Meeting Report for: The 13th International Meeting on Cholinesterases 7th Conference on Paraoxonase

Trichloronate is a racemic organophosphonothioate insecticide that induced delayed neuropathic in hens and human. The avian are species with great susceptibility to organophosphorus poisoning due to their low levels of A-esterases. However, a significant copper-dependent A-esterase activity has been identified in chicken and turkey serum. This study aims at characterizing the trichloronate enantiomers hydrolysis by serum and albumin from chicken (CSA) and turkey (TSA) in the presence of copper by chiral chromatography. A significant Cu(2+)-dependent hydrolysis for both trichloronate enantiomers (38%) was observed in turkey serum and TSA (p<0.05). The hydrolysis is stereoselective for (-)-trichloronate (p < 0.05). TSA incubation (200 mug) showed residual values of 56 muM and 20 muM of (+)-trichloronate and (-)-trichloronate, respectively; while the chicken serum and CSA presented a slight hydrolysis (1-7%) of both enantiomers. This copper-dependent hydrolysis and stereoselectivity of trichloronate by TSA was proportional to the incubation time. The increase of TSA in the assay (200-3000mug) in the presence of 100muM copper did not significantly increase the levels of hydrolysis and stereoselectivity, an opposite effect was observed for turkey serum (100-200mL), which totally inhibited this copper-dependent activity of both isomers. The present study evidences an A-esterase activity of TSA on a thio form OP compound, which is stereoselective and activated by copper.

DDVP-inhibited rat diaphragm AChE reactivation by oxime K027/K203 was dose-dependent. Dose-response relationships were described by 4-parameter exponential and Hill models. Maximum size of DDVP-inhibited AChE reactivation by K-oximes was 2.1-fold. Oxime K027 had 5-fold higher relative reactivating potency compared to oxime K203.

The organophosphorus chemical warfare agents were initially synthesized in the 1930's and are some of the most toxic compounds ever discovered. The standard means of decontamination are either harsh chemical hydrolysis or high temperature incineration. Given the continued use of chemical warfare agents there are ongoing efforts to develop gentle environmentally friendly means of decontamination and medical counter measures to chemical warfare agent intoxication. Enzymatic decontamination offers the benefits of extreme specificity and mild conditions, allowing their use for both environmental and medical applications. The most promising enzyme for decontamination of the organophosphorus chemical warfare agents is the enzyme phosphotriesterase from Pseudomonas diminuta. However, the catalytic activity of the wild-type enzyme with the chemical warfare agents falls far below that seen with its best substrates, and its stereochemical preference is for the less toxic enantiomer of the chiral phosphorus center found in most chemical warfare agents. Rational design efforts have succeeded in the dramatic improvement of the stereochemical preference of PTE for the more toxic enantiomers. Directed evolution experiments, including site-saturation mutagenesis, targeted error-prone PCR, computational design, and quantitative library analysis, have systematically improved the catalytic activity against the chemical warfare nerve agents. These efforts have resulted in greater than 4-orders of magnitude improvement in catalytic activity and have led to the identification of variants that are highly effective at detoxifying both G-type and V-type nerve agents. The best of these variants have the ability to prevent intoxication when delivered as a post-exposure treatment for VX and as a pre-exposure treatment for G-agent intoxication with observed protective factors up to 60-fold. Combining the best variant, H257Y/L303T, with a PCB polymer coating has enabled the development of a long lasting circulating prophylactic treatment that is highly effective against sarin.

Eight derivatives of 4-aminoquinolines differing in the substituents attached to the C(4)-amino group and C(7) were synthesised and tested as inhibitors of human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Both enzymes were inhibited by all of the compounds with inhibition constants (Ki) ranging from 0.50 to 50muM exhibiting slight selectivity toward AChE over BChE. The most potent inhibitors of AChE were compounds with an n-octylamino chain or adamantyl group. The shortening of the chain length resulted in a decrease in AChE inhibition by 5-20 times. Docking studies revealed that the quinoline group within the AChE active site was positioned in the choline binding site, while the C(4)-amino group substituents, depending on their lipophilicity, could establish hydrogen bonds or pi-interactions with residues of the peripheral anionic site. The most potent inhibitors of BChE were compounds with the most voluminous substituent on C(4)-amino group (adamantyl) or those with a stronger electron withdrawing substituent on C(7) (trifluormethyl group). Based on AChE inhibition, compounds with an n-octylamino chain or adamantyl substituent were shown to possess the capacity for further development as potential drugs for treatment of neurodegenerative diseases.

Human butyrylcholinesterase (BChE) is a widely distributed plasma enzyme. For decades, numerous research efforts have been directed at engineering BChE as a bioscavenger of organophosphorus insecticides and chemical warfare nerve agents. However, it has been a grand challenge to cost-efficiently produce BChE in large-scale. Recently reported studies have successfully designed a truncated BChE mutant (with amino-acid substitutions on 47 residues that are far away from the catalytic site), denoted as BChE-M47 for convenience, which can be expressed in E. coli without loss of its catalytic activity. In this study, we aimed to dimerize the truncated BChE mutant protein expressed in a prokaryotic system (E. coli) in order to further improve its thermal stability by introducing a pair of cross-subunit disulfide bonds to the BChE-M47 structure. Specifically, the E377C/A516C mutations were designed and introduced to BChE-M47, and the obtained new protein entity, denoted as BChE-M48, with a pair of cross-subunit disulfide bonds indeed exists as a dimer with significantly improved thermostability and unaltered catalytic activity and reactivity compared to BChE-M47. These results provide a new strategy for optimizing protein stability for production in a cost-efficient prokaryotic system. Our enzyme, BChE-M48, has a half-life of almost one week at a 37 degrees C, suggesting that it could be utilized as a highly stable bioscavenger of OP insecticides and chemical warfare nerve agents.

Casualties caused by nerve agents, potent acetylcholinesterase inhibitors, have attracted attention from media recently. Poisoning with these chemicals may be fatal if not correctly addressed. Therefore, research on novel antidotes is clearly warranted. Pyridinium oximes are the only clinically available compounds, but poor penetration into the blood-brain barrier hampers efficient enzyme reactivation at the central nervous system. In searching for structural factors that may be explored in SAR studies, we synthesized and evaluated neutral aryloximes as reactivators for acetylcholinesterase inhibited by NEMP, a VX surrogate. Although few tested compounds reached comparable reactivation results with clinical standards, they may be considered as leads for further optimization.

Although the three-dimensional structures of mouse and Torpedo californica acetylcholinesterase are very similar, their responses to the covalent sulfonylating agents benzenesulfonyl fluoride and phenylmethylsulfonyl fluoride are qualitatively different. Both agents inhibit the mouse enzyme effectively by covalent modification of its active-site serine. In contrast, whereas the Torpedo enzyme is effectively inhibited by benzenesulfonyl fluoride, it is almost completely resistant to phenylmethylsulfonyl fluoride. A bottleneck midway down the active-site gorge in both enzymes restricts access of ligands to the active site at the bottom of the gorge. Molecular dynamics simulations revealed that the mouse enzyme is substantially more flexible than the Torpedo enzyme, suggesting that enhanced 'breathing motions' of the mouse enzyme relative to the Torpedo enzyme may explain why phenylmethylsulfonyl fluoride can reach the active site in mouse acetylcholinesterase, but not in the Torpedo enzyme. Accordingly, we performed docking of the two sulfonylating agents to the two enzymes, followed by molecular dynamics simulations. Whereas benzenesulfonyl fluoride closely approaches the active-site serine in both mouse and Torpedo acetylcholinesterase in such simulations, phenylmethylsulfonyl fluoride is able to approach the active-site serine of mouse acetylcholinesterase, but remains trapped above the bottleneck in the Torpedo enzyme. Our studies demonstrate that reliance on docking tools in drug design can produce misleading information. Docking studies should, therefore, also be complemented by molecular dynamics simulations in selection of lead compounds. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:CHEMBIOINT:2.

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.

Human butyrylcholinesterase (BChE) is known as a safe and effective protein for detoxification of organophosphorus (OP) nerve agents. Its rationally designed mutants with considerably improved catalytic activity against cocaine, known as cocaine hydrolases (CocHs), are recognized as the most promising drug candidates for the treatment of cocaine abuse. However, it is a grand challenge to efficiently produce active recombinant BChE and CocHs with a sufficiently long biological half-life. In the present study, starting from a promising CocH, known as CocH3 (i.e. A199S/F227A/S287G/A328W/Y332G mutant of human BChE), which has a approximately 2000-fold improved catalytic activity against cocaine compared to wild-type BChE, we designed an N-terminal fusion protein, Fc(M3)-(PAPAP)2-CocH3, which was constructed by fusing Fc of human IgG1 to the N-terminal of CocH3 and further optimized by inserting a linker between the two protein domains. Without lowering the enzyme activity, Fc(M3)-(PAPAP)2-CocH3 expressed in Chinese hamster ovary (CHO) cells has not only a long biological half-life of 105+/-7h in rats, but also a high yield of protein expression. Particularly, Fc(M3)-(PAPAP)2-CocH3 has a approximately 21-fold increased protein expression yield in CHO cells compared to CocH3 under the same experimental conditions. Given the observations that Fc(M3)-(PAPAP)2-CocH3 has not only a high catalytic activity against cocaine and a long biological half-life, but also a high yield of protein expression, this new protein entity reported in this study would be a more promising candidate for therapeutic treatment of cocaine overdose and addiction.

Structure-guided design of novel pharmacologically active molecules relies at least in part on functionally relevant accuracy of macromolecular structures for template based drug design. Currently, about 95% of all macromolecular X-ray structures available in the PDB (Protein Data Bank) were obtained from diffraction experiments at low, cryogenic temperatures. However, it is known that functionally relevant conformations of both macromolecules and pharmacological ligands can differ at higher, physiological temperatures. We describe in this article development and properties of new human acetylcholinesterase (AChE) crystals of space group P31 and a new unit cell, amenable for room-temperature X-ray diffraction studies. We co-crystallized hAChE in P31 unit cell with the reversible inhibitor 9-aminoacridine that binds at the base of the active center gorge in addition to inhibitors that span the full length of the gorge, donepezil (Aricept, E2020) and AChE specific inhibitor BW284c51. Their new low temperature P31 space group structures appear similar to those previously obtained in the different P3121 unit cell. Successful solution of the new room temperature 3.2 A resolution structure of BW284c51*hAChE complex from large P31 crystals enables us to proceed with studying room temperature structures of lower affinity complexes, such as oxime reactivators bound to hAChE, where temperature related conformational diversity could be expected in both oxime and hAChE, which could lead to better informed structure-based design under closer-to-physiological temperature conditions.

A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198.H438.E325) was replaced by S198.H438.N322E in silico. Molecular dynamics for 1.5 mus and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198.H438.N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.

Organophosphates (OPs) irreversibly inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. The reactivation of these inhibited enzymes is paramount for their normal function. Present study evaluates reactivation potency of two newly developed oximes, K456 and K733, against paraoxon (POX)-inhibited human-RBC-AChE and human-plasma-BChE in comparison to reported reactivator, pralidoxime (2-PAM). In vitro studies showed higher intrinsic toxicities of both oximes than 2-PAM for AChE. No substantial reactivation of hBChE was noted by tested concentration. Contrary to 2-PAM, the in silico study predicted lower binding free energies for both oximes. However, the detailed interaction study revealed inability of oximes to interact with catalytic anionic site of AChE and hBChE in contrast to 2-PAM. Both in vitro and in silico studies conclude that K456 and K733 are unlikely to be used as reactivators of paraoxon-inhibited AChE or BChE.

K-oximes were developed as modern drug candidates acting as AChE reactivators. In this study, it has been investigated which interspecies and intergender differences changes could be observed in Wistar rats and Swiss mice, both genders, after the treatment with increasing doses of selected acetylcholinesterase reactivators - asoxime, obidoxime, K027, K048, and K075. After the 24h, a number of died animals was counted and the median lethal dose (LD50) for each oxime was calculated. By using the intramuscular route of administration, asoxime and K027 had the least toxicity in female rats (640.21mg/kg and 686.08mg/kg), and in female mice (565.75mg/kg and 565.74mg/kg), respectively. Moreover, asoxime and K027 showed 3, 4 or 8 times less acute toxicity in comparison to K048, obidoxime and K075, respectively. Beyond, K075 had the greatest toxicity in male rats (81.53mg/kg), and in male mice (57.34mg/kg), respectively. Our results can help to predict likely adverse toxic effects, target organ systems and possible outcome in the event of massive human overexposure, and in establishing risk categories or in dose selection for the initial repeated dose toxicity tests to be conducted for each oxime.

Butyrylcholinesterase (BChE) is a serine hydrolase widely distributed throughout the body. It provides protection against administrated or inhaled poisons by hydrolyzing or sequestering the toxic compounds. The most frequent genetic variant of BCHE gene - K variant (p.A539T) is located in the C-terminal tetramerization domain, outside of the catalytic center. Many studies tried to reveal the nature of the lower activity of BChE K-variant but results and conclusions were often contradictory. The aim of this study is to estimate K allele frequency and its coexisting alterations in BCHE gene in a population of 162 individuals, as well as, assess influence on the enzyme activity in serum. We present three haplotypes of BChE-K variant, two of them coexist in strong linkage disequilibrium with alterations in 5'UTR (rs1126680), intron 2 (rs55781031) or in exon 2 (rs1799807). We demonstrate a negative role of these alterations on enzyme activity. By oneself BCHE-K (with no other alterations in BCHE gene) haplotype exhibits wild type enzyme activity. Based on our previous and presented results we conclude that SNPs localized outside the coding sequence, in 5'UTR or/and in intron 2 of BCHE gene, but not solely in K-variant alteration (p.A539T) itself, are responsible for reduced enzyme activity.

Substance addiction is a chronic, relapsing mental disorder Characterized by compulsive drug seeking, and loss of control over drug intake and relapse after prolonged abstinence. Genetics has been shown to contribute towards an individual's vulnerability to addiction. Acetylecholine (ACh), a cholinergic neurotransmitter hydrolyzed by acetylcholinesterase (AChE), is an essential neurotransmitter and neuromodulator in central and peripheral nervous system and has regulatory influence on numerous neuronal functions including addiction. The present study was carried out to investigate the role of acetylcholinesterase (AChE) in addiction through measurement of enzyme activity and to find potential association of ACHE gene 3'UTR variants rs17228602 and rs17228616 in heroin, hashish and poly drug addicts. Both SNPs are located within microRNA (miRNA) recognition sites with potential to affect miRNA/transcript interaction. A total of 122 addicts of heroin, hashish and polydrug were recruited from local rehabilitation centers to participate in this study. AChE activity was measured in blood by Ellman's method. SNP genotyping was performed by restriction fragment length polymorphism (PCR-RFLP) and Sanger sequencing. The AChE activity was found significantly higher (p<=0.005) in addicted cohort (mean+/-standard error of mean 0.020+/-0.001mumol/L/min; 95% confidence interval (CI) 0.018-0.022) in comparison to non-addicted healthy subjects (0.011+/-0.001mumol/L/min; 95% confidence interval CI 0.010-0.013). A statistically significant association of ACHE rs17228602 SNP with addiction vulnerability in dominant (DM: Odd's ratio OR=2.095, 95% CI=1.157-3.807 p=0.009) and allelic genetic models (OR=1.854 95% CI=1.082-3.187, p=0.016) was observed. However, no statistically significant association of rs17228616 SNP with substance abuse disorder was found. The data presented here shows that AChE could play significant role in substance addiction. Further studies with larger sample size and other variants of AChE are recommended to identify novel therapeutic approaches for cholinergic based treatment of addiction.

Flavonoids, considered as phytoestrogen mainly deriving from fruit and vegetable, are known to have beneficial effects in brain functions. The role of flavonoids in induction of a cholinergic enzyme, acetylcholinesterase (AChE), was being explored here. In cultured PC12cells, twenty-four commonly found flavonoids were tested for its induction on AChE activity. Fourteen flavonoids showed induction, and five of them had robust effect, i.e. daidzin, alpinetin, irisflorentin, cardamonin and lysionotin. The induction of AChE was fully blocked by pre-treatment of G15 (a selective G protein-coupled receptor 30 [GPR 30] antagonist), suggesting a direct involvement of a membrane-bound estrogen receptor, named as GPR 30, in the cultures. In addition, daidzin was further identified to induce expression of tetrameric globular form of proline-rich membrane anchor (PRiMA)-linked AChE. In parallel, application of daidzin in cultured PC12cells significantly induced expression of neurofilaments, markers for neuronal differentiation. Taken together, flavonoids could induce the expression of AChE via GPR 30 in cultured PC12cells, which could be a good candidate for possible treatment of the brain diseases.

Emerging data indicate that prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could interfere with myogenic differentiation in vivo. Acetylcholinesterase (EC3.1.1.7; AChE), an enzyme critical for cholinergic neurotransmission, is abundantly expressed in neurons and mature myotubes, and we recently found that muscle AChE expression was suppressed in parallel with the inhibition of myogenic differentiation upon TCDD treatment in mouse C2C12cells. This TCDD-induced suppression of muscle AChE was proposed to involve an aryl hydrocarbon receptor (AhR)-independent mechanism, but the precise underlying mechanism remains unclear. Considering the widely recognized role of muscular activity in AChE expression and its potential crosstalk with the AhR signaling pathway, we sought to investigate the effect of TCDD on muscle AChE expression in the presence of muscular activity. Therefore, we employed a highly contractile rat primary skeletal muscle culture system in which AChE activity and the expression of genes related to it (AChE T subunit and collagen Q (ColQ)) were increased during the myogenic differentiation process. Although TCDD treatment successfully induced the expression of genes regulated by AhR activation, the treatment exerted no notable effects on myogenic differentiation. Moreover, muscle AChE enzymatic activity and mRNA level remained unchanged following TCDD treatment, and only ColQ mRNA expression was slightly increased after 4-day treatment with TCDD (10(-10)M). The compensatory role of muscle-contraction-related signaling pathways in this newly identified unresponsiveness of muscle AChE to TCDD warrants further investigation.

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: gamma-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 gamma-carbolines and phenothiazine I, gamma-carbolines and carbazoles II, and aminoadamantanes and carbazoles III) as well as inhibitors of both cholinesterases (conjugates of gamma-carbolines and methylene blue IV and bis-gamma-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.

O-Hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) induces delayed neuropathy in hens. It has been used as a tool to identify new A-esterase activities in animal tissues. This study shows the EDTA-resistant, Cu(2+)- and Zn(2+)-dependent hydrolysis of racemic HDCP in domestic and sea bird serum using UV/Vis spectrophotometry and chiral chromatography. The results clearly show a significant (p < 0.05) Cu(2+)- and Zn(2+)-dependent HDCP hydrolysis in the serum of all bird species versus EDTA, except for the Zn(2+)-dependent HDCPase activity from Yucatecan quail serum. The ratio of Cu(2+)/Zn(2+) hydrolysis varied between 1 and 7 (intraspecies) and 15.6 (interspecies). EDTA affected the Cu(2+)- and Zn(2+)-dependent HDCPase activity in the range of 37-95% and 40-50%, respectively. HDCP hydrolysis activated by Cu(2+) was significantly (p < 0.05) stereoselective (R-(+)-HDCP > S-(-)-HDCP) in chicken and sea bird serum. Its R-(+)-HDCP/S-(-)-HDCP ratios were 6.8 and 1.6-2.8, respectively. EDTA-resistant and zinc-dependent HDCP hydrolysis were not stereospecific in all bird sera tested. The present ex vivo study reinforces the idea that bird sera have HDCPase activity that is sensitive to divalent metals, resistant to EDTA and possibly associated with the protein albumin.

Organophosphorus compounds (OP) pose a significant threat. Administration of human butyrylcholinesterase (HuBChE) may reduce or prevent OP toxicity. Thus, we evaluated the safety and efficacy of HuBChE in monkeys using sensitive neurobehavioral tests while concurrently characterizing absorption and elimination in the presence and absence of high-dose soman exposure to predict time course and degree of protection. Eight young adult male cynomolgus macaques were trained on two distinct automated tests of neurobehavioral functioning. HuBChE purified under current Good Manufacturing Practices (CGMP) was injected intramuscularly at 13.1mg/kg, producing an average peak plasma value (Cmax) of over 27 Units/ml. The apparent time to maximum concentration (Tmax) approximated 7h, the elimination half-life approximated 102h, and plasma levels returned to pre-administration (baseline) levels by 14 days. No behavioral disruptions following HuBChE administration were observed on either neurobehavioral test, even in monkeys injected 24h later with an otherwise lethal dose of soman. Thus, HuBChE provided complete neurobehavioral protection from soman challenge. The present data replicate and extend previous results from our laboratory that had used a different route of administration (intravenous), a different species (rhesus macaque), and a different BChE product (non-CGMP material). The addition of two sensitive neurobehavioral tests coupled with the PK/PD results convincingly demonstrates the neurobehavioral safety of plasma-derived HuBChE at therapeutic levels. Protection against an otherwise-lethal dose of soman by a pre-exposure treatment dose that is devoid of side effects establishes a foundation for additional testing using other exposure routes and treatment times, other challenge agents/routes, or other classes of organophosphate scavengers.

The crystal structures of truncated forms of cholinesterases provide good models for assessing the role of non-covalent interactions in dimer assembly in the absence of cross-linking disulfide bonds. These structures identify the four-helix bundle that serves as the interface for formation of acetylcholinesterase and butyrylcholinesterase dimers. Here we performed a theoretical comparison of the structural and energetic factors governing dimerization. This included identification of inter-subunit and intra-subunit hydrogen bonds and hydrophobic interactions, evaluation of solvent-accessible surfaces, and estimation of electrostatic contributions to dimerization. To reveal the contribution to dimerization of individual amino acids within the contact area, free energy perturbation alanine screening was performed. Markov state modelling shows that the loop between the alpha13 and alpha14 helices in BChE is unstable, and occupies 4 macro-states. The order of magnitude of mean first passage times between these macrostates is ~10(-8)s. Replica exchange molecular dynamics umbrella sampling calculations revealed that the free energy of human BChE dimerization is -15.5kcal/mol, while that for human AChE is -26.4kcal/mol. Thus, the C-terminally truncated human butyrylcholinesterase dimer is substantially less stable than that of human acetylcholinesterase. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:CHEMBIOINT:1.

Multitarget ligands (MTL) based on sterically hindered phenol and containing a quaternary ammonium moiety (SHP-n-Q) were synthesized. These compounds are inhibitors of cholinesterases with antioxidant properties. The inhibitory selectivity is 10-fold potent for BChE than for AChE. IC50 of SHP-n-Q for BChE is 20muM. SHP-n-Q and their nanosystems exhibit more pronounced antioxidant properties than the synthetic antioxidant (hindered phenol, butylated hydroxytoluene). These compounds display a low hemolytic activity against human red blood cells. The nanotechnological approach was used to increase the bioavailability of SHP-n-Q derivatives. For water soluble SHP-n-Q derivative, the self-assembled structures have a size close to 100nmat critical association concentration (0.01M). Mixed cationic liposomes based on l-alpha-phosphatidylcholine and SHP-n-Q of 100nm diameter were prepared. The stability, encapsulation efficacy and release from liposomes of a model drug, Rhodamine B, depend on the structure of SHP-n-Q. Cationic liposomes based on l-alpha-phosphatidylcholine and SHP-3-Q show a good stability in time (1year) and a sustained release (>65h). They are promising templates for the development of anti-Alzheimer MT-drug delivery systems.

Organophosphorus compounds have been widely employed to the development of warfare nerve agents and pesticides, resulting in a huge number of people intoxicated annually, being a serious problem of public health. Efforts worldwide have been done in order to design new technologies that are capable of combating or even reversing the poisoning caused by these OP nerve agents. In this line, the bioremediation arises as a promising and efficient alternative for this purpose. As an example of degrading enzymes, there is the organophosphate-degrading (OpdA) enzyme from Agrobacterium radiobacter, which has been quite investigated experimentally due to its high performance in the degradation of neurotoxic nerve agents. This work aims to look into the structural and electronic details that govern the interaction modes of these compounds in the OpdA active site, with the posterior hydrolysis reaction prediction. Our findings have brought about data about the OpdA performance towards different nerve agents, and among them, we may realize that the degradation efficiency strongly depends on the nerve agent structure and its stereochemistry, being in this case the compound Tabun the one more effectively hydrolyzed. By means of the chemical bonds (AIM) and orbitals (FERMO) analysis, it is suggested that the initial reactivity of the OP nerve agents in the OpdA active site does not necessarily dictate the reactivity and interaction modes over the reaction coordinate.

Organophosphorus compounds (OPs) are neurotoxic molecules developed as insecticides and chemical warfare nerve agents (CWNAs). They are covalent inhibitors of acetylcholinesterase (AChE), a key enzyme in central and peripheral nervous systems and are responsible for numerous poisonings worldwide. Many animal models have been studied over the years but finding a suitable in vivo model to account for both acute toxicity and long-term exposure remains a topical issue. Recently, an emerging aquatic animal model harboring a mammalian-like cholinergic nervous system, the freshwater planarian from Platyhelminthes, has been used to investigate neurotoxicity and developmental disruption. Given the tremendous toxicity of OPs, various bioremediation strategies have been considered over the years to counter their poisonous effects. Among these, enzymes have been particularly highlighted as they can degrade OPs in a fast, non toxic and environmentally friendly manner. In this article we investigated the biotechnological potential for decontaminating OPs of the previously reported variant SsoPox-alphasD6 from the hyperstable enzyme SsoPox, isolated from the archaea Sulfolobus solfataricus. The capacity to hydrolyze 4 new substrates (methyl-pirimiphos, quinalphos, triazophos and dibrom) was demonstrated and the degradation products generated by enzymatic hydrolysis were characterized. We further evaluated the capacity of SsoPox-alphasD6 for in vivo protection of freshwater planarians Schmidtea mediterranea (Smed). The use of SsoPox-alphasD6 drastically decreased mortality and enhanced mobility of planarians. Then, an enzyme-based filtration device was developed by immobilizing intact Escherichia coli cells expressing SsoPox-alphasD6 into alginate beads. The efficacy of the device was demonstrated using planarians as biosensors.

Studies with oximes have been extensively developed to design new reactivators with better efficiency, and greater spectrum of action. In this study, we aimed to analyze the influence of the Carbamoyl group position change in two isomeric oximes, K203 and K206, on the reactivation percentage of Mus musculus Acetylcholinesterase (MmAChE), inhibited by different nerve agents. Theoretical calculations were performed to assess the difference for the oxime activity with inhibited AChE-complexes and the factors that govern this difference. Comparing theoretical and experimental data, it is possible to observe that this change between the oximes results in different reactivation percentage for the same nerve agent, due to the different interaction modes and activation energy for the studied systems.

Carbamates are esters of substituted carbamic acids that react with acetylcholinesterase (AChE) by initially transferring the carbamoyl group to a serine residue in the enzyme active site accompanied by loss of the carbamate leaving group followed by hydrolysis of the carbamoyl enzyme. This hydrolysis, or decarbamoylation, is relatively slow, and half-lives of carbamoylated AChEs range from 4min to more than 30 days. Therefore, carbamates are effective AChE inhibitors that have been developed as insecticides and as therapeutic agents. In this report, we review recent data showing that decarbamoylation rate constants are independent of the ester leaving group for a series of carbamic acid esters with the same carbamoyl group and that decarbamoylation rate constants decreased by 800-fold when the alkyl substituents on the carbamoyl group increased in size from N-monomethyl- to N,N-diethyl-. We also review data showing that solvent deuterium oxide isotope effects for decarbamoylation decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE, indicating a shift in the rate-limiting step from general acid-base catalysis to a likely conformational change in the distorted active site in N,N-diethylcarbamoyl AChE. The nature of such a conformational change is suggested from X-ray crystal structures of AChE phosphorylated by paraoxon.

Acetylcholinesterase (EC3.1.1.7; AChE) is a key enzyme in the cholinergic system. Emerging evidence has shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a typical persistent organic pollutant, suppressed neuronal AChE activity via dysregulation of different biosynthesis processes in human and rat neuronal cells. In the nervous system, astrocytes protect neurons from environmental pollutants. As a known target cell of TCDD, the astrocyte might be involved in TCDD effects on neuronal AChE. Therefore, in the present study, we found astrocyte-derived conditioned medium (ACM) could induce AChE activity preferentially in mature neurons in the absence of TCDD. The enzymatic activity of AChE was generally decreased in cultured cortical neurons upon direct treatment with TCDD (0.003-0.01nM). This trend of changes in AChE activity was not significantly altered in immature neurons exposed to ACM produced in the presence of TCDD (TACM group), but reversed in mature neurons. Compared with effects of treatment with ACM plus TCDD (ACMT), a significant differential effect on AChE activity was found in the TACM group in response to TCDD treatment specifically in immature neurons, suggesting the presence of a TCDD-specific active component derived from the astrocyte. Inconsistent alterations in expression and enzymatic activities of the AChE T subunit (AChET) and the proline-rich membrane anchor (PRiMA) were found, suggesting that a mechanism of action beyond the transcriptional level might be involved. These data indicate that the astrocyte might play a protective role in TCDD-induced alterations of neuronal AChE in certain stages of differentiation.

In this study, 68 crystal structures of complexes between acetylcholinesterase (AChE, EC 3.1.1.7) and its ligands, deposited in the PDB, were analyzed by scoring the functions: LigScore1, LigScore2, PLP1, PLP2, Jain, PMF and PMF04. The scores derived from scoring functions were correlated with an inhibition constant for each ligand (Ki or IC50) in a broad range 10(-3) - 10(-12)M. The linear correlation model resulted in the highest coefficient of determination (r(2)) for the PLP2 function, 0.591. The LigScore1 function resulted in the lowest r(2) value of 0.226. The PubChem database was the source of in silico computed ligand properties which were then correlated with an inhibition constant for each ligand. For the purposes of this study, two additional non-PubChem parameters were evaluated: total and relative number of sp(2) hybridized atoms in the ligand. A high coefficient of determination (r(2)>0.5) was calculated for the following parameters: the number of heavy atoms, molecular mass, and number of atoms with sp(2) hybridization. The PLP2 scoring function is a good candidate for drug discovery related to AChE, although a better scoring function could be developed with a higher number of crystal structures of AChE complexes and more reliable kinetic data.

Magnolol, the most abundant bioactive constituent of the Chinese herb Magnolia officinalis, has been found with multiple biological activities, including anti-oxidative, anti-inflammatory and enzyme-regulatory activities. In this study, the inhibitory effects and inhibition mechanism of magnolol on human carboxylesterases (hCEs), the key enzymes responsible for the hydrolytic metabolism of a variety of endogenous esters as well as ester-bearing drugs, have been well-investigated. The results demonstrate that magnolol strongly inhibits hCE1-mediated hydrolysis of various substrates, whereas the inhibition of hCE2 by magnolol is substrate-dependent, ranging from strong to moderate. Inhibition of intracellular hCE1 and hCE2 by magnolol was also investigated in living HepG2 cells, and the results showed that magnolol could strongly inhibit intracellular hCE1, while the inhibition of intracellular hCE2 was weak. Inhibition kinetic analyses and docking simulations revealed that magnolol inhibited both hCE1 and hCE2 in a mixed manner, which could be partially attributed to its binding at two distinct ligand-binding sites in each carboxylesterase, including the catalytic cavity and the regulatory domain. In addition, the potential risk of the metabolic interactions of magnolol via hCE1 inhibition was predicted on the basis of a series of available pharmacokinetic data and the inhibition constants. All these findings are very helpful in deciphering the metabolic interactions between magnolol and hCEs, and also very useful for avoiding deleterious interactions via inhibition of hCEs.

Activation of human butyrylcholinesterase by small quaternary ammonium ions is known. Here, additional ligands in this series are presented: edrophonium and choline, and the reactivator pyridine-2-aldoxime methochloride. Kinetic analysis of the progress curves with these compounds indicates the mechanism of enhanced deacylation by the ligand bound to the catalytic anionic site (Trp82) at the base of the active site. The larger, bis-quaternary ligands examined, as propidium, hexamethonium, decamethonium, and bis-thiocholine, show only competitive inhibition of butyrylcholinesterase, by preventing substrate approach. This hypothesis of enhanced deacylation was tested for reactivation of methanesulfonylfluoride-inactivated butyrylcholinesterase, a complex analogous to organophosphate-aged cholinesterases. The combination of substrate/products and pyridine-2-aldoxime methochloride improved butyrylcholinesterase activity over 2h of continuous measurements, before which time substrate depletion prevailed. Similar reactivation of Torpedo californica acetylcholinesterase was unsuccessful, but both of these cholinesterases regain some activity if they have been inhibited and aged for days by diisopropylfluorophosphate.

Since the development in the 1950's of 2-PAM (Pralidoxime), an antidote that reactivates organophosphate conjugated acetylcholinesterase in target tissues upon pesticide or nerve agent exposure, improvements in antidotal therapy have largely involved congeneric pyridinium aldoximes. Despite seminal advances in detailing the structures of the cholinesterases as the primary target site, progress with small molecule antidotes has yet to define a superior agent. Two major limitations are immediately apparent. The first is the impacted space within the active center gorge, particularly when the active center serine at its base is conjugated with an organophosphate. The reactivating nucleophile will have to negotiate the tortuous gorge terrain to access the phosphorus atom with its most nucleophilic form or ionization state, the oximate anion. A second limitation stems from the antidote crossing the blood-brain barrier sufficiently rapidly, since it is well documented that central acetylcholinesterase inhibition gives rise to cardiovascular and respiratory compromise. The associated hypoxia then leads to a sequelae of events, including poor perfusion of the brain and periphery, along with muscle fasciculation, tremors and eventually seizures. We consider both the barriers confronting and further achievements necessary to enhance efficacy of antidotes.

AIMS: K117 and K127 are bis-pyridinium aldoximes but K117 is a bis-pyridinium bis-aldoxime while K127 has only one single aldoxime in addition to its amide substituent. Is there any difference in pharmacokinetics in these compounds that otherwise have the same chemical structure? Both K117 and K127 are developed as antidotes in acetylcholinesterase and butyrylcholinesterase poisoning in terrorist attacks or intoxication with other organophosphorous compounds. Their distributions have been scouted in the bodies of rats. MAIN METHODS: White male Wistar rats were intramuscularly injected. The animals were sacrificed, tissue samples were homogenized, and either K117 or K127 concentrations were determined using reversed-phase high-performance liquid chromatography. KEY FINDINGS: Both K117 and K127 were present in all tissues that were analyzed including blood (serum), the brains, cerebrospinal fluid, the eyes, livers, kidneys, lungs and testes. Their pharmacokinetics and body distributions are similar. SIGNIFICANCE: Either K117 or K127 meets the essential requirements for antidotes. Dose dependence and kinetics of their distribution were compared to that of other pyridinium aldoximes.

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.

Acetylcholinesterase (AChE) hydrolyzes acetylcholine at cholinergic synapses, and which has various isoforms of AChE, i.e. AChER, AChEH and AChET, deriving from single gene. AChEH exists as a glycophosphatidylinositol (GPI)-linked dimer (G2), presents mainly in plasma membrane of mammalian erythrocyte. Transgenic mice with ACHE gene depletion were employed here to investigate the possible role of AChE in blood cell formation. ACHE knock-out mice were found to suffer normocytic anemia. In erythrocyte of ACHE-/- mice, the amount of hemoglobin, especially alpha-globin, was found to be markedly reduced. In addition, the number of erythrocyte and hematocrit of ACHE-/- mice were significantly lowered. To probe the role of AChE isoforms in erythroid differentiation, erythroblast-like cells (TF-1) over-expressed with different AChE isoforms were induced to differentiate by erythropoietin (EPO): this differentiation induced the expression of each AChE isoform. Only in the TF-1cells over-expressed with AChEH, the EPO-induced transcriptions and protein expressions of alpha- and beta-globins could be significantly enhanced, which therefore suggested that AChEH might regulate the responsiveness of TF-1cells to EPO. The alternation of EPO-induced downstream signaling might be accounted by association of AChE with EPO receptor in cell surface. The findings indicated the significance of AChE in erythroblast maturation, which provided an insight in elucidating possible mechanisms in regulating erythropoiesis.

According to recent research advance, it is interesting to identify new, potent and selective inhibitors of human butyrylcholinesterase (BChE) for therapeutic treatment of both the Alzheimer's disease (AD) and heroin abuse. In this study, we carried out a structure-based virtual screening followed by in vitro activity assays, with the goal to identify new inhibitors that are selective for BChE over acetylcholinesterase (AChE). As a result, a set of new, selective inhibitors of human BChE were identified from natural products with solanaceous alkaloid scaffolds. The most active one of the natural products (compound 1) identified has an IC50 of 16.8nM against BChE. It has been demonstrated that the desirable selectivity of these inhibitors for BChE over AChE is mainly controlled by three key residues in the active site cavity, i.e. residues Q119, A277, and A328 in BChE versus the respective residues Y124, W286, and Y337 in AChE. Based on this structural insight, future rational design of new, potent and selective BChE inhibitors may focus on these key structural differences in the active site cavity.

Bispyridinium oximes with one (K865, K866, K867) or two (K868, K869, K870) ortho-positioned chlorine moiety, analogous to previously known K027, K048 and K203 oximes, and potent reactivators of human acetylcholinesterase (AChE) inhibited by nerve agents, were tested in the reactivation of human butyrylcholinesterase (BChE) inhibited by sarin, cyclosarin, VX, and tabun. A previously highlighted AChE reactivator, dichlorinated bispyridinium oxime with propyl linker (K868), was tested in more detail for reactivation of four nerve agent-BChE conjugates. Its BChE reactivation potency was showed to be promising when compared to the standard oximes used in medical practice, asoxime (HI-6) and pralidoxime (2-PAM), especially in case of sarin and tabun. This finding could be used in the pseudo-catalytic scavenging of the most nerve agents due to its cumulative capacity to reactivate both AChE and BChE.

The novel prophylactic agent 7-methoxytacrine-4-pyridinealdoxime is a hybrid compound formerly designed to keep acetylcholinesterase resistant to organophosphates by reactivating it in case of intoxication by such inhibitors. In rational design, a 5-carbon length-spacer hybrid compound was synthesized to evaluate its inhibitory and reactivation capabilities. In this work, theoretical results were achieved through molecular modelling techniques, taking for granted the enzymatic reactivation reaction through nucleophilic substitution. Based on the near attack conformation approach, docking studies were performed to assess the spacer-length from 1 to 10 carbons long of a series of analogues of 7-methoxytacrine-4-pyridinealdoxime. Consequently, the hybrids with length-spacer of 4 and 5 carbons long were the best assessed and subsequently subjected to further molecular dynamics simulations, complemented by Poisson-Boltzmann surface area calculations. As a result, intermolecular interactions with the different binding sites inside human acetylcholinesterase were elucidated. Besides, thermodynamics and kinetics concepts pointed to the 4-carbon linker as optimum for enzymatic reactivation. Further studies, based on quantum mechanics in conjunction with molecular mechanics, were recommended to the presented near attack conformations to achieve more thermodynamics results between the hybrids with 4- and 5-carbon linkers, like values of activation energy for the reactivation reaction. All of those in silico evaluations could be considered as a set of tools for theoretically investigate novel enzymatic reactivators with different shape of spacers.

Aflatoxin M1 (AFM1) is a mycotoxin produced by Aspergillus fungi and found in contaminated milk, breastfeed and dairy products, being highly toxic and carcinogenic to humans and other mammalian species. It is also produced in the human body as a metabolite of aflatoxin B1 (AFB1), one of the most toxic natural products known. Previous studies have shown that AFM1 is a potential inhibitor of the enzyme acetylcholinesterase (AChE), and therefore, a potential neurotoxic agent. In this work, surface screening (SS) and molecular dynamics (MD) simulation on human acetylcholinesterase AChE (HssAChE) were performed to corroborate literature data regarding preferential binding sites and type of inhibition. Also, an inedited theoretical study on the interactions of AFM1 with human butyrylcholinesterase (HssBChE) was performed. In vitro inhibition tests on both enzymes were done to support theoretical results. MD simulations suggested the catalytic anionic site of HssAChE as the preferential binding site for AFM1 and also that this metabolite is not a good inhibitor of HssBChE, corroborating previous studies. In vitro assays also corroborated molecular modeling studies by showing that AFM1 did not inhibit BChE and was able to inhibit AChE, although not as much as AFB1.