pyridinium triazole-annulated click-chemistry bis-oximes, which were previously identified as potent reactivators for inhibited acetylcholinesterase (AChE). Ex vivo assessment of reactivation potency demonstrated that the combined addition of BChE with one efficient reactivator for BChE and another for AChE achieved > 90% reactivation of cyclosarin-inhibited cholinesterases in whole blood (WB), demonstrating near-complete degradation of a 100-fold excess of cyclosarin within 6 min. These results confirm that oxime-assisted catalysis is feasible for NA bioscavenging in blood and underscore BChE’s potential as a target for developing therapies against NA poisoning.

There is evidence that the expression of acetylcholinesterase (AChE) is associated with the development of certain cancers. However, there are still insufficient studies to reveal the correlation between AChE expression and cancer development and prognosis from the perspective of pan-cancer. In this study, a comprehensive bioinformatics analysis of the pan-cancer expression signature of AChE was conducted through a data-driven approach. Pan-cancer datasets were obtained through online databases, including the Cancer Genome Atlas and Genotypic Tissue Expression databases. The correlation between ACHE expression and cancer prognosis was predicted by survival analysis. The results showed differences in the expression levels of the ACHE gene in cancer and paired normal tissues. However, the alterations in cancer tissue ACHE expression were different depending on the cancer type, e.g., higher ACHE expression was found in pancreatic adenocarcinoma, and lower ACHE expression was found in glioblastoma multiforme (GBM). In addition, ACHE expression was found to have dual prognostic significance, e.g., cutaneous melanoma with higher levels of ACHE expression had better overall survival, while the opposite outcome was found in uveal melanoma. The function of ACHE-expressing cells in primary and metastatic tumors was predicted using single-cell RNA sequencing datasets from patients with GBM. Differences in cell type preferences for ACHE expression were found in primary and metastatic GBM, and different functional enrichment features were identified, suggesting a potential role of ACHE expression in GBM progression. Overall, our study highlights the potential of AChE as a target for cancer research.

Toxic organophosphates like the nerve agent sarin readily cross the blood-brain barrier (BBB) and inhibit acetylcholinesterase (AChE), a pivotal enzyme in regulating neurotransmission by hydrolysis of acetylcholine (ACh). Elevated levels and prolonged residence time of ACh initiate seizures and activation of glial cells leading to neuroinflammation. Furthermore, AChE inhibition induces life-threatening symptoms if not treated promptly with atropine and an oxime reactivator of inhibited AChE. The oximes approved for therapy (e.g. 2-PAM) poorly cross the BBB due to their permanent positive charge and do not restore synaptic AChE activity, leaving the brain vulnerable to long-term damage. In this study, we investigated whether treatment with the centrally-active oxime RS194B acts protectively on the brain of mice exposed to sarin. We compared the levels of specific proteins expressed in neuronal and glial cells of mice treated with RS194B after sarin exposure with those of sarin-exposed mice, mice treated with 2-PAM, and untreated control mice. The level of Iba-1 protein was investigated as a measure of microgliosis, and GFAP of astrogliosis, whereas neuronal cell viability was assessed by detecting NeuN immunoreactivity. Our results indicated that sarin-induced gliosis was suppressed in mice treated with RS194B, in contrast to mice treated with 2-PAM. Treatment with RS194B re-established the physiological function of AChE, thus correcting the neurochemical imbalance of ACh that initiates seizures and leads to neuroinflammation. Overall, our results highlight the significance of restoring synaptic AChE activity extending beyond merely mitigating cholinergic crisis.

The development of small-molecule ligands targeting cholinesterases remains a central focus in neuropharmacology, particularly for the treatment of neurodegenerative disorders and organophosphate poisoning. This review highlights the rational design, synthesis, and biological profiling of diverse classes of heterocyclic compounds - including oxazoles, heterostilbenes, triazoles, and bicyclo[3.2.1]octane/octadiene derivatives - as reversible inhibitors and reactivators of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Novel amino-oxazolostilbenes and their photoproducts exhibited selective BChE inhibition, while naphtoxazole and triazole-containing scaffolds demonstrated promising dual-target or BChE-selective profiles. Several uncharged oximes, such as thienostilbene and heterostilbene oximes, showed potential for reactivating cyclosarin-inhibited BChE, supporting their further development as CNS-permeable antidotes. Additionally, resveratrol-based triazoles and carbamates revealed enhanced BChE inhibition, antioxidant activity, and favorable selectivity. Collectively, these findings underscore the therapeutic potential of structurally diverse cholinesterase ligands and provide a framework for the discovery of multifunctional agents for Alzheimer's disease and chemical threat countermeasures.

Given the scarcity of experimental studies on A-series nerve agents (NAs), this paper provides ground-breaking insights and, for the first time, describes the inhibition and reactivation of human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibited by these NAs using standard oximes. Furthermore, we present a detailed assessment of the toxicity profile of A-series NAs, based on both in vitro and in vivo studies. Our findings demonstrate that A-230, A-232, and A-234 are the most potent inhibitors of AChE and BChE among the A-series, with inhibitory potency comparable to the G-series NAs cyclosarin and soman. A-242 and A-262 inhibited both enzymes with potency similar to that of tabun and VX. Reactivation assays identified HI-6 oxime as the most effective reactivator in mitigating A-230-AChE conjugate-induced toxicity, achieving complete restoration of AChE activity within 4 h. Obidoxime and TMB-4 showed moderate reactivation capacity over 24 h, while 2-PAM was ineffective, indicating limited reactivation potential for counteracting A-230-inhibited AChE. HI-6 also achieved partial reactivation of AChE inhibited by A-232 and A-234. A-242 and A-262-AChE conjugates exhibited minimal susceptibility to oxime reactivation. These results confirm that both inhibition and reactivation are finely tuned processes, dependent on the structural characteristics of all reactants. In other words, while standard oximes show reasonable potency in reactivating AChE inhibited by agents like sarin and VX, they lack universal efficacy across different NA-AChE conjugates. The reactivation of BChE inhibited by A-agents was negligible when using standard oximes. Additionally, we modelled a near-attack conformation of HI-6 within AChE phosphylated by A-230, providing insights into the structural requirements necessary for more effective reactivation. Beyond enzyme studies, we also assessed hepatotoxicity and neurotoxicity in vitro, and evaluated the acute subcutaneous toxicity of A-series agents in mice. The toxicity of A-230, A-232, and A-234 was comparable to VX, while A-242 and A-262 were similar in toxicity to sarin. These findings significantly advance our understanding of the toxicological properties of phosphoramidates and underscore the urgent need to develop more effective medical countermeasures against A-agents exposure.

Environmental contaminants, such as pesticides, can inhibit the enzymatic activity of acetylcholinesterase (AChE), an enzyme necessary for neurotransmission. The inhibitory effects of structurally diverse pesticides on AChE may result from either reversible or covalent interactions. Therefore, assessing their potency typically requires different assay design to determine either dissociation constants or rate constants, respectively. To avoid complex kinetic experiments and enable comparison of potencies across structurally diverse pesticides, we optimized an AChE inhibitor detection system using an endpoint Ellman assay in 96-well plates. Given the significant interspecies variability in sensitivity to inhibitors, we investigated AChE inhibition using both electric eel AChE (eeAChE) and human AChE (hAChE). After confirming the repeatability, reproducibility, and solvent compatibility of the detection system using the reversible inhibitor BW284c51, we determined the inhibition potency of selected organophosphorus (OP) pesticides based on IC50 values. We found that chlorpyrifos, fenamiphos and ethoprophos were more potent inhibitors of hAChE than eeAChE. In contrast, phosalone and methamidophos showed similar inhibitory effects on both enzymes. The potencies aligned well with previously reported inhibitory rate constants. In conclusion, since OPs acts as progressive inhibitors of AChE, our optimized assay offers a simplified yet effective method for assessing their inhibitory potency. It also allows for comparative evaluation of various environmental pollutants based solely on IC50 values, eliminating the need for complex kinetic studies.

In both non-human primate and rodent models, the RS194B oxime is currently the most efficacious single administration post-exposure treatment against highly toxic organophosphate agents; rapidly reversing severe symptoms within 1-2 h and preventing death. This exceptional protective efficacy, which results from its ability to rapidly cross the BBB and remove the conjugated OP moiety from the active serine of OP-inhibited-AChE and BChE, allows for studies using severely OP-intoxicated macaques. We have compared here the reactivation of RBC-AChE and soluble BChE in the circulation to determine the relevance of each enzyme in survival; as an important aid in further oxime development. The results indicate that RS194B oxime administration to severely intoxicated macaques following exposure to inhaled sarin and paraoxon, and orally to diethyl-phosphorothioate insecticides, chlorpyrifos and parathion, results in very rapid AChE reactivation (>60 % in 1 h), sufficient by itself for protection, while the observed lower and slower increases in BChE activity play an insignificant role. Unexpectedly, increases in BChE activity appear to be biphasic in treated macaques, comprising an early oxime-dependent increase followed by a later increase observed after oxime has been eliminated from the blood; the latter increase also being observed in sarin-exposed and untreated animals. This previously unreported oxime-independent BChE recovery was observed in all OP-exposed macaques and is compatible with release/secretion of native BChE from stores in the liver as a result of OP-mediated damage; such increases being too fast to represent water-mediated reactivation or biosynthesis.

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.

Glyphosate has remained the leading herbicide on the global market to date, despite the continuous debate between consumers, scientific community, and regulatory agencies over its carcinogenicity, genotoxicity, environmental persistence, and the role in the development of neurodegenerative disorders. Chemically, glyphosate belongs to a large family of organophosphorus pesticides, which exert a neurotoxic effect by inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), enzymes of the cholinergic system essential for maintaining neurotransmission. Although research shows that glyphosate is a weak cholinesterase inhibitor in fish and mammals compared to other OP compounds, no conclusive data exist concerning the inhibition of human AChE and BChE. In our study we analysed its inhibitory potency on human AChE and BChE, by establishing its IC50 and reversible inhibition in terms of dissociation inhibition constants. Glyphosate concentration of 40 mmol/L caused near total inhibition of enzyme activity (approx. 10 % activity remaining). Inhibition dissociation constants (K i) of glyphosate-AChE and -BChE complexes were 28.4±2.7 mmol/L and 19.3±1.8 mmol/L, respectively. In conclusion, glyphosate shows a slight binding preference for BChE but exhibits inhibition only in a high concentration range. Our results are in line with studies reporting that its neurotoxic effect is not primarily linked to the cholinergic system.

Olesoxime, a cholesterol derivative with an oxime group, possesses the ability to cross the blood–brain barrier, and has demonstrated excellent safety and tolerability properties in clinical research. These characteristics indicate it may serve as a centrally active ligand of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), whose disruption of activity with organophosphate compounds (OP) leads to uncontrolled excitation and potentially life-threatening symptoms. To evaluate olesoxime as a binding ligand and reactivator of human AChE and BChE, we conducted in vitro kinetic studies with the active metabolite of insecticide parathion, paraoxon, and the warfare nerve agents sarin, cyclosarin, tabun, and VX. Our results showed that both enzymes possessed a binding affinity for olesoxime in the mid-micromolar range, higher than the antidotes in use (i.e., 2-PAM, HI-6, etc.). While olesoxime showed a weak ability to reactivate AChE, cyclosarin-inhibited BChE was reactivated with an overall reactivation rate constant comparable to that of standard oxime HI-6. Moreover, in combination with the oxime 2-PAM, the reactivation maximum increased by 10–30% for cyclosarin- and sarin-inhibited BChE. Molecular modeling revealed productive interactions between olesoxime and BChE, highlighting olesoxime as a potentially BChE-targeted therapy. Moreover, it might be added to OP poisoning treatment to increase the efficacy of BChE reactivation, and its cholesterol scaffold could provide a basis for the development of novel oxime antidotes.