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Homocysteine (Hcy) is an independent cardiovascular disease (CVD) risk factor, whose mechanisms are poorly understood. We aimed to explore mild hyperhomocysteinemia (HHcy) effects on oxidative status, inflammatory, and cholinesterase parameters in aged male Wistar rats (365 days old). Rats received subcutaneous Hcy (0.03 μmol/g body weight) twice daily for 30 days, followed by euthanasia, blood collection and heart dissection 12 h after the last injection. Results revealed increased dichlorofluorescein (DCF) levels in the heart and serum, alongside decreased antioxidant enzyme activities (superoxide dismutase, catalase, glutathione peroxidase), reduced glutathione (GSH) content, and diminished acetylcholinesterase (AChE) activity in the heart. Serum butyrylcholinesterase (BuChE) levels also decreased. Furthermore, nuclear factor erythroid 2-related factor 2 (Nrf2) protein content decreased in both cytosolic and nuclear fractions, while cytosolic nuclear factor kappa B (NFκB) p65 increased in the heart. Additionally, interleukins IL-1β, IL-6 and IL-10 showed elevated expression levels in the heart. These findings could suggest a connection between aging and HHcy in CVD. Reduced Nrf2 protein content and impaired antioxidant defenses, combined with inflammatory factors and altered cholinesterases activity, may contribute to understanding the impact of Hcy on cardiovascular dynamics. This study sheds light on the complex interplay between HHcy, oxidative stress, inflammation, and cholinesterases in CVD, providing valuable insights for future research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The ESTHER database, dedicated to ESTerases and alpha/beta-Hydrolase Enzymes and Relatives (https://bioweb.supagro.inra.fr/ESTHER/general?what=index), offers online access to a continuously updated, sequence-based classification of proteins harboring the alpha/beta hydrolase fold into families and subfamilies. In particular, the database proposes links to the sequences, structures, ligands and huge diversity of functions of these proteins, and to the related literature and other databases. Taking advantage of the promiscuity of enzymatic function, many engineered esterases, lipases, epoxide-hydrolases, haloalkane dehalogenases are used for biotechnological applications. Finding means for detoxifying those protein members that are targeted by insecticides, herbicides, antibiotics, or for reactivating human cholinesterases when inhibited by nerve gas, are still active areas of research. Using or improving the capacity of some enzymes to breakdown plastics with the aim to recycle valuable material and reduce waste is an emerging challenge. Most hydrolases in the superfamily are water-soluble and act on or are inhibited by small organic compounds, yet in a few subfamilies some members interact with other, unrelated proteins to modulate activity or trigger functional partnerships. Recent development in 3D structure prediction brought by AI-based programs now permits analysis of enzymatic mechanisms for a variety of hydrolases with no experimental 3D structure available. Finally, mutations in as many as 34 of the 120 human genes compiled in the database are now linked to genetic diseases, a feature fueling research on early detection, metabolic pathways, pharmacological treatment or enzyme replacement therapy. Here we review those developments in the database that took place over the latest decade and discuss potential new applications and recent and future expected research in the field., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Sets of 346 herbicides in use and 163 no longer in use were collected from open access online sources and compared in silico with cholinesterases inhibitors (ChI) and drugs in terms of physicochemical profile and estimated toxic effects on human health. The screening revealed at least one potential adverse consequence for each herbicide class assigned according to their mode of action on weeds. The classes with most toxic warnings were K1, K3/N, F1 and E. The selection of 11 commercial herbicides for in vitro biological tests on human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), the enzymes involved in neurotoxicity and detoxification of various xenobiotics, respectively, was based mainly on the structural similarity with inhibitors of cholinesterases. Organophosphate anilofos and oxyacetanilide flufenacet were the most potent inhibitors of AChE (25 μM) and BChE (6.4 μM), respectively. Glyphosate, oxadiazon, tembotrione and terbuthylazine were poor inhibitors with an estimated IC 50 above 100 μM, while for glyphosate the IC 50 was above 1 mM. Generally, all of the selected herbicides inhibited with a slight preference towards BChE. Cytotoxicity assays showed that anilofos, bensulide, butamifos, piperophos and oxadiazon were cytotoxic for hepatocytes (HepG2) and neuroblastoma cell line (SH-SY5Y). Time-independent cytotoxicity accompanied with induction of reactive oxygen species indicated rapid cell death in few hours. Our results based on in silico and in vitro analyses give insight into the potential toxic outcome of herbicides in use and can be applied in the design of new molecules with a less impact on humans and the environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

G and V-type nerve agents represent the most toxic chemical warfare agents. Their primary toxicity was the consequence of the covalent inhibition of the pivotal acetylcholinesterase (AChE), which induces overstimulation of cholinergic receptors and overaccumulation of cholines, eventually leading to death by respiratory arrest. The inhibitory and reactivation kinetics of cholinesterase (ChE) are essential for the toxicology and countermeasures of nerve agents. Medical defensive research on V-type nerve agents (V agents) has been mainly reported on VX and VR. Here we demonstrated the first systematical kinetic analysis between the type of ChE [native or recombinant human AChE and butyrylcholinesterase (BChE)] and three V agents, including VX, VR, and Vs, another isomer of VX, and highlighted the effects of native and recombinant ChE differences. The spontaneous reactivation and aging kinetics data of Vs-inhibited BChEs were firstly reported here. The results showed that AChE was more easily inhibited by three V agent compared to BChE, regardless of whether it is native or recombinant. The increased inhibitory potency order on AChE was VX, Vs, then VR, and on BChE was VX, then Vs and VR. The difference between native and recombinant ChE could influence the inhibition, aging, and spontaneous reactivation kinetics of three V agents, whether AChE or BChE, which was systematically revealed for the first time. For inhibition kinetics, the k i of three V agents for recombinant AChE was significantly higher than native AChE, and the stronger the inhibitory potency of V agents, the more pronounced difference in k i . In terms of aging and spontaneous reactivation kinetics, recombinant ChE was found to be more prone to spontaneous reactivation, but more resistant to aging compared to native ChE, particularly for AChE. The performed covalent molecular docking results partially explained the effects of differences between native and recombinant ChE on enzyme kinetics from the perspective of binding energy and conformation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The risk of exposure to toxic metals is a known concern to human populations. The overexposure to Mn can lead to a pathological condition, with symptoms similar to Parkinson's disease. Although toxicity of Mn has been reported, studies in neonates are scarce but necessary, as Mn can cross biological barriers. The present study evaluated if chronic perinatal exposure to Mn at low doses lead to neurotoxic effects in mice, after direct and indirect exposure. Couples of mice were exposed to Mn (0.013, 0.13, and 1.3 mg kg -1 .day -1 ) for 60 days prior to mating, as well as during gestation and lactation. The offspring was distributed into two groups: animals that were not exposed after weaning - parental exposure only (PE); and animals subject to additional 60-day exposure through gavages after weaning - parental and direct exposure (PDE). Neurological effects were evaluated by Mn quantification, behavior tests and biochemical markers in the brain. PDE animals had alterations in short/long-term memory and increased anxiety-like behavior. Exposure to Mn triggered a decrease of glutathione-s-transferase and increase of cholinesterase activity in different regions of the brain. These findings highlight the risk of exposure to low doses of Mn over a generation and at early stages of development., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Chlorpyrifos is a extensively used organophosphate pesticide (OP). In this study, we closely looked into neurotoxicity of CPF and effect of vitamin B1, by checking the levels of cholinesterases, determining the activity of parameters of oxidative stress, inflammation and also level of apoptotic regulator. The study was performed on a total of 80 male Japanese quails (Coturnix japonica), (two control and 6 experimental groups, n = 10). Three group of quails were given by gavage chlorpyrifos (CPF) for 7 consecutive days at doses of 1.50 mg/kg b.w., 3.00 mg/kg b.w., and 6.00 mg/kg b.w. Another three groups were treated with 10 mg/kg b.w. of vitamin B1 i.m. 30 min after CPF application (in above mentioned doses). Our study have proved that all doses of CPF significantly inhibited cholinesterases in brain, while vitamin B1 reactivated them. CPF has led to an increase in the concentration of malondialdehyde (MDA), and activity of catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST), while tiamin changed the activity of antioxidant enzymes: CAT, SOD, GST. CPF stimulated apoptosis by decreasing B-cell lymphoma (Bcl-2) in brain, while application of vitamin B1 caused an increase of this parameter. CPF amplified inflammatory effect by elevating levels of inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX-2). Thiamine proved its anti-inflammatory property by decreasing the expression of iNOS and interleukin-1(IL-1) and interleukin-6(IL-6). This study is highly pertinent because there is little defense currently available to humans and animals to prevent toxic effects of pesticides., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Competing substrate kinetic analysis of human butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) from the time-course of enzyme-catalyzed substrate hydrolysis, using spectrophotometric assays is described. This study is based on the use of a chromogenic reporter "visible" substrate (substrate A), whose complete hydrolysis time course is retarded by a competing "invisible" substrate (substrate B). For BChE, four visible substrates were used, two thiocholine esters, benzoylthiocholine and butyrylthiocholine, and two aryl-acylamides, o-nitro trifluoro acetaminide and 3-(acetamido)-N,N,N-trimethylanilinium. Three different competing invisible substrates were used, phenyl acetate, acetylcholine and butyrylcholine. For AChE, two visible substrates were used, acetylthiocholine and 3-(acetamido)-N,N,N-trimethylanilinium. For AChE, acetylcholine was competing with visible substrates. The ratio (R) of bimolecular rate constants, k cat /K m , for all couples of substrates, invisible/visible (B/A) covered all possible limit situations, R ≪ 1, R ≈ 1 and R ≫ 1. The kinetic approach, based on the method developed by Golicnik and Masson allowed determination of binding and catalytic parameters of cholinesterases for both visible and invisible substrates. This analysis was applied to michaelian and non-michaelian catalytic behaviors (activation and inhibition by excess substrate). Reevaluation of catalytic parameters obtained for acetylcholine and butyrylcholine more than 50 years ago was made. The method is fast, reliable, and particularly suitable for poorly soluble substrates and for substrates B when no direct spectrophotometric assays exist. Moreover, replacing substrate B by a reversible inhibitor, mechanism of cholinesterase inhibition was possible to study. It is therefore, useful for screening libraries of new substrates and inhibitors, and/or screening of new cholinesterase mutants. This method can be applied to any other enzymes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Acetylcholinesterase (AChE) is an enzyme which terminates the cholinergic neurotransmission, by hydrolyzing acetylcholine at the nerve and nerve-muscle junctions. The reversible inhibition of AChE was suggested as the pre-treatment option of the intoxications caused by nerve agents. Based on our derived 3D-QSAR model for the reversible AChE inhibitors, we designed and synthesized three novel compounds 8-10, joining the tacrine and aroylacrylic acid phenylamide moieties, with a longer methylene chain to target two distinct, toplogically separated anionic areas on the AChE. The targeted compounds exerted low nanomolar to subnanomolar potency toward the E. eel and human AChE's as well as the human BChE and showed mixed inhibition type in kinetic studies. All compounds were able to slow down the irreversible inhibition of the human AChE by several nerve agents including tabun, soman and VX, with the estimated protective indices around 5, indicating a valuable level of protection. Putative noncovalent interactions of the selected ligand 10 with AChE active site gorge were finally explored by molecular dynamics simulation suggesting a formation of the salt bridge between the protonated linker amino group and the negatively charged Asp74 carboxylate side chain as a significant player for the successful molecular recognition in line with the design strategy. The designed compounds may represent a new class of promising leads for the development of more effective pre-treatment options., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Within the alpha/beta hydrolase fold superfamily of proteins, the COesterase group (carboxylesterase type B, block C, cholinesterases …) diverged from the other groups through simultaneous integration of an N-terminal, first disulfide bond and a significant increase in the protein mean size. This first disulfide bond ties a large Cys loop, which in the cholinesterases is named the omega loop and forms the upper part of the active center gorge, essential for the high catalytic activity of these enzymes. In some non-catalytic members of the family, the loop may be necessary for heterologous partner recognition. Reshuffling of this protein portion occurred at the time of emergence of the fungi/metazoan lineage. Homologous proteins with this first disulfide bond are absent in plants but they are found in a limited number of bacterial genomes. In prokaryotes, the genes coding for such homologous proteins may have been acquired by horizontal transfer. However, the cysteines of the first disulfide bond are often lost in bacteria. Natural expression in bacteria of CO-esterases comprising this disulfide bond may have required compensatory mutations or expression of new chaperones. This disulfide bond may also challenge expression of the eukaryote-specific cholinesterases in prokaryotic cells. Yet recently, catalytically active human cholinesterase variants with enhanced thermostability were successfully expressed in E. coli. The key was the use of a peptidic sequence optimized through the Protein Repair One Stop Shop process, an automated structure- and sequence-based algorithm for expression of properly folded, soluble and stable eukaryotic proteins. Surprisingly however, crystal structures of the optimized cholinesterase variants expressed in bacteria revealed co-existing formed and unformed states of the first disulfide bond. Whether the bond never formed, or whether it properly formed then broke during the production/analysis process, cannot be inferred from the structural data. Yet, these features suggest that the recently acquired first disulfide bond is difficult to maintain in E. coli-expressed cholinesterases. To explore the fate of the first disulfide bond throughout the cholinesterase relatives, we reanalyzed the crystal structures of representative COesterases members from natural prokaryotic or eukaryotic sources or produced as recombinant proteins in E. coli. We found that in most cases this bond is absent., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is a multifactorial neurodegenerative process whose effective treatment will require drugs that can act simultaneously on multiple pathogenic targets. Here, we present an overview of our previous multitarget studies of five groups of novel hybrid structures that combine, through spacers, five pharmacophores that have been found promising for AD treatment: γ-carbolines, carbazoles, tetrahydrocarbazoles, phenothiazines, and aminoadamantanes. Biological activity of the compounds was assessed by a battery of assays. These included inhibitory potency against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) as indicators of potential for cognition enhancement and against carboxylesterase (CaE) to exclude unwanted inhibition of this biotransformation pathway. Displacement of propidium from the peripheral anionic site of AChE was determined as a predictor of anti-aggregation activity. Binding to the two sites of the NMDA subtype of the glutamate receptor was conducted as an additional indicator of potential cognition enhancement and neuroprotection. Propensity to protect against mitochondrial triggers of cell death was evaluated by tests of mitochondrial potential and calcium-induced swelling as indicators of mitochondrial permeability transition. Antioxidant potential was measured to evaluate the tendency to prevent oxidative stress. Potential for disease modification was gauged by the ability to stimulate microtubule assembly. Finally, binding modes of conjugates to AChE and BChE were studied using quantum mechanical-assisted molecular docking. We found selective BChE inhibitors (conjugates of γ-carbolines and phenothiazine I, γ-carbolines and carbazoles II, and aminoadamantanes and carbazoles III) as well as inhibitors of both cholinesterases (conjugates of γ-carbolines and methylene blue IV and bis-γ-carbolines with ditriazole-containing spacers V). These compounds combined potentials for cognition enhancement, neuroprotection, and disease modification. None of the conjugates exhibited high potency against CaE, thereby precluding potential drug-drug interactions from CaE inhibition. Thus, the studied compounds exhibited positive characteristics of multitarget drugs, indicating their potential for the next generation of AD therapeutics., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The efficacy and pharmacokinetics of the aqueous co-formulation contents of the Trobigard™ (atropine sulfate, obidoxime chloride) auto-injector were evaluated in a sarin exposed guinea pig model. Two subcutaneous (sc) sarin challenge doses were evaluated in guinea pigs instrumented with brain and heart electrodes for electroencephalogram (EEG) and electrocardiogram (ECG). Sarin challenge doses were chosen to reflect exposure subclasses with sublethal (moderate to severe clinical signs) and lethal consequences. The level of protection of intramuscular human equivalent doses of the co-formulation was defined by (1) the mitigation of signs and symptoms at a sublethal level and (2) the increase of survival time at the supralethal sarin dose levels. Pharmacokinetics of both atropine sulfate and obidoxime were proportional at 1 and 3 human equivalent doses, and only a small increase in heart rate was observed briefly as a side effect. At both sarin challenge doses, 54 μg/kg and 84 μg/kg, the co-formulation treatment was effective against sarin-induced effects. Survival rates were improved at both sarin challenge levels, whereas clinical signs and changes in EEG activity could not in all cases be effectively mitigated, in particular at the supralethal sarin challenge dose level. Reactivation of sarin inhibited cholinesterase was observed in blood, and higher brain cholinesterase activity levels were associated with a better clinical condition of the co-formulation treated animals. Although the results cannot be directly extrapolated to the human situation, pharmacokinetics and the effects over time related to plasma levels of therapeutics in a freely moving guinea pig could aid translational models and possibly improve prediction of efficacy in humans., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this 28 day-study, we evaluated the effects of the insecticide chlorpyrifos orally administered to Wistar rats at doses 0.160, 0.015, and 0.010 mg/kg b. w./day. Following treatment, total cholinesterase activity and activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were measured. Oxidative stress responses were evaluated using a battery of endpoints to establish lipid peroxidation, changes in total antioxidant capacity, level of reactive oxygen species (ROS), glutathione (GSH) level and activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase. Using HPLC-UV DAD analysis, levels of the parent compound and its main metabolite 3,5,6-trichloro-2-pyridinol in plasma and brain tissue were measured. The genotoxic effect was estimated using alkaline comet assay in leukocytes and brain tissue. The exposure did not result in significant effects on total cholinesterase, AChE and BChE activity in plasma and brain tissue. Lipid peroxidation slightly increased both in plasma and brain tissue. Total antioxidant capacity, ROS and GSH levels were marginally influenced by the exposure. Treatment led to significant increases of GSH-Px activity in blood, SOD activity in erythrocytes and a slight increase of catalase activity in plasma. HPLC-UV DAD analysis revealed the presence of both the parent compound and its main metabolite in the plasma of all of the experimental animals and brain tissue of the animals treated at the two higher doses. All of the tested doses of chlorpyrifos were slightly genotoxic, both to leukocytes and brain tissue. Our results call for further research using other sensitive biomarkers of effect, along with different exposure scenarios., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The conformational rigidity of Drosophila melanogaster AChE, was checked by kinetic means on recombinant enzyme with the substitutions of two important amino acids, one at the catalytic anionic site (W83A), one at the peripheral anionic site (W321A) and the double mutant with both tryptophans substituted by alanines (W83A/W321A). It was hypothesized that the individual mutations would affect only the binding affinities of substrate molecules at each site and that a predictable effect would show up in the corresponding double mutant. Simple inspection revealed that bell shaped curves of activity at wide substrate concentration range in the catalytic anionic site mutants carry much less information than the analogous asymmetric ones of the wild type and peripheral anionic site mutant. Therefore, a concurrent kinetic analysis of the curves for all four enzymes was undertaken by constraining mutation independent parameters: unchanged affinity at the catalytic/peripheral anionic site of the opposite mutant in comparison to the parameters for wild type enzyme. Additionally, the parameters for W83A mutated enzyme were employed for the characterization of double mutant (W83A/W321A) protein by setting the dissociation constant for the substrate at the peripheral anionic site as determined for W321A mutant. Simultaneous analysis exactly reproduced the behavior of the double mutant without any significant change of previously reported values for the wild type enzyme (Stojan et al., 2004). This kinetic behavior is completely in line with the crystallographic evidence of structural rigidity in cholinesterases., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

The first report in literature of the isolation of coumarin was in the year 1820. After this report, other papers were published demonstrating the isolation and synthesis of coumarin and analogues. These compounds have been studying along the years for several different pathologies. One of these pathologies was Alzheimer's disease (AD), being the main cause of dementia in the contemporary world. There are two hypotheses to explain the pathogenesis mechanism and disease symptoms, then having the "amyloid hypothesis" and the "cholinergic hypothesis". Some drugs for AD are based on the theory of "cholinergic hypothesis", which objective is to increase the concentration of ACh in the synaptic cleft by the inhibition of cholinesterases. Over the last twenty years, many studies with coumarins compounds were reported as cholinesterases inhibitors. The aim of the present review is to discuss the studies and development of new compounds for AD treatment., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Our aim was to investigate the possible effects of regular drinking of Rosmarinus officinalis L. leaf infusion on behavior and on AChE activity of mice. Rosemary tea (2% w/w) phytochemical profile was investigated through LC/DAD/ESI-MS(n). Adult male mice were randomly divided into two groups: "Rosemary-treated" that received orally the rosemary tea for 4weeks and "control" that received drinking water. The effects of regular drinking of rosemary tea on behavioral parameters were assessed by passive avoidance, elevated plus maze and forced swimming tests. Moreover, its effects on cerebral and liver cholinesterase (ChE) isoforms activity were examined colorimetricaly. Phytochemical analysis revealed the presence of diterpenes, flavonoids and hydroxycinnamic derivatives in rosemary tea; the major compounds were quantitatively determined. Its consumption rigorously affected anxiety/fear and depression-like behavior of mice, though memory/learning was unaffected. ChE isoforms activity was significantly decreased in brain and liver of "rosemary treated" mice. In order to explain the tissue ChE inhibition, principal component analysis, pharmacophore alignment and molecular docking were used to explore a possible relationship between main identified compounds of rosemary tea, i.e. rosmarinic acid, luteolin-7-O-glucuronide, caffeic acid and known AChE inhibitors. Results revealed potential common pharmacophores of the phenolic components with the inhibitors. Our findings suggest that rosemary tea administration exerts anxiolytic and antidepressant effects on mice and inhibits ChE activity; its main phytochemicals may function in a similar way as inhibitors., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Within the autonomic system, acetylcholine signaling contributes simultaneously and interactively to cognitive, behavioral, muscle and immune functions. Therefore, manipulating cholinergic parameters such as the activities of the acetylcholine hydrolyzing enzymes in body fluids or the corresponding transcript levels in blood leukocytes can change the global status of the autonomic system in treated individuals. Specifically, cholinesterase activities are subject to rapid and effective changes. The enzyme activity baseline increases with age and body mass index and depends on gender and ethnic origin. Also, the corresponding DNA (for detecting mutations) and RNA (for measuring specific mRNA transcripts) of cholinergic genes present individual variability. In leukocytes, acetylcholine inhibits the production of pro-inflammatory cytokines, suggesting relevance of cholinergic parameters to both the basal levels and to disease-induced inflammation. Inversely, acetylcholine levels increase under various stress stimuli, inducing changes in autonomic system molecules (e.g., pro-inflammatory cytokines) which can penetrate the brain; therefore, manipulating these levels can also effect brain reactions, mainly of anxiety, depression and pain. Additionally, neurodegenerative diseases often involve exacerbated inflammation, depression and anxiety, providing a focus interest group for cholinergic manipulations. In Alzheimer's disease, the systemic cholinergic impairments reflect premature death of cholinergic neurons. The decline of cholinesterases in the serum of Parkinson's disease and post- stroke patients, discovery of the relevant microRNAs and the growing range of use of anticholinesterase medications all call for critical re-inspection of established and novel approaches for manipulating cholinergic parameters., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Cholinesterases do not follow the Michaelis-Menten kinetics. In the past, many reaction schemes were suggested to explain their complex interactions during the substrate turnover. Covalent catalysis was recognized very early and therefore, double intermediate traditional reaction scheme for the hydrolysis of good substrates at low concentrations was postulated. However, at intermediate and high substrate concentrations homotropic pseudocooperative effects take place in all cholinesterases, due to the nature of their buried active center. In this study, the significance and usefulness of experimental data obtained at low substrate concentrations, where only one substrate molecule accesses the active site at a time, are to be specified for the overall mechanistic evaluations. Indeed, different interpretations are expected when data are processed with equations derived from different reaction schemes. Consequently, the scheme with two substrate binding sites which comprises the structurally evidenced fully occupied active site as ultimate cause for substantially decreased cholinesterase activity at extremely high substrate concentrations is considered here. A special emphasis is put on butyrylcholinesterase, the enzyme with the largest active site among cholinesterases, where the pseudocooperative effects appear at much higher concentrations than in acetylcholinesterases., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

In the past four decades of cholinesterase (ChE) research, we have seen substantive evolution of the field from one centered around substrate and inhibitor kinetic profiles and compound characterizations to the analysis of ChE structure, first through the gene families and then by X-ray crystallographic determinations of the free enzymes and their complexes and conjugates. Indeed, these endeavors have been facilitated by recombinant DNA technologies, structure determinations and parallel studies in related proteins in the α/β-hydrolase fold family. This approach has not only contributed to a fundamental understanding of structure and function of a large family of hydrolase-like proteins possessing functions other than catalysis, but also has been used to develop new practical strategies for scavenging and antidotal activity in cases of organophosphate insecticide or nerve agent exposure., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Incoherent neutron scattering is one of the most powerful tools for studying dynamics in biological matter. Using the cold neutron backscattering spectrometer IN16 at the Institut Laue Langevin (ILL, Grenoble, France), temperature dependence of cholinesterases' dynamics (human butyrylcholinesterase from plasma: hBChE; recombinant human acetylcholinesterase: hAChE and recombinant mouse acetylcholinesterase: mAChE) was examined using elastic incoherent neutron scattering (EINS). The dynamics was characterized by the averaged atomic mean square displacement (MSD), associated with the sample flexibility at a given temperature. We found MSD values of hAChE above the dynamical transition temperature (around 200K) larger than for mAChE and hBChE, implying that hAChE is more flexible than the other ChEs. Activation energies for thermodynamical transition were extracted through the frequency window model (FWM) (Becker et al. 2004) [1] and turned out to increase from hBChE to mAChE and finally to hAChE, inversely to the MSDs relations. Between 280 and 316K, catalytic studies of these enzymes were carried out using thiocholine esters: at the same temperature, the hAChE activity was systematically higher than the mAChE or hBChE ones. Our results thus suggest a strong correlation between dynamics and activity within the ChE family. We also studied and compared the ChEs thermal inactivation kinetics. Here, no direct correlation with the dynamics was observed, thus suggesting that relations between enzyme dynamics and catalytic stability are more complex. Finally, the possible relation between flexibility and protein ability to grow in crystals is discussed., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Research on cholinesterases and effects of their inhibition in the USSR and Russia since 1930-1940s till present is exposed in historical aspects. The first physiological and toxicological effects of cholinesterase inhibition were reported by Alexander Ginetsinsky during World War II, when academic institutions were evacuated from Leningrad to Kazan. The main scientific schools that initiated research on chemistry, enzymology and physiology of cholinesterases and their inhibitors were leaded by Alexandr and Boris Arbuzovs, Victor Rozengart, Viktor Yakovlev, Michael Michelson, Martin Kabachnik, Mikhail Voronkov, Ivan Knunyants, Alexandr Bretskin and others. They investigated the main physiological effects of cholinesterase inhibitors, and analyzed the catalytic mechanisms of cholinesterases and related enzymes. Their contributions are landmarks in the history of cholinesterase research. At the present time revival of research on cholinesterases in different universities and institutes is vivid, in particular at the Moscow State University, research institutes of Russian Academy of Sciences and Kazan Scientific Center., (Copyright © 2013. Published by Elsevier Ireland Ltd.)

It is known than the most potent homologues in various series of O,O-dialkylphosphates are the dibutyl or diamyl derivatives toward mammalian cholinesterases (ChEs) (both Acetyl- and Butyryl-ChEs), and the dimethyl or diethyl ones toward insect AChEs. To investigate the ChE interaction with organophosphorus inhibitors (OPIs) in more detail, we have performed in silico docking of the series of O,O-dialkylfluorophosphates into active center of different ChEs - both from mammals (human and mouse AChEs and horse BChE), and from insects (spring grain aphid AChE belonging to AChE-1 type, and housefly AChE belonging to AChE-2 type). According to the modeling results, one radical is directed to the anionic site W84, another to the acyl pocket. In addition to well-known residues 288 and 290 (Torpedo AChE sequence numbering), we showed an essential influence of residue 400 - a short alkyl residue in mammalian ChEs and phenylalanine in insect ChEs. Phenylalanine in this position creates sterical hindrance for proper orientation of the OPI molecule, which increases the distance between the catalytic serine gamma-oxygen and phosphorus, and decreases the angle of nucleophylic attack. This suggestion was supported by docking of dibutylfluorophosphate into the active center of AChEs with in silico mutations. Thus, we suggest both the angle of nucleophylic attack and the distance between the catalytic serine and phosphorus atom as measures of productivity of OPI binding., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Cholinesterases emerged from a family of enzymes and proteins with adhesion properties. This family is absent in plants and expanded in multicellular animals. True cholinesterases appeared in triploblastic animals together with the cholinergic system. Lineage specific duplications resulted in two acetylcholinesterases in most hexapods and in up to four genes in nematodes. In vertebrates the duplication leading to acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) is now considered to be an ancient event which occurred before the split of osteichthyes. The product of one or the other of the paralogues is responsible for the physiological hydrolysis of acetylcholine, depending on the species lineage and tissue considered. The BChE gene seems to have been lost in some fish lineages. The complete genome of amphioxus (Branchiostoma floridae: cephalochordate) contains a large number of duplicated genes or pseudogenes of cholinesterases. Sequence comparison and tree constructions raise the question of considering the atypical ChE studied in this organism as a representative of ancient BChE. Thus nematodes, arthropods, annelids, molluscs, and vertebrates typically possess two paralogous genes coding for cholinesterases. The origin of the duplication(s) is discussed. The mode of attachment through alternative C-terminal coding exons seems to have evolved independently from the catalytic part of the gene., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Serum albumin displays an esterase activity that is capable of hydrolysing the anti-cholinesterase compounds carbaryl, paraoxon, chlorpyrifos-oxon, diazoxon and O-hexyl, O-2,5-dichlorphenyl phosphoramidate. The detoxication of all these anti-cholinesterase compounds takes place at significant rates with substrate concentrations in the same order of magnitude as expected during in vivo exposures, even when these substrate concentrations are between 15 and 1300 times lower than the recorded K(m) constants. Our data suggest that the efficacy of this detoxication system is based on the high concentration of albumin in plasma (and in the rest of the body), and not on the catalytic efficacy itself, which is low for albumin. We conclude the need for a structure-activity relationship study into the albumin-associated esterase activities because this protein is universally present in vertebrates and could compensate for reduced levels of other esterases, i.e., lipoprotein paraoxonase, in some species. It is also remarkable that the biotransformation of xenobiotics can be reliably studied in vitro, although conditions as similar as possible to in vivo situations are necessary., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

It is generally accepted that inhibition of acetylcholinesterase (AChE) is the most important acute toxic action of organophosphorus compounds, leading to accumulation of acetylcholine followed by a dysfunction of cholinergic signaling. However, the degree of AChE inhibition is not uniformly correlated with cholinergic dysfunction, probably because the excess of essential AChE varies among tissues. Moreover, the cholinergic system shows remarkable plasticity, allowing modulations to compensate for dysfunctions of the canonical pathway. A prominent example is the living (-/-) AChE knockout mouse. Clinical experience indicates that precipitous inhibition of AChE leads to more severe poisoning than more protracted yet finally complete inhibition. The former situation is seen in parathion, the latter in oxydemeton methyl poisoning. At first glance, this dichotomy is surprising since parathion is a pro-poison and has to be activated to the oxon, while the latter is still the ultimate inhibitor. Also oxime therapy in organophosphorus poisoning apparently gives perplexing results: Oximes are usually able to reactivate diethylphosphorylated AChE, but the efficiency may be occasionally markedly smaller than expected from kinetic data. Dimethylphosphorylated AChE is in general less amenable to oxime therapy, which largely fails in some cases of dimethoate poisoning where aging was much faster than expected from a dimethylphosphorylated enzyme. Similarly, poisoning by profenofos, an O,S-dialkyl phosphate, leads to a rapidly aged enzyme. Most surprisingly, these patients were usually well on admission, yet their erythrocyte AChE was completely inhibited. Analysis of the kinetic constants of the most important reaction pathways, determination of the reactant concentrations in vivo and comparison with computer simulations may reveal unexpected toxic reactions. Pertinent examples will be presented and the potentially underlying phenomena discussed., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

A consensus exists that cholinesterase inhibitors (ChEIs) are efficacious for mild to moderate Alzheimer's Disease (AD). Unfortunately, the number of non-responders is large and the therapeutic effect is usually short-lasting. In experimental animals, ChEIs exert three main actions: inhibit cholinesterase (ChE), increase extracellular levels of brain acetylcholine (ACh), improve cognitive processes, particularly when disrupted in models of AD. In this overview we shall deal with the cognitive processes that are improved by ChEI treatment because they depend on the integrity of brain cholinergic pathways and their activation. The role of cholinergic system in cognition can be investigated using different approaches. Microdialysis experiments demonstrate the involvement of the cholinergic system in attention, working, spatial and explicit memory, information encoding, sensory-motor gating, skill learning. No involvement in long-term memory has yet been demonstrated. Conversely, memory consolidation is facilitated by low cholinergic activity. Experiments on healthy human subjects, notwithstanding caveats concerning age, dose, and different memory tests, confirm the findings of animal experiments and demonstrate that stimulation of the cholinergic system facilitates attention, stimulus detection, perceptual processing and information encoding. It is not clear whether information retrieval may be improved but memory consolidation is reduced by cholinergic activation. ChEI effects in AD patients have been extensively investigated using rating scales that assess cognitive and behavioural responses. Few attempts have been made to identify which scale items respond better to ChEIs and therefore, presumably, depend on the activity of the cholinergic system. Improvement in attention and executive functions, communication, expressive language and mood stability have been reported. Memory consolidation and retrieval may be impaired by high ACh levels. Therefore, considering that in AD the degeneration of the cholinergic system is associated with alteration of other neurotransmitter systems and a diffuse synaptic loss, a limited efficacy of ChEIs on memory processes should be expected., (Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.)

The enigma of the cholinergic function concerns the role of ChEs and other components of the cholinergic system in non-transmittive, non-synaptic phenomena. The notion that such unorthodox, non-classical phenomena must exist is clearly supported by several lines of evidence, such as the presence of ChEs and other cholinergic components early before neurogenesis, and indeed in unfertilized and fertilized eggs and in the sperm of many species, and their presence throughout phylogenesis, including non-motile, monocellular organisms, fungi and plants and many anervous and ephemeral tissues. The "flexibility" of ChEs, expressed in their polymorphism and their changeability during ontogenesis also speaks for the notion of non-classical functions of ChEs. Today, there is direct evidence that such functions do indeed exist, as for example, the evidence as to the role of ChEs and other cholinergic components in processes of cell proliferation and differentiation of synaptic and myoneural structures. Also, ChEs participate in cell communications as examplified by immunity processes, as well as pathological states, including Alzheimer's disease and states induced by "insults" such as stress and exposure to agents such as antiChEs. Finally, consistent with the non-classical roles of ChEs and cholinergic components are the morphogenetic and teratologic effects of antiChEs, including OP compounds and cholinergic agonists and antagonists. The structural homology between ChEs on the one hand, and adhesion molecules and protohormones on the other may explain some of this phenomenology. It is proposed that the phylogenetic ubiquity of ChEs and their basic capacities that are important for evolutionary phylogenesis, such as the capacity to promote cell adhesion and cell communication speaks for ChEs as "Ur" proteins., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Phosphylated cholinesterases (ChE) can undergo a side reaction that progressively decreases their reactivatability. This process, termed "aging", results from dealkylation of the adduct and depends on the structure of the organophosphyl moiety. Aged ChEs are resistant to reactivation by oximes. Owing to the toxicological importance of OPs, the molecular mechanism of aging has been the subject of research for decades. It was not clear whether aging involves the same bond breakage regardless the type of OP or is a scission of P-O-C bonds (P-O or O-C) in phosphates/phosphonates, P-N-C bonds in phosphoramidates, and P-S-C bonds in phosphonothionates. It was assumed that the resulting negatively charged atom on phosphorus of the aged adduct prevented nucleophilic attack by oximates, but studies on negatively charged model molecules do not support this hypothesis. Decrease in conformational flexibility of aged enzymes may contribute to their non-reactivatability by preventing proper adjustment of reactivators in the active site gorge. MALDI-TOF mass spectrometry of phosphylated human butyrylcholinesterase (hBChE) in water and in (18)O-water provided evidence that aging results from O-C breakage, i.e. O-dealkylation. In contrast, the isomalathion-BChE conjugate ages mostly through P-S bond cleavage, but a minor product results from O-C and/or S-C breakage. The crystal structures of hBChE and hAChE inhibited by tabun showed that aging of tabun-ChE conjugates results from O-dealkylation. However, depending on the nature of O-alkyl and N-alkyl chains, aging of BChE inhibited by other phosphoramidates results either from O-C breakage or deamination, i.e. P-N breakage. It was found that dealkylation of branched alkoxy involves a transient carbocation. Dealkylation of OP-ChE conjugates is accompanied by enzyme conformational changes. Urea, organic solvent, heat and pressure denaturation of human BChE showed that the conformational stability of aged OP-BChE conjugates is dramatically increased compared to native enzyme. Determination of the three-dimensional structure of BChE and AChE conjugated to different OPs showed that aged adducts form a salt bridge with the protonated catalytic histidine. Structure alteration of aged enzymes is accompanied by exit of water molecules from the enzyme's active site gorge. In addition, neutron scattering studies provided evidence that the structural dynamics of aged BChE is dramatically altered compared to native enzyme. Knowledge of the molecular basis of aging will help to design reactivators of aged ChEs, molecules capable of slowing the aging process, and pseudocatalytic ChE-based bioscavengers., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

This study compared the ability of nine oximes (HI-6, HLö7, MMB-4, TMB-4, carboxime, ICD585, ICD692, ICD3805, and 2-PAM) to reactivate in vivo cholinesterase (ChE) in blood, brain, and peripheral tissues in guinea pigs intoxicated by one of four organophosphorus nerve agents. Two bis-pyridinium compounds without an oxime group, SAD128 and ICD4157, served as non-oxime controls. Animals were injected subcutaneously with 1.0 x LD(50) of the nerve agents sarin, cyclosarin, VR or VX and treated intramuscularly 5 min later with one of these oximes. Toxic signs and lethality were monitored; tissue ChE activities were determined at 60 min after nerve agent. Some animals exposed to sarin or cyclosarin, with or without non-oxime treatment, died within 60 min; however, no animal treated with an oxime died. For VR or VX, all animals survived the 60 min after exposure, with or without non-oxime or oxime therapy. The four nerve agents caused differential degrees of inhibition in blood, brain regions and peripheral tissues. The tested oximes exhibited differential potency in reactivating nerve agent-inhibited ChE in various peripheral tissues, but did not affect ChE activity in the brain regions. There was no direct relation between blood and peripheral tissues in the reactivating efficacy of oxime treatments. ChE inhibited by sarin was the most susceptible to oxime reactivation while cyclosarin the least susceptible. There was no difference in the ChE reactivating potency between the dimethanesulfonate and dichloride salts of HI-6. MMB-4 significantly reactivated the ChE inhibited by these four nerve agents in blood and all three peripheral tissues of the guinea pig, and among all the oximes tested it was the most effective in vivo ChE reactivator against all four nerve agents., (Published by Elsevier Ireland Ltd.)

The classification of the cholinesterases into 'true' and 'pseudo' became obsolete when, some 60 years ago, the author and his co-workers showed that both enzymes had a broad specificity and differed mainly in their acyl group specificity. The importance of complementarity between enzyme and substrate was shown by the high rate of hydrolysis of carbon analogues of choline esters and this enabled pioneer studies of the intermolecular forces between the enzymes' active centres and their substrates to be carried out., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Wild-type human butyrylcholinesterase (BuChE) has proven to be an efficient bioscavenger for protection against nerve agent toxicity. Human acetylcholinesterase (AChE) has a similar potential. A limitation to their usefulness is that both cholinesterases (ChEs) react stoichiometrically with organophosphosphorus (OP) esters. Because OPs can be regarded as pseudo-substrates for which the dephosphylation rate constant is almost zero, several strategies have been attempted to promote the dephosphylation reaction. Oxime-mediated reactivation of phosphylated ChEs generates a turnover, but it is too slow to make pseudo-catalytic scavengers of pharmacological interest. Alternatively, it was hypothesized that ChEs could be converted into OP hydrolases by using rational site-directed mutagenesis based upon the crystal structure of ChEs. The idea was to introduce a nucleophile into the oxyanion hole, at an appropriate position to promote hydrolysis of the phospho-serine bond via a base catalysis mechanism. Such mutants, if they showed the desired catalytic and pharmacokinetic properties, could be used as catalytic scavengers. The first mutant of human BuChE that was capable of hydrolyzing OPs was G117H. It had a slow rate. Crystallographic study of the G117H mutant showed that hydrolysis likely occurs by activation of a water molecule rather than direct nucleophilic attack by H117. Numerous BuChE mutants were made later, but none of them was better than the G117H mutant at hydrolyzing OPs, with the exception of soman. Soman aged too rapidly to be hydrolyzed by G117H. Hydrolysis was however accomplished with the double mutant G117H/E197Q, which did not age after phosphonylation with soman. Multiple mutations in the active center of human and Bungarus AChE led to enzymes displaying low catalytic activity towards OPs and unwanted kinetic complexities. A new generation of human AChE mutants has been designed with the assistance of molecular modelling and computational methods. According to the putative water-activation mechanism of G117H BChE, a new histidine/aspartate dyad was introduced into the active center of human AChE at the optimum location for hydrolysis of the OP adduct. Additional mutations were made for optimizing activity of the new dyad. It is anticipated that these new mutants will have OP hydrolase activity.

Phosphyloximes (POX) are generated upon the reactivation of organophosphorus (OP)-inhibited cholinesterases (ChEs) by pyridinium oximes. These POXs are known to be potent inhibitors of the ChEs following reactivation. However, they can also decompose to give an OP derivative and a cyano derivative of the oxime when a base abstracts the benzylic proton. Using density functional theory, thermodynamic properties were calculated for the reactivation and decomposition pathways of three different oximes (2-PAM, 3-PAM and 4-PAM) with six different OPs (cyclosarin, paraoxon, sarin, tabun, VR and VX). For reactivation purposes, 2-PAM is predicted to be more efficient than 3- and 4-PAM. Based on atomic charges and relative energies, 2-POXs were found to be more inclined towards the decomposition process.

Presented below is a brief description of research supported by the National Institutes of Health (NIH) on cholinesterases that was discussed at the IXth International Meeting on Cholinesterases in Suzhou, China. It is a partial description of the research conducted by researchers at academic and other institutions supported by the NIH, and by some of the researchers in NIH intramural laboratories. It does not represent a comprehensive survey of all research supported by the NIH related to cholinesterases, but rather a brief discussion of some of the studies discussed at the IXth International Meeting on Cholinesterases. The article describes exciting basic, translational and clinical research on therapies for neurological and other diseases. In addition, cholinesterases that may treat substance abuse are discussed, and pesticide and chemical warfare agents that inhibit cholinesterases are highlighted as part of the NIH portfolio. It is the intent of this article to share with the international community some of the research being supported by the NIH on cholinesterases that complements many of the studies being conducted elsewhere. The information was obtained only from published articles or from abstracts available to the public within the NIH CRISP database (http://crisp.cit.nih.gov/).

Cholinesterases have been intensively studied for a long time, but still offer many fascinating and fundamental questions regarding their evolution, activity, biosynthesis, folding, post-translational modifications, association with structural proteins (ColQ, PRiMA and maybe others), export or degradation. They constitute an excellent model to study these processes, particularly because of the sensitivity and specificity of enzymic assays. In addition, a number of provocative ideas concerning their proposed non-conventional, or non-catalytic functions deserve to be further documented.

Cholinesterases (ChEs) including acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are abundant in the nervous system and other tissues. Here we describe two different aspects of ChEs and the cholinergic system. The first aspect concerns the role of cholinergic transmission in central pattern generation in the neonatal rat spinal cord and the second one describes the involvement of ChEs in the pathologies of dystrophin-deficient mutant (mdx) mice, the animal model of Duchenne muscular dystrophy. Thus, this study is divided into two distinct parts. In the first part we show that AChE is abundant in ventral horn neurons, central canal-adjacent and partition neurons in all the observed segments (L2, L5, S1, and S2). AChE was also found in the intermediolateral and sacral parasympathetic nuclei of L2 and S1, respectively. Blocking the AChE by edrophonium produced non-stationary bursting in spinal cord preparations of developing rats. Cross-wavelet/coherence analyses of the data revealed epochs of locomotor-like activity (left-right and flexor-extensor alternation) followed by other rhythmic or non-rhythmic bursting patterns. Addition of exogenous ACh stabilized the rhythm and increased the incidence of locomotor-like pattern in the preparations. Thus, the cholinergic system in the spinal cord is capable of producing and modulating functional rhythmic bursts. Moreover, bath-applied edrophonium and exogenous ACh were found as potent means of modulation of the locomotor rhythm produced by stimulation of sacrocaudal afferents (SCAs). We show that a subclass of sacral neurons with contralateral funicular projections to the thoracolumbar cord is associated with the cholinergic system. This group of neurons may play a major role in the observed enhancement of the SCA-induced motor rhythm. In the second part we show that adult mdx-muscles are malformed with distorted neuromuscular junctions (nmjs) and impaired regulation of acetylcholine receptors. Examination of circulating ChE levels revealed that in mdx-sera, while AChE activity was elevated, BuChE activity was markedly lower than in wild-type (wt) sera. Thus, BuChE to AChE ratio in mouse sera decreased from 6:1 in wt control to 3:1 in mdx. Because serum ChE levels may be modulated by gonadal steroids, it is possible that lack of dystrophin in mdx-mice may affect this regulation. Further studies are in progress to determine the potential endocrine regulation of ChEs in circulation and at the nmjs of mdx- and wt-mice. These studies will help clarify whether the hormonal regulation is impaired in the mdx mutant, and whether changes in circulating ChE reflect or influence the functional deficits observed in excitable tissues of diseased states.

Kinetic parameters of the effect of tacrine as a cholinesterase inhibitor have been studied in two different sources: snake venom (Bungarus sindanus) acetylcholinesterase (AChE) and human serum butyrylcholinesterase (BChE). Tacrine inhibited both venom acetylcholinesterase (AChE) as well as human serum butyrylcholinesterase (BChE) in a concentration-dependent manner. Kinetic studies indicated that the nature of inhibition was mixed for both enzymes, i.e. Km values increase and Vmax decrease with the increase of the tacrine concentration. The calculated IC50 for snake venom and for human serum were 31 and 25.6 nM, respectively. Ki was observed to be 13 nM for venom acetylcholinesterase (AChE) and 12 nM for serum butyrylcholinesterase (BChE). KI (constant of AChE-ASCh-tacrine complex into AChE-ASCh complex and tacrine) was estimated to be 20 nM for venom and 10 nM for serum butyrylcholinesterase (BChE), while the gammaKm (dissociation constant of AChE-ASCh-tacrine complex into AChE-tacrine complex and ASCh) were 0.086 and 0.147 mM for snake venom AChE and serum BChE, respectively. The present results suggest that this therapeutic agent used for the treatment of Alzheimer's disease can also be considered an inhibitor of snake venom and human serum butyrylcholinesterase. Values of Ki and KI show that tacrine had more affinity with these enzymes as compared with other cholinesterases from the literature.

Studies cited by Cowan et al. [J. Appl. Toxicol. 23, 177 (2003)] indicate existence of inflammatory and cholinergic pathways in both nerve agents and sulfur mustard (HD) injury. Increase in AChE synthesis and neurite extension was noted after exposure to HD [K.W. Lanks et al., Exp. Cell Res. 355 (1975)]. Moreover, anti-inflammatory drugs reduce the dermal, respiratory and ocular damage caused by exposure to HD. On the other hand, recent studies have noted the involvement of neuro-inflammatory processes during exposure to the nerve agents sarin or soman [Cowan et al., 2003]. The use of various anti-inflammatory drugs in addition to the classical antidotal drugs (e.g. atropine and oximes) caused decrease in certain toxic symptoms and inflammation-induced brain damage. Our new bifunctional drugs (Scheme 1) are based on CNS-permeable molecular combination of pseudo-reversible AChE inhibitor (pyridostigmine, PYR) coupled via a hydrophobic spacer (octyl or decyl hydrocarbon chain) to a non-steroidal anti-inflammatory drug (NSAID) such as Ibuprofen or Diclofenac (Scheme 1). This study evaluates the efficacy of certain bifunctional compounds against HD and soman poisoning in mice in vivo.

Among the large variety of reversible inhibitors that bind to cholinesterases (ChE), only a few exhibit exquisitely strong binding reflected in low femtomolar to picomolar equilibrium dissociation constants. These tight binding inhibitors owe their high affinity to distinctive modes of interaction with the enzyme: naturally occurring snake toxins, the fasciculins, share a large 1000 angstroms2 complementary surface for its complex with acetylcholinesterases (AChE; EC 3.1.1.7); transition state analogs trifluoroacetophenones form a covalent bond with the active serine; disubstituted 1,2,3-triazole inhibitors formed in situ are selected by AChE for optimal interaction surface over the length of the active center gorge. All these inhibitors bind with higher affinity to AChEs than to the closely related butyrylcholinesterases (BuChE; EC 3.1.1.8). Selectivity of individual inhibitors towards BuChE increases with increasing their molecular size. Interaction kinetics for all three classes of compounds reveal very slow rates of dissociation of the AChE-inhibitor complexes or conjugates combined with very fast association rates. The influence of conformational flexibility of the active center gorge on the affinity of inhibitor binding was demonstrated by comparing binding properties of a series of disubstituted 1,2,3-triazoles having systematically varied structures. Analysis of the linear free energy relationships of binding to both mouse and Electrophorus AChE reveals independent contributions of individual structural elements of inhibitors to their binding with the triazole ring emerging as an independently contributing pharmacophore. These tight binding inhibitor interactions reveal useful information not only on the conformational flexibility of ChEs, but also on the diversity of modes of interaction that achieve inhibition.

The hydrolysis of substrates by cholinesterases does not follow the Michaelis-Menten reaction mechanism. In addition to the inhibition by excess substrate, these enzymes often show an unexpectedly high activity at low substrate concentrations. It seems that these deviations are the consequence of an unusual architecture of the active site, buried deep inside the core of the molecule. Kinetic data and structural evidence allow for a detailed prediction of the events during a very fast substrate turnover. Recently, we presented a procedure which provides an unbiased framework for mathematical modelling of the complex cholinesterase reaction [J. Stojan, M. Golicnik, D. Fournier, Rational polynomial equation as an unbiased approach for the kinetic studies of Drosophila melanogaster acetylcholinesterase reaction mechanism, Biochim. Biophys. Acta 1703 (2004) 53-61]. It is based on regression analysis of a rational polynomial using classical initial rate data. Here, we extend the use of the rational polynomial rate equation for finding and comparing some selected homeomorphic reaction schemes useful for the mechanistic interpretation of cholinesterase kinetic data.

Organophosphate (OP) compounds exert inhibition on cholinesterase (ChE) activity by irreversibly binding to the catalytic site of the enzymes. For this reason, they are employed as insecticides for agricultural, gardening and indoor pest control. The biological function of the ChE enzymes is well known and has been studied since the beginning of the XXth century; in particular, acetylcholinesterase (AChE, E.C. 3.1.1.7) is an enzyme playing a key role in the modulation of neuromuscular impulse transmission. However, in the past decades, there has been increasing interest concerning its role in regulating non-neuromuscular cell-to-cell interactions mediated by electrical events, such as intracellular ion concentration changes, as the ones occurring during gamete interaction and embryonic development. An understanding of the mechanisms of the cholinergic regulation of these events can help us foresee the possible impact on environmental and human health, including gamete efficiency and possible teratogenic effects on different models, and help elucidate the extent to which OP exposure may affect human health. The chosen organophosphates were the ones mainly used in Europe: diazinon, chlorpyriphos, malathion, and phentoate, all of them belonging to the thionophosphate chemical class. This research has focused on the comparison between the effects of exposure on the developing embryos at different stages, identifying biomarkers and determining potential risk factors for sensitive subpopulations. The effects of OP oxonisation were not taken into account at this level, because embryonic responses were directly correlated to the changes of AChE activity, as determined by histochemical localisation and biochemical measurements. The identified biomarkers of effect for in vitro experiments were: cell proliferation/apoptosis as well as cell differentiation. For in vivo experiments, the endpoints were: developmental speed, size and shape of pre-gastrula embryos; developmental anomalies on neural tube, head, eye, heart. In all these events, we had evidence that the effects are mediated by ion channel activation, through the activation/inactivation of acetylcholine receptors (AChRs).

The purpose of this project was to determine and compare the time-related changes in blood, brain, and tissue acetylcholinesterase (AChE) activity during the first hour after exposure to six organophosphorus nerve agents (GA, GB, GD, GF, VR, and VX) in Hartley guinea pigs. Animals were pretreated with atropine methyl nitrate (1.0mg/kg, i.m.) to minimize peripheral toxic effects 15 min before they were given a 1.0 x LD50 subcutaneous dose of a nerve agent. At 0, 5, 10, 15, 30, and 60 min after nerve agent, animals were humanely euthanized. Blood was collected and brain regions (brainstem, cortex, hippocampus, midbrain, cerebellum, striatum, and spinal cord) and peripheral tissues (diaphragm, skeletal muscle, and heart) were dissected and processed for AChE activity. All six nerve agents produced maximum inhibition of AChE in red blood cells between 5 and 10% of the control within 10 min after exposure. In whole blood, differential effects were observed among the agents: GB, GD, and GF produced more rapid and greater inhibition than did GA, VR, and VX. GF was the most rapid, producing a maximum inhibition to 5% of the control in 5 min, while VR and VX were slower reaching maximum inhibition to 30% of the control at 15 min. The enzyme activity in the majority of the brain regions was more markedly inhibited by the G-agents than by the V-agents. The G-agents caused rapid AChE inhibition, reaching maximum levels (20-30% of control) at 15 min and GA produced the most rapid effects. V-agents produced much slower and less AChE inhibition, reaching maximum (35-60% of control) at 30 min. In the diaphragm, VR, VX, and GD produced more rapid and greater AChE inhibition than other G-agents; GA produced the slowest and least inhibition. In the skeletal muscle, VX induced the most rapid and severe inhibition, while GA the least inhibition. In the heart, all agents produced very rapid inhibition, and GD produced the most severe inhibition of AChE activity. These observations suggest that G-agents and V-agents are tissue compartment specific in their ability to inhibit AChE activity.

We investigated whether transcriptional inducers could enhance the expression of acetylcholinesterase (AChE) in cell lines to achieve protection against organophosphate (OP) poisoning. Trichostatin A (TSA), an inhibitor of histone deacetylase that de-condenses chromatin and increases the binding of transcription factors and mRNA synthesis, induced three- to four-fold extracellular and 8-10-fold intracellular AChE expression at the optimal dose of 165-333 nM in Neuro 2A cells. Pre-treatment with TSA protected against OP exposure. Thus, transcriptional inducers, such as TSA, up-regulate AChE, which then can scavenge the OP and protect the cells from OP-induced toxicity, and are potential novel ways to treat chemical warfare nerve agent (CWNA) exposure.

Cholinesterases (ChEs) are classified as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) according to their substrate specificity and sensitivity to selected inhibitors. The activities of AChE in red blood cells (RBC-AChE) and BChE in serum can be used as potential biomarkers of suppressed and/or heightened activity in the central and peripheral nervous systems. Exposure to organophosphate (OP) chemical warfare agents (CWAs), pesticides, anesthetics, and a variety of drugs such as cocaine, as well as some neurodegenerative and liver disease states, selectively reduces AChE or BChE activity. In humans, the toxicity of pesticides is well documented. Therefore, blood cholinesterase activity can be exploited as a tool for confirming exposure to these agents and possible treatments. Current assays for measurement of RBC-AChE and serum BChE require several labor-intensive processing steps, suffer from wide statistical variation, and there is no inter-laboratory conversion between methods. These methods, which determine only the serum BChE or RBC-AChE but not both, include the Ellman, radiometric, and deltapH (modified Michel) methods. In contrast, the Walter Reed Army Institute of Research Whole Blood (WRAIR WB, US Patent #6,746,850) cholinesterase assay rapidly determines the activity of both AChE and BChE in unprocessed (uncentrifuged) whole blood, uses a minimally invasive blood sampling technique (e.g., blood from a finger prick), and is semi-automated for high-throughput using the Biomek 2000 robotic system. To date, the WRAIR whole blood assay was used to measure AChE and BChE activities in human blood from volunteers in FDA clinical trials. In the first FDA study, 24 human subjects were given either 30 mg PB orally (n = 19) or placebo (n = 5). Blood samples were obtained pre-dosing and 2.5, 5, 8, and 24 h post-dosing. The samples were analyzed for AChE and BChE activity using the WRAIR WB robotic system, and for PB concentration by HPLC. We found that maximal inhibition of AChE (26.2%) and concentration of PB (17.1 ng/mL) occurred at 2.5 h post-PB dosing. AChE activity returned to almost 100% of pre-dose values by 6 h. A dose-dependent linear correlation was found between the amount of PB measured in the blood and the inhibition of AChE. Following soman (GD) exposure, recovered AChE activity was similar to levels that were reversibly protected by the PB administration. Therefore, the WRAIR ChE WB data clearly supports the conclusion that PB is an effective pre-treatment drug for nerve agent exposure (GD). In the second FDA human study for the treatment of Alzheimer's disease, the WRAIR ChE WB assay was used to determine the RBC-AChE and serum BChE profile of healthy elderly volunteers receiving Huperzine A. Huperzine A is a plant-derived reversible and selective AChE inhibitor compared to BChE, and is a more potent inhibitor of AChE than PB. Huperzine A is available as a nutraceutical, a natural supplement reported to improve memory, and has a variety of neuroprotective effects. Individuals received an increasing dose regimen of huperzine A (final dose 200 microg after 4 weeks), which produced more than 50% inhibition of RBC-AChE. Huperzine A was well tolerated by these patients at doses that sequestered more RBC-AChE than PB, and thus warrants further study as a prophylaxis for OP poisoning in addition to Alzheimer's therapy. Due to the documented use of OPs by terrorists and in warfare around the globe, Federal, State, and local authorities need a reliable, fast, inexpensive, and standard method for confirming such an assault in order to initiate appropriate containment, decontamination, and treatment measures. This assay is ideal for prescreening military personnel for atypical ChE activities that would preclude their deployment to areas of potential CWA exposure. The WRAIR WB ChE assay will fulfill the requirement for rapid and reliable monitoring of such exposure in military and civilian populations.

Kinetics of hydrolysis of acetylthiocholine (ATCH) and acetylcholine (ACH) by butyrylcholinesterase (BCHE) and acetylcholinesterase (ACHE) are studied. ATCH is used for testing of enzymatic hydrolysis of ACH in vitro, because mechanism of ATCH hydrolysis is qualitatively similar to ACH and its reaction course can be quantitatively on-line measured by two independent methods: spectrophotometrical (determination of thiocholine - product of ATCH hydrolysis - using Ellman's method) and electrochemical (determination of acetic acid - product of ATCH hydrolysis - by pH-stat method). All tested hydrolyses correspond to the Michaelis-Menten's equation with the second irreversible step up to the total exhaustion of the substrate. The correlations were made by means of differential and integral kinetic equations describing Michaelis-Menten model. The optimal values of Michaelis constant (KM), maximum velocity (Vm), kinetic constants of single reaction steps and absolute concentration of the used enzyme were calculated for each experiment.