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Amanita muscaria, the red fly agaric, is the most famous of all Amanita. The initial history of this fascinating mushroom dates back to at least the 13^th century. The use of mushrooms began in antiquity and is associated with mysticism. The collection and consumption of mushrooms and other plants containing psychoactive substances is now very popular, especially among young people who are experimenting with drugs. Ibotenic acid and muscimol are the main active ingredients of this mushroom, but other substances are likely to be involved in the psychotropic effects. A. muscaria also contains some other non-psychotropic substances that are interesting not only for their chemical structure but also for their biological activity. Current knowledge about chemistry, pharmacology and toxicology regarding this fungus is reviewed in this article.

Brazilian pepper tree (Schinus terebinthifolius Raddi) from the Anacardiaceae family is native to Central and South America. In folk medicine, S. terebinthifolius has been used as a remedy for ulcers, respiratory problems, wounds, rheumatism, gout, diarrhea, skin ailments and arthritis, as well as to treat tumors and leprosy. The Brazilian pepper tree has various biological properties and it is a source of many bioactive compounds. Several classes of compounds can be found in extracts obtained from these plants, such as terpenes and flavonoids. Essential oils obtained by steam distillation or extraction by solvents from berries or leafs is rich in monoterpenes and shows interesting antioxidant activity. It has displayed good-to-very strong in vitro antifungal actions against numerous fungi, as well as Candida. The essential oil and leaves have demonstrated in vitro antibacterial activity against numerous bacterial strains.

The state of the lipid interface is known to influence activity of membrane-bound enzymes. Indeed, many enzymes exhibit changes in activity at phase transitions in the membrane to which they are attached. We utilized a Langmuir trough in which detergent-soluble Torpedo californica acetylcholinesterase (DS-TcAChE)¹ was anchored to the solvent face of a phospholipid monolayer in order to study this phenomenon. A peak in activity was observed at the compressibility maximum accompanying the transition between the ordered and fluid phases. Neither molecular nor physical alterations affected this correlation qualitatively, as shown by varying lipid type, pH over 2 units, temperature over 20°C, and lateral pressure over 10 mN/m. Thus the only consistent correlation is between the thermodynamic state of the interface and the measured activity. Our data are consistent with a theory in which the interface state and its corresponding fluctuations control catalytic activity². It was earlier demonstrated that pH-pulses initiated by local acidification of the monolayer propagate, in analogy to sound, at velocities up to 1.4 m/s³. We have now shown that such a pulse, by transiently modifying compressibility, can concomitantly and reversibly enhance the activity of DS-TcAChE attached to the monolayer. Our data demonstrate a feasible mechanism for signaling between widely separated biological entities that differs fundamentally from the molecular mechanisms currently accepted, and is also very much faster.

The Glu197 of butyrylcholinesterase (BChE) has been long considered as deprotonated in various studies, e.g. discovering the dynamical characters, interpreting the binding properties of inhibitors, and proposing hypotheses for BChE-catalyzed reaction mechanism. By performing a series of 100 ns molecular dynamics simulations, we accidently discovered that Glu197 needed to be protonated to have the structures simulated appropriately, whereas the deprotonated Glu197 eventually caused the collapse of catalytic triad with long enough simulation time.[1] we found that a highly conserved water molecule required Glu197 to be protonated in order to form an important hydrogen bond network, which supported His438 to be preserved within the catalytic triad. Interestingly, catalytic triad and Glu197 have been long recognized for possibly deviating largely from their crystal structure positions, which could be catalytic deficient and is generally considered as the result from difference between crystal and aqueous environment. Here, our results suggest that the large deviations of catalytic triad and Glu197 from crystal structure are caused by inappropriate protonation state of Glu197. This finding of the unexpected protonation state of Glu197 shall provide an important clue that has been long missing for the better understanding of BChE related puzzles or even reconsideration of some BChE-catalyzed reaction mechanisms.

To investigate how protein dynamics facilitates substrate entering and product exiting the phosphotriesterase active site, over 60 distinct, independent, unrestricted, unbiased, isobaric–isothermal, microsecond molecular dynamics simulations of zinc-containing phosphotriesterase in complex with a substrate analog¹ were performed using the second-generation cationic dummy atom model for the zinc divalent cation, forcefield FF12MC², and PMEMD of AMBER 16 with a periodic boundary condition at 1 atm and 277 K, 300 K, and 340 K. In-depth conformational analysis of these simulations with an aggregated simulation time of over 76 microseconds revealed atomic and dynamic details on the phosphtriesterase catalysis and its requirement of two cations, which offers insight into re-engineering of phosphotriesterase to develop an improved scavenger against phosphorous-containing inhibitors of acetylcholinesterase.

Tabun represents a phosphoramide class of organophosphosphates that are covalent inhibitors of acetylcholinesterase (AChE), an essential enzyme in neurotransmission. The currently used therapy in excessive cholinergic stimulation consists of the muscarinic antagonist of acetylcholine stimulation, an anti-seizure drug when indicated and an oxime as the reactivator of inhibited AChE. Since common oximes are particularly ineffective in tabun exposure, we probed the reactivation of phosphoramidate conjugates in more depth by using mutants of AChE and pyridinium oximes to reveal the structural subtleties and yield more information on the architecture of the active centre gorge needed for the reactivation of phosphoramidate agents used in terrorism and as pesticides. Our results indicated that the replacement of aromatic residues with aliphatic ones at the acyl pocket and choline binding site mostly interfered with the stabilization of the oxime’s pyridinium ring(s) in the proper orientation of the oxime group toward the phosphorylated active site serine. The peripheral binding site mutation resulted in a 2-5 fold increase in the reactivation rates by bis-pyridinium oximes when compared to the AChE wild type. In the case of mono-pyridinium oximes, we reported a 150-fold enhancement of the maximal reactivation rate for the choline binding site mutation, while the molecular recognition seemed to remain preserved. Therefore, our results emphasized the positive effect of several mutations on oxime embedding and orientation into a position for productive interactions with the tabun-phosphorylated active site serine indicating a future potential for further development of pseudo-catalytic bioscavengers based on AChE mutants.

Background: Memantine is the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, used in the treatment of Alzheimer’s disease. Memantine pretreatment assured protection of skeletal muscles from poisoning with nerve agents and an interaction between memantine and AChE was proposed [1].  Aim: Memantine and its main metabolite (1-amino-3-hydroxymethyl-5-methyl adamantine, Mrz 2/373) were used to ascertain their interaction with erythrocyte acetylcholinesterase (AChE) in vitro. The effect of these two compounds on the kinetics of the soman-induced AChE inhibition and on the aging of the soman-AChE complex was also investigated.  Methods: Bovine AChE activity was measured titrimetrically and the effect on aging of the soman-AChE complex was studied [2].  Results: Memantine and Mrz 2/373 exerted concentration-dependent inhibition of AChE, with Mrz 2/373 being a more potent inhibitor than the parent compound. Addition of soman 2.5x10^-8 mol/l induced gradual AChE inhibition that became almost 100% after 20 min. Memantine (0.1, 0.5 and 1 mmol/l) and Mrz 2/373 (0.1 and 1 mmol/l) concentration-dependently slowed down the AChE inhibition. Neither memantine nor Mrz 2/373 prevented the aging of the soman-AChE complex. After 5 min incubation with AChE and soman, AChE activity was 11%, 36% and 30% in control medium and after adding of 1 mmol/l of memantine and Mrz 2/373, respectively. Conclusion: Since high micromolar and low millimolar concentrations of memantine can be achieved in rats [3], it is quite possible that memantine and Mrz 2/373 can prevent AChE from inhibition by soman, which could, along with known memantine’s neuroprotective activity, explain its potent antidotal effect in soman poisoning.

Chronic illness from exposure to organophosphorus toxicants is hypothesized to involve modification of unknown proteins.  Tyrosine readily reacts with organophosphorus toxicants in proteins that have no active site serine.  We developed a monoclonal antibody, depY, that specifically recognizes diethoxyphospho-tyrosine in proteins and peptides, independent of the surrounding amino acid sequence ¹. Our goal was to identify diethoxyphosphorylated proteins in human HEK293 cell lysate treated with chlorpyrifos oxon.  Cell lysates treated with chlorpyrifos oxon were examined by ELISA and capillary electrophoresis Western blot.  Tryptic peptides were analyzed by liquid chromatography-tandem mass spectrometry.  The depY antibody recognized diethoxyphospho-tyrosine containing proteins by ELISA and Western blotting.  Mass spectrometry identified 40 diethoxyphospho-tyrosine peptides from 24 proteins in immunopurified samples, but found only 9 diethoxyphospho-tyrosine peptides from 6 proteins when the same sample was not immunopurified on depY. The most abundant proteins in the cell lysate, Histone H4, Heat shock 70 kDa protein 1A/1B, Heat shock protein HSP 90 beta, and Alpha-enolase, were represented by several diethoxyphospho-tyrosine peptides.  It was concluded that use of immobilized depY improved the number of diethoxyphospho-tyrosine peptides identified in a complex mixture.  The mass spectrometry results confirmed the specificity of depY for diethoxyphospho-tyrosine peptides independent of the context of the modified tyrosine, which means depY could be used to analyze modified proteins in any species.

Efforts to develop a single enzyme capable of catalyzing the hydrolysis of a broad spectrum of organophosphorus (OP) compounds into non-toxic products have produced multiple candidate enzymes on different structural scaffolds.  While protection against multiple OPs from a single enzyme has been obtained, no single enzyme has been identified that can provide protection against all G- and V-type OP nerve agents.  The most promising candidate enzyme platform is the bacterially produced recombinant variant of organophosphorus hydrolase (OPH) from B. diminuta.  In vivo protective efficacy of candidate OPH scavengers as prophylactics was tested in guinea pigs by administering the enzyme via a carotid catheter, followed 20 minutes later by a subcutaneous injection of increasing doses of the OP nerve agents GA, GB, GD, GF, VX, VR, or VM.  A stage-wise, adaptive dosing experimental design was used to determine the median lethal dose (LD₅₀) of each OP in the context of enzyme prophylaxis.  We report that a combination of two different OPH variants is capable of providing protection against at least 2 x LD₅₀s of all of the OPs tested.  The results indicate that broad spectrum prophylactic protection against OP intoxication can be provided with a cocktail of two different catalytic scavengers with appropriate catalytic activity.  Formulation of the enzymes to promote circulatory stability will be discussed.

Organophosphate hydrolase (OPH) mutants have shown potential use as a medical countermeasure against organophosphorus compounds (OPs). OPH is typically expressed in bacteria as a homodimer. Two separate subunits (35 kDa each) self-assemble through non-covalent bonding at the enzyme face close to the putative active site. OPH homodimers do not secrete expediently from mammalian cells. This causes potential problems when trying to express the protein from a heterologous plasmid or viral delivery system. To enhance secretion of OPH from mammalian cells, we sought to increase protein solubility without catastrophic detriment to activity and without addition of fusion proteins. To this end, we designed OPH to be expressed as a tethered monomer by joining two OPH subunits with a poly-glycine linker. We created the single polypeptide OPH with a tether 10 or 35 amino acids in length between the two halves, and named them T10 and T35 respectively. Western blot analysis and paraoxon hydrolysis assays revealed that T10 was being produced and retained some activity against paraoxon. This was a surprise as we expected T10 to have no enzymatic activity. T35 monomer (75 kDa) was also being produced and retained 71% of specific activity against paraoxon compared to untethered OPH. T10 and T35 showed no significant decrement in activity against the nerve agent sarin. Both constructs showed high molecular weight aggregates greater than 250 kDa in dynamic light scattering and native polyacrylamide gels. These tethered constructs are the first attempts known for producing OPH as a single polypeptide.

Light–induced isomerization of enzyme ligands allows controlling specific biological processes in time and space. Photoisomerisable azobenzene-based inhibitors allow photo-control of acetylcholine (ACh) signalling by regulating acetylcholinesterase (AChE), the enzyme that catalyses ACh hydrolysis in the central and peripheral nervous system. By regulating AChE, this family of inhibitors would allow spatial and temporal regulation of ACh levels in the synaptic cleft. Adequate regulation of ACh levels is an essential part of Alzheimer’s disease (AD) treatment and other common pathologies. Win this work we present the crystal structures of AChE in complex with three different azobenzene derived inhibitors, we confirmed AzoTHA-1 as the only photoactive compound and we determined its structure in its cis- and trans- isomeric forms bound to AChE. Three-dimensional structures, supported by online UV-Vis spectroscopy and kinetic data, explain why only AzoTHA-1 is an effective photoactive AChE inhibitor and suggest possible ways to improve photoactive drugs. We utilised S/WAXS to follow photo-isomerisation induced-changes in the wide-angle scattering region to demonstrate that photoisomerisation of the inhibitor induces its release from AChE’s active site.

Acetylcholinesterase (AChE) is anchored onto cell membranes by a transmembrane protein PRiMA (Proline-Rich Membrane Anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. Several lines of evidence suggest that the dimer formation probably represents an intermediate in the assembly of the tetramer. In addition, the assembly of AChE tetramers with PRiMA requires the presence of a C-terminal “t-peptide” in the AChE catalytic subunit (AChE_T). This protein assembly could be affected by chaperons. AChE inhibitors (AChEIs) are the most established treatment strategy for Alzheimer's disease (AD). Many AChEIs are membrane permeable, and thus which could act as chemical chaperons in affecting the protein assembly of PRiMA-linked AChE in the endoplasmic reticulum (ER). In cultured neuroblastoma or cortical neuron, application of AChEIs, including tacrine (Cognex), rivastigmine (Exelon), but not donepezil (Aricept) and galantamine (Razadyne), caused an accumulation of the unfolded AChE being retained in ER fraction: the AChEI-bound enzyme was not able to transport to Golgi/plasma membrane fraction. As a result, the transcripts encoding AChE and PRiMA were decreased by 50% in the AChEI-treated cultures. In parallel, an increase of ubiquitin-associated enzyme degradation was revealed. The treatment of AChEI in the cultures induced the expression of apoptotic markers, e.g. cleaved caspase 3. In parallel, the apoptotic cell number and mitochondrial membrane potential (MMP) were increased in a dose-dependent manner. The AChEI-bound enzyme retained intracellularly could induce a result of ER stress, as indicated by increased expressions of BiP and CHOP in the treated cultures. The AChEI-induced ER stress resulted with an activation of cAMP signaling, which could regulate the expressions of miR132 and miR212. These findings provide guidance for the drug design and discovery in AD based on inhibition of AChE.

Intoxications with organophosphate or carbamate shut down control of breathing in minutes. These central apneas are reversed by atropine the well-known antidote of acetylcholinesterase (AChE) inhibitors. But how the excess of ACh triggers the crisis remains unclear. If the buildup of ACh on the post-synaptic receptors at cholinergic synapses is critical, we expected that mice in which the synaptic transmission is adapted to the deficit of AChE should resist to intoxication with carbamates. AChE is specifically anchored in the synapses by ColQ at the neuromuscular junction (NMJ) and by PRiMA in central nervous system (CNS). We have thus intoxicated mice with paraoxon, physostigmine or pyridostigmine and recorded in great details the modifications of breathing in double chamber plethysmography. Physostigmine triggers very long end inspiration pauses (EIP) in WT whereas pyridostigmine provokes only short EIP. The duration of EIP was changed with physostigmine or pyridostigmine in PRiMA KO mice when the brain was adapted to a huge excess of ACh. Surprisingly, when AChE is absent at the NMJ, EIP were much shorter with physostigmine. If AChE in the respiratory center is a key target, we expected long EIP when AChE is normal in the brain and reduced in muscles. Altogether these observations do not support that the change of the synaptic transmission explains the central shutdown control of breathing when cholinesterases are inhibited. In addition, we observed that methacholine provokes similar alteration of breathing when injected subcutaneously to mice. I will discuss a novel model to reconciliate these observations.

Acetylcholinesterase (AChE, EC3.1.1.7) plays an important role in the cholinergic neurotransmission in central and peripheral nervous systems, which has been widely recognized as a biomarker for monitoring pollution of organophosphate and carbamate pesticides. Recently, a broad spectrum of environmental toxic substances has been found to decrease AChE activity in various species. Dioxin is one of the emerging environmental AChE disruptors, which is a typical persistent organic pollutant with multiple toxic effects on the nervous system. We have reported that dioxin suppresses the expression of neuronal AChE via aryl hydrocarbon receptor (AhR), in which both transcriptional and posttranscriptional regulations could be involved. Moreover,, muscular AChE expression was also disturbed by dioxin exposure. During myogenic differentiation of C2C12 cells, the mRNA expression of AChE T subunit and the enzymatic activity of AChE were significantly suppressed by dioxin exposure in parallel with the disturbances on the myotube formation. However, the addition of AhR antagonist was not able to reverse the suppressive effect of dioxin, suggesting a distinct role of AhR during the myogenic differentiation process. These results further support the notion that dioxin is a novel environmental AChE disruptor which acts on the biosynthesis processes via multiple molecular mechanisms.

Acetylcholinesterase (AChE) plays hydrolytic role to terminate cholinergic transmission in vertebrate. AChE is intensively reported to exist in different tissues, and may participate in differentiation process. Here, AChE was demonstrated to participate in osteoblastic differentiation. In rat-derived bone tissues and primary cultured osteoblasts, the expression of AChE was increased in parallel with bone development, as well as osteoblastic differentiation. Transcriptional expression and protein of AChE in differentiating osteoblast could be enhanced by application of Wnt3a. Runx2, a downstream transcription factor in Wnt/β-catenin signaling pathway, played crucial role in Wnt3a-induced AChE expression in osteoblasts. This was confirmed by identification of Runx2-binding site in the ACHE gene promoter, over-expression of Runx2 and deletion of the Runx2-binding site in the ACHE promoter. Bone defect was observed in ACHE-/- mice. The non-enzymatic role of AChE in osteoblast was determined by over-expression system and application of AChE inhibitors. By transcriptomics, AChE was found to influence gene expressions of Wnt/β-catenin signaling components, and may participate in osteoblastic function, e.g. affecting osteoclastogenesis and cell adhesion of osteoblast. A notion of non-cholinergic role of AChE in osteoblast, as well as an insight for elucidating other possible mechanisms in regulation of bone formation was provided.

Combination of tacrine/huprine, connected through a different linker tether length, with tryptophan led to the generation of a novel, highly-potent family of multi-target directed ligands targeting key molecular mechanisms of Alzheimer’s disease. Based on in vitro biological profile, the 6-chloro-tacrine-(CH₂)₆-L-tryptophan heterodimer S-K1035 was found to be the most potent inhibitor of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE) within the series, with nanomolar IC₅₀ values (6.31 and 9.07 nM, respectively). Moreover, S‑K1035 showed good ability to inhibit Aβ₄₂ self-aggregation and hAChE-induced Aβ₄₀ aggregation. The X-ray crystallographic analysis of TcAChE in complex with S-K1035 highlighted the utility of the hybridization approach used in the structure based drug design. S‑K1035 also exerted moderate inhibition against neuronal nitric oxide synthase (nNOS). In vivo studies displayed low toxicity profile compared to parent tacrine. S-K1035 also significantly ameliorated performances of scopolamine-treated animals.

Today, treatment of Alzheimer's Disease (AD) mainly involves acetylcholinesterase inhibitors (AChEIs). AChEIs display solely a symptomatic benefit, alleviating the cognitive disorders associated to AD through a temporary restoration of the cholinergic neurotransmission impaired by the neurodegeneration. The gradual loss of efficiency for AChEIs led to associate them to drugs exhibiting potential disease-modifying properties. The “Multi-Target-Directed Ligands” (MTDLs) was used in the recent years with a great potential benefit towards multiple targets implicated in the complex AD,¹ as well as other neurodegenerative syndromes, which involve multiple pathogenic factors. Our contribution to the field led recently to the discovery of Donecopride, the first 5-HT₄R partial agonist, which possesses important acetylcholinesterase (AChE) inhibition properties currently under preclinical development.^2,3 Based on this experience, we have recently developed a novel pleiotropic prodrugs approach to generate promising in vivo active compounds. Based on the structure of rivastigmine, novel MTDLs were designed, acting as prodrugs, able to temporarily covalently bind and inhibit AChE (for a symptomatic effect). and to secondarily release a drug able to selectively reach another AD target (for a potential disease-modifying effect) This concept was applied to several secondary targets, including different 5-HT receptors of interest⁴ for the treatment of AD. The concept, the synthetic development, in vitro and in vivo evaluation of these candidates and our undisclosed results will be presented for the first time in this communication.

Alzheimer’s disease (AD) is a complex and progressive neurodegenerative disorder. The available therapy is limited to the symptomatic treatment and its efficacy remains unsatisfactory ^[1]. In view of the prevalence and expected increase in the incidence of AD, the development of an effective therapy is crucial for public health. Due to the multifactorial etiology of this disease, the multi-target-directed ligand (MTDL) approach is a promising method in search for new drugs for AD. Aiming at developing new MTDLs, this project consists on the development of new multifunctional agents, which will act simultaneously on the different players in AD pathology. The project aims at developing MTDLs by combining an AChE inhibitory activity with an alpha-7 nAChR activation ^[2].

In response to the complex and still not fully understood pathomechanism of Alzheimer's disease, many researchers have turned towards the promising paradigm of designing ligands with a multi-target nature¹. One of the possible benefits of this approach in Alzheimer's disease is an opportunity to merge activity against cholinesterases, which are used in the current symptomatic therapies, with disease-modifying targets associated with β-amyloid and tau protein pathways². Optimization of ligand with respect to several biological targets while maintaining good physicochemical parameters is not an easy task.  Computer modeling can be a huge help in this task. Computer modeling in the design of biologically active substances can be used to effectively search through the huge, available chemical space, or provide support for drawing conclusions of results obtained during the study³. In the work presented here, we would like to describe how the molecular modeling methods were used to design and obtain new series of 1-benzylamino-2-hydroxyalkyl derivatives that are effective against both acetyl- and butyrylcholinesterase as valid, symptomatic targets with an anti-aggregating properties against Tau protein, β-amyloid and inhibition properties against β-secretase (BACE-1) as disease-modified targets⁴.

Cocaine abuse is a major medical and health problem. There is no FDA-approved medication for treatment of cocaine overdose and addiction. Statistical data show that 92% of cocaine users also consume alcohol. The risk of immediate death is 18 - 25 times greater for cocaine co-ingested with alcohol than for cocaine alone. Alcohol can react with cocaine to get a series of toxious compounds in body including cocaine, cocaethylene, norcocaine, norcocaethylene and benzoylecgonine. In combination of our “virtual screening of transition states” computational protocol and artificial intelligence, a novel approach was used to design BChE mutants as multiple functional cocaine hydrolases (mfCocHs) for treatment of toxicity caused by concurrent use of cocaine and alcohol. Comparing the kinetic parameters of native human BChE and mfCocH against cocaine as well as its four toxic/harmful metabolites (i.e. norcocaine, cocaethylene, norcocaethylene and benzoylecgonine) determined by us, the most effective mfCocH has at least a ~1000-fold improved catalytic efficiency against three of the substrates (cocaine, norcocaine, and cocaethylene), ~100-fold and ~10-fold improved catalytic efficiency against norcocaethylene and benzoylecgonine, respectively. In vivo studies have revealed that the mfCocH can effectively hydrolyze cocaine and its four metabolites in rats produced from the concurrent abuse of cocaine and alcohol in both addiction and overdose models. The mfCocHs was powerful antidote to treat cocaine (w/ or w/o alcohol) induced toxicity, even from the lethal toxicity after co-administrated 1 g/kg alcohol (IP) and 180 mg/kg cocaine (IP), at any time point as long as the subject is alive before treatment.

Bis-pyridinium mono-aldoxime (BPMA) compounds are potential antidotes against organophosphorus inhibitors of either acetylcholinesterase or these of butyrylcholinesterase. From the points of drug distribution and pharmacokinetics essential characteristics were determined (concentration versus time curves). Experimental results of pharmacokinetics of BPMA will be detailed with special focus on drug distribution and HPLC analysis of oxime K117. The concentration of BPMAs decreases fast in the body of rats, and thus they fulfil the basic requirement for antidotes: elimination should be as fast as possible. Their elimination curve should be characterized by the term „tenth-life” rather than half-life. BPMA compounds penetrate into the brain in considerable amounts of their concentration in the serum. As blood-brain penetration can have vital importance, time of the maximum extent of blood-brain barrier should also be conceived as a novel pharmacokinetic parameter.

Progress in the development of biodegradable ionic liquids (ILs) [1] allowed finding sustainable fragments to assist the synthesis of sustainable molecules by means of “benign by design” approach. Based on our recent experience in creating micellar catalytic systems for decomposition of organophosphates [2, 3] we have elaborated the following oxime-functionalized low-toxic biodegradable ILs as potential AcChE reactivators: amide/ester linked (amino acid free) IL (I) as well as L-alanine (II) and L-phenylalanine (III) containing compounds with pyridinium aldoxime moiety in cationic part. Variation of amino acid variation (e.g. Me for I and phenyl for II) can help us to analyze a role of hydrophobicity of IL’s cation in AcChE reactivation. The reactivation capacity of novel ILs were evaluated towards AcChE inhibited by typical toxic organophosphate agents. The regularities of antidotal activity of studied compounds are to use in the further improvement of their structures.

Several years, there are ideas how to use reactivators of BChE in prophylaxis of OP-poisoning. They could be applied in combination with human BChE as a pseudo-catalytic scavenging system. However, the effective hBChE reactivator is still missing. The aim of this project is to find highly active and plausibly universal reactivator of hBChE. In the first phase, a database of about 6 mil. structures (ZINC Lead Like) was screened by rigid molecular docking. The receptor (hBChE) was found in the PDB database (pdb code 3DJY, hBChE inhibited by tabun) and prepared for docking. For the second phase, over one hundred molecules were selected. These structures were docked to hBChE with flexible residues within the active site. After manual inspection, over twenty molecules were chosen. Such molecules were modified (e.g. addition of oxime moiety, pK_a optimization) and redocked to hBChE with flexible residues. Finally, two novel compounds were recommended for synthesis. The newly designed compounds will be further synthesized and evaluated on the model of OP-inhibited hBChE and hAChE. They could be used for development of new series of hBChE reactivators.

An endogenous tetrameric wild-type human BChE expressed in Expi293 cells hydrolyzes the neutral substrate N-methylindoxyl acetate (NMIA) with the same K_m as wild-type huBChE (0.14 mM) [1]. For this enzyme, the steady state is preceded by a pre-steady state phase of several minutes in 10 mM Bis-Tris, pH 7 at 25°C. Thermal inactivation of this BChE is biphasic. Kinetic constants (k₁ and k₂) for thermal inactivation shows differences between this mutant and plasma derived wtBChE: the Expi293 is more stable at 55°C and less stable at 60°C than natural wtBChE [2]. At 55°C half-life times of the first and the second phases are 11 min and 43 min for plasma wtBChE; 14 min and 36 min for the Expi293 wtBChE, respectively. At 60°C, the corresponding values are 6 min and 14 min for natural wtBChE; 3 min and 11 min for Expi293 wtBChE. The endogenous enzyme is more stable in urea: urea-induced denaturation is 10 % slower than for the wtBChE and the urea concentration at the mid-point of denaturation is 4.1M for wtBChE and 4.6M for the endogenous enzyme. A bistable dynamic behavior of the endogenous BChE is also observed from pre-steady state behavior for hydrolysis of 1 mM NMIA, showing either long lags or bursts under the same conditions while plasma BChE shows only lags. Molecular mechanic simulations have been undertaken to determine the molecular basis of bistability of this wild-typeBChE.

A human embryonic kidney cell line (Expi293), adapted for suspension growth in serum-free medium, secretes a tetrameric butyrylcholinesterase (BChE). Expression levels are very low, but are increased 10-fold upon treatment with polyethylenimine. DNA sequencing shows that this enzyme is wild-type BCHE. This endogenous BChE displays catalytic properties very close to that of natural huBChE with butyrylthiocholine and N-methylindoxyl acetate as substrates [1]. Several endogenous co-secreted esterases self-reactivate after inhibition by echothiophate, paraoxon, cresyl saligenin phosphate (CBDP), racemic coumarin(CM)-soman, CM-tabun and CM-VX. Overall reactivation rate constants, k_r, of diethylphosphorylated enzymes after inhibition by echothiophate and paraoxon are 0.171 min^-1 and 0.059 min^-1, respectively, suggesting multiple OP-hydrolyzing enzymes. After phosphonylation by CM-soman, CM-tabun and CM-VX, k_r values range from 0.0375 min^-1 to 0.0078 min^-1. k_r of CBDP-inhibited enzyme is 0.028 min^-1. Interestingly, an apparent aging rate is observed after phosphylation. The aging rate of the soman-phosphonylated enzyme(s) is approximately 2-fold slower than for wtBChE (half-time =16 min against 9 min for wtBChE [2]). The half-time for aging after inhibition by CBDP is 31 min whereas aging of wtBChE-CBDP is almost instantaneous [3]. Diethylphosphorylated enzyme(s) inhibited by paraoxon and echiothiophate age(s) with apparent k_a =0.162 min^-1 and 0.057 min^-1, respectively. This difference also supports the multiple enzyme hypothesis.  Further studies are in progress to indentify the different OP-reacting enzymes produced by this Expi293 cell line.

In efficacy testing of experimental oximes, traditionally reactivation of OP-inhibited acethylcholinesterase (AChE) has been analysed by comparing the obtained effects of the single dose with the control [1]. However, quantitative analysis of in vivo dose-response data by benchmark dose (BMD) approach would improve both identification and quantification of the effect and it will allow more rigorous comparison of different oximes efficacies [2]. Thus, we have evaluated in vivo dose-response relationship for two promising experimental oximes, K203 and K027, concerning reactivation of diaphragmal AChE inhibited by dichlorvos (DDVP). To compare the oximes effects, BMD-covariate method was used to estimate oxime dose (with 90% confidence intervals) that elicits a pre-specified effect size of 50% (1.5-fold increase in AChE activity compared to DDVP-treated group). Wistar rats (5/group) were treated with oxime (0/1.25/2.5/5/25/50% LD₅₀ im) immediately after DDVP challenge (75% LD₅₀ sc). Activity of AChE was measured in rat diaphragm homogenates by modified Ellman´s method 60 min after the treatment. Dose-response modeling was done by PROAST software (version 65.5, RIVM, Nederlands). Exponential model m5-ab (y=a[c-(c-1)exp(-bx^d)]) was selected as best estimate with parameters: a_K203=0.1525, a_K027=0.1498, b_K203=0.008472, b_K027=0.03941, c=2.117 and d=0.8916. Derived BMD₅₀ were K203=117 (56, 209) and K027=21 (10, 37) µmol/kg bw, indicating that oxime K027 induces the same effect size with 5.5-times lower dose compared to oxime K203. Moreover, obtained confidence intervals of BMDs did not overlap allowing the conclusion that more potent dose-response relationship belongs to experimental oxime K027.

The novel 7-methoxytacrine-4-pyridinealdoxime agent, named hybrid 5C, is a hybrid compound comprised of a linkage between 7-methoxytacrine (7-MEOTA-4-PA) and reactivator 4-pyridinealdoxime (4-PA) moieties through a 5-carbon length-spacer. This compound was formerly designed as a prophylactic agent for intoxication by organophosphates (OP), able to form a complex with acetylcholinesterase (AChE) and reactivate this enzyme in case of OP inhibition. In order to check if the 5 carbons spacer is the ideal to maximize the interactions of this compound inside AChE, we performed in this work docking, molecular dynamics and mmpbsa studies on a series of analogues of hybrid 5C, varying the spacer-length from 1 to 10 carbons long. Our results helped to elucidate the interactions of these compounds with the different binding sites inside human AChE (HssAChE) and pointed to the 4 and 5 carbons long as the best spacers for optimizing these interactions.

Newly developed oximes, when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure the different organs. In this work, we focused our attention on an investigation of morphological lesions produced by increasing doses of asoxime, obidoxime, K027, K048, and K075 were selected as experimental reactivators. The whole experiment was conducted on Wistar rats. All rats were sacrificed 24 hrs and 7 days after single im application of 0.1 LD₅₀, 0.5 LD₅₀ and 1.0 LD₅₀ of each reactivator. Tissue alterations were carefully quantified by semiquantitative grading scales - cardiac, diaphragm, muscular, pulmonary, gastric, hepatic and splenic damage score, respectively. Morfological structure of different tissues treated with of 0.1 LD₅₀ of all reactivators were similar to those evaluated in the control groups. Focal and reversible degenerative and vascular changes, were established in tissue samples after treatment with 0.5 LD₅₀ of asoxime, obidoxime and K027 (p < 0.01 vs. control group). Acute alterations were developed in tissue samples within 7 days following treatment with 0.5 LD₅₀ and 1.0 LD₅₀ of all reactivators. The most severe tissue alterations were found in rats treated with .0 LD₅₀ of K048 and K075 (p < 0.001 vs. control and asoxime group, respectively). Our results showed that all AChE reactivators given by a single, high, unitary dose regimen, have adverse effect not only on the main visceral and muscular tissues, but on the whole rat as well, but the exact cause-effect relationship causing cellular injury remains to be established in further investigation.

Mono- and bis-pyridinium aldoximes are the only causal antidotes that are designated for the treatment of organophosphate (OP) poisoning. Intoxication by OPs is caused either by pesticides or by the nerve agents, the latter belong to group of chemical warfare agents. These compounds irreversibly inhibit enzyme acetylcholinesterase (AChE) that is no more able to fulfill its physiological function. Mono- and bis-pyridinium aldoximes are able to restore catalytic function of AChE. The reactivating ability of aldoximes is limited by several drawbacks like low blood-brain barrier permeation, low reactivation potency against specific nerve agents etc. In order to obtain efficient treatment of OP, the introduction of novel AChE reactivators raised as an important issue. For over 60 years of intensive research, none of the reactivators reached sufficient activity. Herein, we present novel mono quaternary reactivators that possess excellent in vitro activity to restore AChE activity after intoxication with different nerve agents as well as pesticides. The molecular docking simulations, total synthesis and biological evaluation will be discussed.

Even though reactive oxygen/nitrogen species (ROS/RNS) are physiologically generated in biological systems, their excessive production may cause severe damage of cellular components. Excessive production of ROS/RNS can occur in response to various stressors such as xenobiotics, radiation or pathological processes. Oxidative stress has also been reported to cause adverse effects of some therapeutic drugs including acetylcholinesterase (AChE) oxime reactivators which are used in therapy of organophosphate poisoning.  In this study, we determined the effect of obidoxime, methoxime, asoxime, pralidoxime and trimedoxime on redox homeostasis in cultured human hepatoma (HepG2) cells. The cells were incubated with oximes at concentration corresponding with their IC₅₀ for 1, 4 and 24 hours. Intracellular ROS levels were determined using two fluorescent probes (2',7'‑dichlorodihydrofluorescein diacetate and dihydroethidium). Malondialdehyde and 3‑nitrotyrosine were measured using LC-MS/MS. Additionally, non-protein thiols and non-protein disulfides were evaluated to reflect antioxidant capacity. Individual reactivators displayed distinct quantitative and/or qualitative changes in redox homeostasis reflecting different role of oxidative stress in their intrinsic toxicity. Future perspectives are to test new AChE reactivators synthetized at Department of Toxicology and Military Pharmacy in order to minimalize their unwanted side effect related to oxidative stress.