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Background
Based on in vitro and in vivo rat experiments, the newly developed acetylcholinesterase (AChE) reactivator, K203, appears to be much more effective in the treatment of tabun poisonings than currently fielded oximes. Methods
To determine if this reactivating efficacy would extend to humans, studies were conducted in vitro using human brai...
Context in source publication
Context 1
... K203 ((E)-1-(4-carbamoylpyridinium)-4-(4-hydroxyimino- methylpyridinium)-but-2-ene dibromide) was prepared at the Toxicology Department according to the published method [26]. The structure of K203 and structure of ox- imes tested for comparison (pralidoxime, obidoxime, asox- ime (HI-6)) are shown in Fig. 3. Purity of all tested reactivators was measured using TLC (DC-Alufolien Cellu- lose F; Merck, Germany; mobile phase BuOH-CH 3 COOH- H 2 O 5-1-2; detection by solution of Dragendorff reagent), HPLC (P200 gradient pump Spectra-Physics Analytical, Fremont, USA; a 7125 injection valve -10 ul loop, Rheo- dyne, Cotati, USA; an UV1000 ...
Citations
... There are reports on the analysis of these compounds using theoretical and spectroscopic methods [9,10,13,15,16], and scarce in vitro research [17], yet there is, to the best of our knowledge, no information about any extensive in silico examinations of Novichoks' mechanism of action and their potential reactivators. In reactivating the enzyme from OPNAs, several oxime-based compounds [18][19][20][21][22] as well as several engineered OPNA-degrading enzymes [14] are effective, and purely theoretical studies were conducted (e.g., [23,24]). Regarding Novichoks however, there is a lack of both QM and molecular mechanics (MM) investigations of Novichok-AChE interactions, including the noncovalent ones; yet, obviously, the noncovalent interplay between the ligand and the enzyme contributes to and facilitates the irreversible phosphonylation of the AChE catalytic-triad serine e.g., by adjusting the ligand electron density distribution therefore stabilizing the ligand in the enzyme active centre gorge and making it thus more susceptible to nucleophilic enzyme attack. ...
In silico studies were performed to assess the binding affinity of selected organophosphorus compounds toward the acetylcholinesterase enzyme (AChE). Quantum mechanical calculations, molecular docking, and molecular dynamics (MD) with molecular mechanics Generalized–Born surface area (MM/GBSA) were applied to assess quantitatively differences between the binding energies of acetylcholine (ACh; the natural agonist of AChE) and neurotoxic, synthetic correlatives (so-called “Novichoks”, and selected compounds from the G- and V-series). Several additional quantitative descriptors like root-mean-square fluctuation (RMSF) and the solvent accessible surface area (SASA) were briefly discussed to give—to the best of our knowledge—the first quantitative in silico description of AChE—Novichok non-covalent binding process and thus facilitate the search for an efficient and effective treatment for Novichok intoxication and in a broader sense—intoxication with other warfare nerve agents as well.
... In a different study, 50 different forms of the reactivator compound were tested, and the one that interacted best with the AChE active site was chosen. According to the study's findings, both reactivators exhibit consistent action on several AChE species when tested; the findings were confirmed using DFT calculations [36,37]. ...
Acetylcholinesterase (AChE) is a cholinergic enzyme that plays an essential role in the autonomic nervous system. This enzyme is often the target of many nerve agents. When this enzyme is inhibited, its function to hydrolyze acetylcholine is stopped, accumulating the acetylcholine in the tissue and causing prolonged stimulation. Some of the significant nerve agents include sarin (GB), soman (GD), tabun (GA), and venomous agent (VX). In order to determine which compound is the most stable and has the best affinity, the nerve agent venomous agent (VX), sarin (GB), soman (GD), and tabun (GA) are docked to the acetylcholinesterase (AChE) enzyme. After that, toxicity tests will be performed on 17 targets for the compound that was chosen. Autodock Vina 1.2.0 is the software used for the docking procedure. should use the Pymol program version 2.5.4 for analysis and the Ligplot software version 2.2 for visualization of the docking findings. The 'Tox Prediction' algorithm from Insilico was used to determine the toxicity of various substances. Based on the results of molecular docking, the free binding energy of Donepezil, sarin (GB), soman (GD), tabun (GA), and venomous agent (VX) in kcal/mol are -12,3, -4.8, -6.0, -5,1, and -6.3 respectively. Finally, four ligands bind strongly to the receptor Donepezil at RMSD 0.327 Å, and the venomous agent (VX) compound binds the most strongly compared to the other test ligands. Furthermore, in the toxicity test of Compound VX, which exhibits neurotoxic activity, no toxic activity was observed on specific organs and targets.
... Three-dimensional and two-dimensional visuals were obtained using BIOVIA Discovery Studio Visualizer software to clearly show the location of UA in the target pocket of the enzyme and to highlight the types of these interactions (Almaz et al., 2021;Biovia, 2019). MolDock scoring in MVD software results from the piecewise linear potential (PLP), a reduced potential whose settings are regulated by enzyme-ligand complexes (da Cunha et al., 2008;Kuca et al., 2018). The docking scoring values, E score , are determined by Equation 2: ...
... It consists of a Coulomb potential presenting a dielectric constant in the range (D(r) ¼ 4r). Considering that the electrostatic energy unit can be provided in kilocalories per molecule, the numerical amount of 332.0 is significant (Equation 3) (Kuca et al., 2018;Silva et al., 2016). E intra relates to the internal energy of the ligand: ...
... If several torsions can be resolved, each torsional energy is significant and an associated average is used. The last measure, Eclash, imposes a penalty equal to 1,000 if the distances between heavy atoms are less than 2.0 Å, ignoring irrelevant ligand conformations (Equation 4) (Kuca et al., 2018;Silva et al., 2016). The discovery algorithm in MVD combines a differential evolution optimization method with a gap estimation algorithm throughout the search process, allowing potential binding modes (poses) to be identified quickly and precisely (Borman, 2004;da Cunha et al., 2010;Kuca et al., 2018;Silva et al., 2016). ...
Ursolic acid (UA), which has many biological properties such as anti-cancer, anti-inflammatory and antioxidant, and regulates some pharmacological processes, has been isolated from the flowers, leaves, berries and fruits of many plant species. In this work, UA was purified from the methanol-chloroform crude extract of Nepeta species (N. aristata, N. baytopii, N. italica, N. trachonitica, N. stenantha) using a silica gel column with chloroform or ethyl acetate solvents via bioactivity-guided isolation. The most active sub-fractions were determined under bioactivities using antioxidant and DNA protection activities and enzyme inhibitions. UA was purified from these fractions and its structure was elucidated by NMR spectroscopy techniques. The highest amount of UA was found in N. stenantha (8.53 mg UA/g), while the lowest amount of UA was found in N. trachonitica (1.92 mg UA/g). The bioactivities of UA were evaluated with antioxidant and DNA protection activities, enzyme inhibitions, kinetics and interactions. The inhibition values (IC50) of α-amylase, α-glucosidase, urease, CA, tyrosinase, lipase, AChE, and BChE were determined between 5.08 and 181.96 µM. In contrast, Ki values of enzyme inhibition kinetics were observed between 0.04 and 0.20 mM. In addition, Ki values of these enzymes for enzyme-UA interactions were calculated as 0.38, 0.86, 0.45, 1.01, 0.23, 0.41, 0.01 and 2.24 µM, respectively. It is supported that UA can be widely used as a good antioxidant against oxidative damage, an effective DNA protector against genetic diseases, and a suitable inhibitor for metabolizing enzymes.Communicated by Ramaswamy H. Sarma.
... Therefore, the development of novel oxime reactivators is still ongoing process (Santos et al., 2022). Several notable experimental bisquaternary pyridinium-based oximes involve K027 (Kuča et al., 2003) which acts against a broad range of pesticides (Lorke and Petroianu, 2019) or K203 (Musilek et al., 2007) with a promising reactivating activity against tabun-inhibited AChE (Kuca et al., 2018). Č adež et al. in 2021 evaluated other remarkable oxime-based reactivators with potential for further development of antidotes against OP pesticide poisoning (Čadež et al., 2021) involving bispyridinium triazole oxime 14 A (Kovarik et al., 2019) and zwitterionic oxime RS194B (Somin and Kuznetsov, 1968). ...
This article reviews available data regarding the possible association of organophosphorus (OP) pesticides with neurological disorders such as dementia, attention deficit hyperactivity disorder, neurodevelopment, autism, cognitive development, Parkinson's disease and chronic organophosphate-induced neuropsychiatric disorder. These effects mainly develop after repeated (chronic) human exposure to low doses of OP. In addition, three well defined neurotoxic effects in humans caused by single doses of OP compounds are discussed. Those effects are the cholinergic syndrome, the intermediate syndrome and organophosphate-induced delayed polyneuropathy. Usually, the poisoning can be avoided by an improved administrative control, limited access to OP pesticides, efficient measures of personal protection and education of OP pesticide applicators and medical staff.
... In this context, theoretical approach, such as molecular docking and MM/GBSA calculations were employed for investigating the molecular interactions between the ligands and enzymes. Molecular docking methods are quick and efficient tools to identify the binding of ligand molecules with the protein [15,24]. To achieve this, the 5-LOX inhibitory potential of 23 phytocompounds was assessed by performing docking of compounds at the active site of 5-LOX. ...
Inflammation is caused by a cascade of events, one of which is the metabolism of arachidonic acid, that begins with oxidation by the enzyme 5-lipoxygenase. 5-Lipoxygenase (5-LOX) plays an important role in the inflammation process by synthesizing leukotrienes and several lipid mediators and has emerged as a possible therapeutic target for treatment of inflammatory diseases such as asthma and rheumatoid arthritis. Most of the existing 5-LOX inhibitors are synthetic and exhibit adverse side effects. In view of this, there is need to search for an alternate source of 5-LOX inhibitor with minimal side effects. The essential oil of several species of Curcuma has received considerable attention in recent times in traditional system of medicine especially for treating various inflammatory disorders. Therefore, the present study was carried out to screen the most potential 5-LOX inhibitors from essential oil components of Curcuma species and elucidate their mechanisms of action through computational biology approaches. Twenty-three phytoconstituents derived from the essential oil of Curcuma species were docked and their predictive binding energies were calculated to select the best possible ligand for 5-LOX. The top 8 ranked compounds from docking was tested for drug-likeness properties, bioactivity score, and toxicity analysis. The phytoconstituents such as α -turmerone, β -turmerone, α -terpineol and dihydrocarveolshowed the best binding affinity with 5-LOX and displayed favorable physicochemical properties. Molecular dynamics simulation in POPC lipid bilayers was carried out to understand the intrinsic dynamics and flexibility of the 5-LOX (apo) and 5-LOX-complex ( α -terpineol, α -turmerone, β -turmerone and dihydrocarveol) systems. The molecular dynamic results showed that these 4 phytoconstituents interacted stably with the 5-LOX active site residues and the important bonds that were observed in the initial ligand docked compounds did not alter during the course of simulation. In general, our integrative computational approach demonstrated that the natural compounds like α -turmerone, β -turmerone, α -terpineol, and dihydrocarveol could be considered for designing specific anti-inflammatory drugs using structure-based drug design.
... On the other hand, this leads to the fact that, e.g., practically used insecticides turn out to be toxic to a human being [17]. In addition, the ability of compounds to reactivate inhibited AChE was found to depend on a organism [18][19][20][21]. This brings about the need for further structural studies of AChE using other methods. ...
Acetylcholinesterase (AChE) is the object of many studies due to the fact that it plays an important role in the vital activity of organisms. In particular, when new AChE inhibitors are developed, much attention is paid to the specificity of their action. One of the approaches used to study the specificity is to compare AChE taken from various organisms. In this work, crystallographic data are used to investigate the active sites of AChE (ASAs) in the free (uncomplexed) state for the following five organisms: Homo sapiens (HS), Mus musculus (MM), Torpedo californica (TC), Electrophorus electricus (EE), and Drosophila melanogaster (DM). The structural fractal analysis (SFA) proposed by us earlier is used as a research method. This method is based on the calculation and comparison of the fractal dimensions of molecular structures. SFA demonstrates that there are no significant structural differences between the active sites of human AChE and other AChEs. However, differences are found for the MM/EE pair. Further analysis of individual AARs has revealed two different areas of active sites. Ser203, Trp236, Phe338, and Tyr341 are found to belong to a variable region, and the remaining AARs belong to a conservative region of the ASAs. The fraction of "variability" is low, 0.8%.
... For ii) tabun, K870 was the only reactivator that restored ChE activity in the blood of tabun-poisoned animals. Reactivation of tabun-inhibited ChE activity was previously found to be troublesome Kuca et al., 2018), as evidenced by many studies aimed at finding an anti-tabun oxime (Calic et al., 2006;Kovarik et al., 2009;Katalinic et al., 2015). K870 reactivated tabun-inhibited ChE more efficiently than most previously developed K-oximes, including K361, K378, K454, K456, K458, K733, K920, and K923, but it failed to surpass the unchlorinated oxime K203 . ...
Oxime reactivators are causal antidotes for organophosphate intoxication. Herein, the toxicity, pharmacokinetics, and reactivation effectiveness of o-chlorinated bispyridinium oxime K870 are reported. Oxime K870 was found to have a safe profile at a dose of 30 mg/kg in rats. It exhibited rapid absorption and renal clearance similar to those of other charged oximes after intramuscular administration. Its isoxazole-pyridinium degradation product was identified in vivo. Although it showed some improvement in brain targeting, it was nevertheless rapidly effluxed from the central nervous system. Its reactivation effectiveness was evaluated in rats and mice intoxicated with sarin, tabun, VX, and paraoxon and compared with pralidoxime and asoxime. K870 was found to be less effective in reversing tabun poisoning compared to its parent unchlorinated oxime K203. However, K870 efficiently reactivated blood acetylcholinesterase for all tested organophosphates in rats. In addition, K870 significantly protected against intoxication by all tested organophosphates in mice. For these reasons, oxime K870 seems to have a broader reactivation spectrum against multiple organophosphates. It seems important to properly modulate the oximate forming properties (pKa) to obtain more versatile oxime reactivators.
... For tabun-AChE reactivation, oximes with but-2-ene linker (7, 7a, 7c) achieved the most productive positioning and orientation within the gorge of tabun-AChE [26]. This is in very good agreement with previous studies for oxime K203 (7) [4,5,[28][29][30]. Looking on the effect of substituents, two 3,5-positioned chlorine atoms at pyridinium ring with oxime group favoured the reactivation in the case of all tested OPs, except tabun, in which case the second chloro-moiety seems to somewhat reduce the reactivation ability. ...
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.
... Similarly, in a study by Kuca et al. (2018), the effect of K203, a new generation K oxime, on human cadaver brain tissue homogenates in which the AChE enzyme was inactivated by tabun was investigated. In this study, the AChE enzyme reactivation effect of pyridinium oximes, pralidoxime, obidoxime, and asoxime, which are still used in the treatment of OP poisoning, was compared with the effect of K203. ...
... I.e. K027 (5), K048 (6) and K203 (7) (Figure 2) have been presented to be potent reactivators of OP-inhibited AChE in vitro or in vivo [18][19][20][21] . ...
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.
