Recombinant Acetylcholinesterase Research Articles

Interaction of exogenous acetylcholinesterase and butyrylcholinesterase with amyloid-β plaques in human brain tissue

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are associated with amyloid-β (Aβ) plaques and exhibit altered biochemical properties in human Alzheimer's disease (AD), as well as in the transgenic 5XFAD mouse model of AD amyloidosis. In the brains of the 5XFAD mouse model devoid of BChE enzyme (5XFAD/BChE-KO), incubation of tissue sections with exogenous BChE purified from human plasma (pl-BChE) leads to its association with Aβ plaques and its biochemical properties are comparable to those reported for endogenous BChE associated with plaques in both human AD and in 5XFAD mouse brain tissue. We sought to determine whether these observations in 5XFAD/BChE-KO mice also apply to human brain tissues. To do so, endogenous ChE activity in human AD brain tissue sections was quenched with 50 % aqueous acetonitrile (MeCNaq) leaving the tissue suitable for further studies. Quenched sections were then incubated with recombinant AChE (r-AChE) or pl-BChE and stained for each enzymes' activity. Exogenous r-AChE or pl-BChE became associated with Aβ plaques, and when bound, had properties that were comparable to the endogenous ChE enzymes associated with plaques in AD brain tissues without acetonitrile treatment. These findings in human AD brain tissue extend previous observations in the 5XFAD/BChE-KO mouse model and demonstrate that exogenously applied r-AChE and pl-BChE have high affinity for Aβ plaques in human brain tissues. This association alters the biochemical properties of these enzymes, most likely due a conformational change. If incorporation of AChE and BChE in Aβ plaques facilitates AD pathogenesis, blocking this association could lead to disease-modifying approaches to AD. This work provides a method to study the mechanism of AChE and BChE interaction with Aβ plaque pathology in post-mortem human brain tissue.

A-series agent A-234: initial in vitro and in vivo characterization

A-series agent A-234 belongs to a new generation of nerve agents. The poisoning of a former Russian spy Sergei Skripal and his daughter in Salisbury, England, in March 2018 led to the inclusion of A-234 and other A-series agents into the Chemical Weapons Convention. Even though five years have already passed, there is still very little information on its chemical properties, biological activities, and treatment options with established antidotes. In this article, we first assessed A-234 stability in neutral pH for subsequent experiments. Then, we determined its inhibitory potential towards human recombinant acetylcholinesterase (HssAChE; EC 3.1.1.7) and butyrylcholinesterase (HssBChE; EC 3.1.1.8), the ability of HI-6, obidoxime, pralidoxime, methoxime, and trimedoxime to reactivate inhibited cholinesterases (ChEs), its toxicity in rats and therapeutic effects of different antidotal approaches. Finally, we utilized molecular dynamics to explain our findings. The results of spontaneous A-234 hydrolysis showed a slow process with a reaction rate displaying a triphasic course during the first 72 h (the residual concentration 86.2%). A-234 was found to be a potent inhibitor of both human ChEs (HssAChE IC50 = 0.101 ± 0.003 µM and HssBChE IC50 = 0.036 ± 0.002 µM), whereas the five marketed oximes have negligible reactivation ability toward A-234-inhibited HssAChE and HssBChE. The acute toxicity of A-234 is comparable to that of VX and in the context of therapy, atropine and diazepam effectively mitigate A-234 lethality. Even though oxime administration may induce minor improvements, selected oximes (HI-6 and methoxime) do not reactivate ChEs in vivo. Molecular dynamics implies that all marketed oximes are weak nucleophiles, which may explain the failure to reactivate the A-234 phosphorus-serine oxygen bond characterized by low partial charge, in particular, HI-6 and trimedoxime oxime oxygen may not be able to effectively approach the A-234 phosphorus, while pralidoxime displayed low interaction energy. This study is the first to provide essential experimental preclinical data on the A-234 compound.

Optimization of Expression and Purification of Recombinant One of Central Nervous System Enzyme (Acetylcholinesterase)

In general, the use of natural enzymes is very limited and they are not economical to use in industry. To solve this problem, the recombinant expression systems approach has been used. Optimization of production processes in heterologous expression systems is one of the most important activities in today's competitive industries. One of the key factors for the commercialization of recombinant proteins is the achievement of a low-cost culture medium available for the production of suitable cell biomass that can be used on an industrial scale. Commercialization of recombinant products requires process economics and cost reduction, and reducing the cost of the product is possible by using a cheaper culture medium, so decided optimization of expression and purification of recombinant acetylcholinesterase (EC.3.1.1.7), because it’s one of the body's vital enzymes, that by hydrolyzing acetylcholine, which is a neurotransmitter, terminates neural messages in the area of cholinergic synapses. Because the enzyme acetylcholinesterase produced from natural sources is not suitable for industrial purposes and semi-mass production due to low production efficiency and high costs of enzyme preparation and purification processes, optimizing the growth conditions of microorganisms capable of the production and secretion of the enzyme acetylcholinesterase are very important, both in terms of yield and economic costs. The present study seeks to increase productivity and reduce the cost of producing this enzyme by optimizing the growth medium and expression of pichia pastoris yeast cells that have genes related to this enzyme and also by optimizing the purification and mass production of this enzyme.

Nutritional evaluation of probiotics enriched rabadi beverage (PERB) and molecular mapping of digestive enzyme with dietary fibre for exploring the therapeutic potential

Advent of the silent pandemic of obesity, which is associated with onset of various non-communicable and communicable disorders; led for the necessity to develop a functional food product with good nutritional profile to improve the overall health. Therefore, the current study was aimed to develop a probiotic enriched rabadi beverage. The beverage was prepared by using pearl millet, buttermilk and fortified with L. rhamnosus, Lactobacillus sp and S. faecalis. Subsequently, analysed for different parameters. Molecular mapping was also performed to show the inhibition profiling between cellulose and the human digestive enzymes. There was a significant difference for pH, acidity, viscosity, dry matter, and organic matter of PERB for autoclaved and non-autoclaved batches in all the used strains. However, a non-significant difference was observed in terms of different fermentation temperatures. The ash content varied significantly (0.32 ± 0.02%) in beverages fortified with S. faecalis fermented at 350 C, whereas moisture content had a non-significant effect on all the strains at different fermentation temperatures. Molecular mapping revealed the inhibition profiling in terms of binding affinity (kcal/mol) for various human digestive enzymes (Recombinant Acetylcholinesterase: −8.1, lysosomal acid-alpha-glucosidase: −6.6, and pancreatic alpha-amylase: −6.4) with cellulose. The study concluded that the PERB has a good amount of ADF, NDF, and hemicellulose content as well as indicated the significance of dietary fibre (cellulose) in regulating the digestibility through the interaction of gastrointestinal enzymes in order to explore the therapeutic potential of the PERB. Therefore, the developed PERB would serve as a potential source for better nutrition and overall health.

Halogen substituents enhance oxime nucleophilicity for reactivation of cholinesterases inhibited by nerve agents

The fluorinated bis-pyridinium oximes were designed and synthesized with the aim of increasing their nucleophilicity and potential to reactivate phosphorylated human recombinant acetylcholinesterase (AChE) and human purified plasmatic butyrylcholinesterase (BChE) in relation to chlorinated and non-halogenated oxime analogues. Compared to non-halogenated oximes, halogenated oximes showed lower pKa of the oxime group (fluorinated < chlorinated < non-halogenated) along with higher level of oximate anion formation at the physiological pH, and had a higher binding affinity of both AChE and BChE. The stability tests showed that the fluorinated oximes were stable in water, while in buffered environment di-fluorinated oximes were prone to rapid degradation, which was reflected in their lower reactivation ability. Mono-fluorinated oximes showed comparable reactivation to non-halogenated (except asoxime) and mono-chlorinated oximes in case of AChE inhibited by sarin, cyclosarin, VX, and tabun, but were less efficient than di-chlorinated ones. The same trend was observed in the reactivation of inhibited BChE. The advantage of halogen substituents in the stabilization of oxime in a position optimal for in-line nucleophilic attack were confirmed by extensive molecular modelling of pre-reactivation complexes between the analogue oximes and phosphorylated AChE and BChE. Halogen substitution was shown to provide oximes with additional beneficial properties, e.g., fluorinated oximes gained antioxidative capacity, and moreover, halogens themselves did not increase cytotoxicity of oximes. Finally, the in vivo administration of highly efficient reactivator and the most promising analogue, 3,5-di-chloro-bispyridinium oxime with trimethylene linker, provided significant protection of mice exposed to sarin and cyclosarin.

In vitro studies of a series of synthetic compounds for their anti-acetylcholinesterase activities identified arylpyrano[2,3-f]coumarins as hit compounds

Alzheimer's disease is one of the most widespread neurodegenerative diseases in the world. Until now, the drugs used to control this disease have been developed to target acetylcholinesterase (AChE). With the aim to identify new pharmacophores that can promisingly inhibit this enzyme, a library of 72 compounds belonging to chalcones, pyranochromenes, chromenes, pyranopyrazoles and dihydropyridines, was evaluated against AChE. Nineteen of these compounds are newly reported and among the tested compounds, seven, bearing a pyranochromene skeleton, showed inhibitory effects with IC50 values ranging from 9.01 to 32.39 µM. The most potent was identified as 8-amino-10-(4-bromophenyl)-5‑hydroxy-4-methyl-2-oxo-2,10-dihydropyrano[2,3-f]chromeno-9-carbonitrile. In silico studies using human recombinant acetylcholinesterase (PDB: 4EY7) showed important proximities between the NH2 group of this compound and E202 in AChE, between W86 and the OH-5 group, between W86 and the aromatic bicyclic system (π-π stacking, 4.2 Å), and between Y133 and both the OH-7 and the NH2 groups. These pyranochromenes emerged as a promising prototype for the development of new candidates against AChE.

Derivatives of montanine-type alkaloids and their implication for the treatment of Alzheimer's disease: Synthesis, biological activity and in silico study

Alzheimeŕs disease (AD) is the most common neurodegenerative disorder, characterized by neuronal loss and cognitive impairment. Currently, very few drugs are available for AD treatment, and a search for new therapeutics is urgently needed. Thus, in the current study, twenty-eight new derivatives of montanine-type Amaryllidaceae alkaloids were synthesized and evaluated for their ability to inhibit human recombinant acetylcholinesterase (hAChE) and butyrylcholinesterase (hBuChE). Three derivatives (1n, 1o, and 1p) with different substitution patterns demonstrated significant selective inhibitory potency for hAChE (IC50 < 5 µM), and one analog, 1v, showed selective hBuChE inhibition activity (IC50 = 1.73 ± 0.05 µM). The prediction of CNS availability, as disclosed by the BBB score, suggests that the active compounds in this survey should be able pass through the blood–brain barrier (BBB). Cytotoxicity screening and docking studies were carried out for the two most pronounced cholinesterase inhibitors, 1n and 1v.

Enhanced Secretory Expression and Surface Display Level of Bombyx mori Acetylcholinesterase 2 by Pichia pastoris Based on Codon Optimization Strategy for Pesticides Setection.

The cholinesterase-based spectrophotometric assay, also called enzyme inhibition method, is a good choice for rapid detection of organophosphate pesticides (OPs) and carbamate pesticides (CPs). Obviously, the cholinesterase is the core reagent in enzyme inhibition method. In our previous work, a recombinant acetylcholinesterase 2 from Bombyx mori (rBmAChE2) was expressed in yeast successfully and exhibited great sensitivity. However, the yield of rBmAChE2 is not desirable. In this study, a codon optimization strategy was employed to enhance the yield of rBmAChE2 in Pichia pastoris GS115. Results showed that by replacing 6 key rare codons and increasing the percentage of bases G and C up to 46.85%, codon adaptation index (CAI) of Bombyx mori acetylcholinesterase 2 (bmace2) gene was improved from 0.70 to 0.81. After being transformed into Pichia pastoris GS115 via electroporation, the expression transformant can produce 139.7 U/mL secretory codon-optimized rBmAChE2 (opt-rBmAChE2) in the culture supernatant, 3.62 times higher than that of strain bearing the wild-type bmace2 gene. Meanwhile, opt-rBmAChE2 displayed on the yeast surface was up to 2280.02 U/g, 2.8 times higher than wild-type displayed rBmAChE2. In addition, either secretory or surface-displayed opt-rBmAChE2 maintained the similar sensitivities to the wild-type rBmAChE2 for tested inhibitors. Furthermore, the detection limits of the opt-rBmAChE2-based enzyme inhibition method for 10 kinds of OPs or CPs (0.01-2.69 mg/kg) were lower than most of the indexes present in current standard method (GB/T 5009.199-2003) or the maximum residue limits (GB 2763-2019) in China. The results might contribute to the utilization of rBmAChE2 for pesticide residue screening detection in practice.

Effects of essential oils on native and recombinant acetylcholinesterases of Rhipicephalus microplus.

This study reports the action of essential oils (EO) from five plants on the activity of native and recombinant acetylcholinesterases (AChE) from Rhipicephalus microplus. Enzyme activity of native susceptible AChE extract (S.AChE), native resistant AChE extract (R.AChE), and recombinant enzyme (rBmAChE1) was determined. An acetylcholinesterase inhibition test was used to verify the effect of the EO on enzyme activity. EO from Eucalyptus globulus, Citrus aurantifolia, Citrus aurantium var.dulcis inhibited the activity of S.AChE and R.AChE. Oils from the two Citrus species inhibited S.AChE and R.AChE in a similar way while showing greater inhibition on R.AChE. The oil from E. globulus inhibited native AChE, but no difference was observed between the S.AChE and R.AChE; however, 71% inhibition for the rBmAChE1 was recorded. Mentha piperita oil also inhibited S.AChE and R.AChE, but there was significant inhibition at the highest concentration tested. Cymbopogon winterianus oil did not inhibit AChE. Further studies are warranted with the oils from the two Citrus species that inhibited R.AChE because of the problem with R. microplus resistant to organophosphates, which target AChE. C. winterianus oil can be used against R. microplus populations that are resistant to organophosphates because its acaricidal properties act by mechanism(s) other than AChE inhibition.

Pyridinium-2-carbaldoximes with quinolinium carboxamide moiety are simultaneous reactivators of acetylcholinesterase and butyrylcholinesterase inhibited by nerve agent surrogates

The pyridinium-2-carbaldoximes with quinolinium carboxamide moiety were designed and synthesised as cholinesterase reactivators. The prepared compounds showed intermediate-to-high inhibition of both cholinesterases when compared to standard oximes. Their reactivation ability was evaluated in vitro on human recombinant acetylcholinesterase (hrAChE) and human recombinant butyrylcholinesterase (hrBChE) inhibited by nerve agent surrogates (NIMP, NEMP, and NEDPA) or paraoxon. In the reactivation screening, one compound was able to reactivate hrAChE inhibited by all used organophosphates and two novel compounds were able to reactivate NIMP/NEMP-hrBChE. The reactivation kinetics revealed compound 11 that proved to be excellent reactivator of paraoxon-hrAChE better to obidoxime and showed increased reactivation of NIMP/NEMP-hrBChE, although worse to obidoxime. The molecular interactions of studied reactivators were further identified by in silico calculations. Molecular modelling results revealed the importance of creation of the pre-reactivation complex that could lead to better reactivation of both cholinesterases together with reducing particular interactions for lower intrinsic inhibition by the oxime.

Acetylcholinesterase Inhibition of Diversely Functionalized Quinolinones for Alzheimer's Disease Therapy.

In this communication, we report the synthesis and cholinesterase (ChE)/monoamine oxidase (MAO) inhibition of 19 quinolinones (QN1-19) and 13 dihydroquinolinones (DQN1-13) designed as potential multitarget small molecules (MSM) for Alzheimer’s disease therapy. Contrary to our expectations, none of them showed significant human recombinant MAO inhibition, but compounds QN8, QN9, and DQN7 displayed promising human recombinant acetylcholinesterase (hrAChE) and butyrylcholinesterase (hrBuChE) inhibition. In particular, molecule QN8 was found to be a potent and quite selective non-competitive inhibitor of hrAChE (IC50 = 0.29 µM), with Ki value in nanomolar range (79 nM). Pertinent docking analysis confirmed this result, suggesting that this ligand is an interesting hit for further investigation.

Structural and functional characterization of an organometallic ruthenium complex as a potential myorelaxant drug

In addition to antibacterial and antitumor effects, synthetic ruthenium complexes have been reported to inhibit several medicinally important enzymes, including acetylcholinesterase (AChE). They may also interact with muscle-type nicotinic acetylcholine receptors (nAChRs) and thus affect the neuromuscular transmission and muscle function. In the present study, the effects of the organometallic ruthenium complex of 5-nitro-1,10-phenanthroline (nitrophen) were evaluated on these systems. The organoruthenium-nitrophen complex [(η6-p-cymene)Ru(nitrophen)Cl]Cl; C22H21Cl2N3O2Ru (C1-Cl) was synthesized, structurally characterized and evaluated in vitro for its inhibitory activity against electric eel acetylcholinesterase (eeAChE), human recombinant acetylcholinesterase (hrAChE), horse serum butyrylcholinesterase (hsBChE) and horse liver glutathione-S-transferase. The physiological effects of C1-Cl were then studied on isolated mouse phrenic nerve-hemidiaphragm muscle preparations, by means of single twitch measurements and electrophysiological recordings. The compound C1-Cl acted as a competitive inhibitor of eeAChE, hrAChE and hsBChE with concentrations producing 50 % inhibition (IC50) of enzyme activity ranging from 16 to 26 μM. Moreover, C1-Cl inhibited the nerve-evoked isometric muscle contraction (IC50 = 19.44 μM), without affecting the directly-evoked muscle single twitch up to 40 μM. The blocking effect of C1-Cl was rapid and almost completely reversed by neostigmine, a reversible cholinesterase inhibitor. The endplate potentials were also inhibited by C1-Cl in a concentration-dependent manner (IC50 = 7.6 μM) without any significant change in the resting membrane potential of muscle fibers up to 40 μM. Finally, C1-Cl (5–40 μM) decreased (i) the amplitude of miniature endplate potentials until a complete block by concentrations higher than 25 μM and (ii) their frequency at 10 μM or higher concentrations. The compound C1-Cl reversibly blocked the neuromuscular transmission in vitro by a non-depolarizing mechanism and mainly through an action on postsynaptic nAChRs. The compound C1-Cl may be therefore interesting for further preclinical testing as a new competitive neuromuscular blocking, and thus myorelaxant, drug.

Synthesis, characterization, crystal structure and evaluation of four carbazole-coumarin hybrids as multifunctional agents for the treatment of Alzheimer’s disease

Coupling of two distinct pharmacophores, carbazole and coumarin, endowed with different biological properties, afforded four hybrid compounds. The structures of the carbazole-coumarin hybrids were characterized by FT-IR, NMR, HRMS and single-crystal X-ray diffraction studies. All of these compounds exhibited significant acetylcholinesterase inhibitory activities. Among them, compound 4-((5-(9H-carbazol-9-yl)pentyl)oxy)-2H-chromen-2-one (3d) exhibited the best inhibition activity with IC50 of 3.75 μM for acetylcholinesterase from electric eel and 70.51 μM for human recombinant acetylcholinesterase. Moreover, the compound 7-(3-(9H-carbazol-9-yl propoxy)-4-methyl-2H-chromen-2-one (3a) had the best antioxidant activity. The docking studies demonstrated that compound 3d could interact with both the catalytic active site and the peripheral anionic site of acetylcholinesterase. These attributes imply carbazole-coumarin hybrids as multifunctional agents for the Alzheimer’s disease treatment.

Mouse Model for Assessing the Subchronic Toxicity of Organophosphate Pesticides

The development of antidotes to organophosphate poisons is an important aspect of modern pharmacology. Recombinant acetylcholinesterase and butyrylcholinesterase are effective DNA-encoded acceptors of organophosphate poisons and, in particular, pesticides. Here, we present the results of a study on the effectiveness of recombinant butyrylcholinesterase (BChE) in modeling organophosphate poisoning caused by oral administration of paraoxon at a dose of 2 mg / kg. The study showed a high activity of BChE as a protective agent for subchronic anticholinesterase poisoning in an in vivo model. The administration of BChE in a dose of 20 mg / kg allows one to avoid mortality, and also contributed to rapid recovery after model poisoning.

Stripe‐shaped Electrochemical Biosensor for Organophosphate Pesticide

AbstractDetection of organophosphate pesticides is indispensable to improve the quality of the water and therefore required by the European Union law. Here, we propose a novel architecture of a pesticide biosensor where the enzyme and the electrode are placed into neighboring zones in the stripe form. This separation permits to use the optimal conditions for their immobilization resulting in undisrupted mechanical stability of the biosensor. The enzyme, acetylcholinesterase, generates thiocholine which is further oxidized at the carbon nanoparticles film electrode, exhibiting high electrocatalytic activity. The whole device was tested as a biosensor for malaoxon employing a wild or recombinant acetylcholinesterase. The latter allowed for incubation time decrease down to 10 minutes and lowered the detection limit to 0.25 nM. Moreover, the encapsulated enzyme activity was visualised by a scanning electrochemical microscopy.

Novel deoxyvasicinone derivatives as potent multitarget-directed ligands for the treatment of Alzheimer's disease: Design, synthesis, and biological evaluation

A series of multitarget ligands was designed by introducing several structurally diverse aminoacetamide groups at position 6 of the deoxyvasicinone group, with the aim of obtaining novel multifunctional anti-Alzheimer's disease agents using deoxyvasicinone as the substrate. In vitro studies showed that almost all of the derivatives were potent inhibitors of human recombinant acetylcholinesterase (hAChE) and human serum butyrylcholinesterase (hBChE), with IC50 values in the low nanomolar range, and exhibited moderate to high inhibition of Aβ1−42 self-aggregation. In particular, compounds 12h, 12n, and 12q showed promising inhibitory activity for hAChE, with IC50 values of 5.31 ± 2.8, 4.09 ± 0.23, and 7.61 ± 0.53 nM, respectively. Compounds 12h and 12q also exhibited the greatest ability to inhibit hBChE, with IC50 values of 4.35 ± 0.32 and 2.35 ± 0.14 nM, respectively. Moreover, enzyme kinetics confirmed that compound 12q caused a mixed type of AChE inhibition, by binding to both the active sites (PAS and CAS) of AChE. Remarkably, compound 12q also demonstrated the highest potential inhibitory activity for Aβ1−42 self-aggregation (63.9 ± 4.9%, 10 μM), and it was also an excellent metal chelator.

Recombinant Acetylcholinesterase purification and its interaction with silver nanoparticle

Although the use of silver nanoparticles (AgNPs) has substantial benefits, their entrance into the environment, food chain, and human body and their toxicity have come under serious scrutiny. Multiple noncovalent attractive forces between AgNPs and bio-macromolecules are responsible for immediate corona formation upon exposure to biological tissue. Here, the influence of AgNPs with neuro-enzyme Acetylcholinesterase (AChE) was investigated. AgNPs to enzyme ratio had an effect on the enzyme and features of the treated samples. It was also observed that time increments had a positive effect on the size of AgNPs and caused an increase in their initial size. In other words, smaller AgNPs resulted in size increments after interaction with enzymes, while the larger ones showed size decrements. The nano-crystalline AgNPs were identified in x-ray powder diffraction analyses before and after treatment with AChE. The (220) crystalline plane is related to the internal crystallinity of cubic Ag. The results show that the interaction between AChE and AgNPs could lead not only to a decrease in AChE activity, but also to a reduction in the crystallinity and stability of AgNPs. The circular dichroism demonstrates that the secondary structure of AChE also declined after 30 min of incubation with AgNPs at 37 °C.

Acetylcholinesterase inhibitory activity of stigmasterol & hexacosanol is responsible for larvicidal and repellent properties of Chromolaena odorata

BackgroundChromolaena odorata, has been traditionally known for its insect repellent property. Aim of this study was to determine larvicidal tendency of C. odorata on Culex quinquefasciatus and isolate compounds responsible for this activity and to determine the mechanism of action of these compounds. MethodsC. odorata plant extract was screened for mosquito larvicidal activity. The extract was fractionated using chromatography and the bioactive fraction showing larvicidal activity was identified. The chemical nature of the compounds in the bioactive fraction was determined using NMR and Mass spectrometry. ResultsWe identified phytosterols and alkanols to be the compounds regulating larvicidal activity in the bioactive fraction of the plant extract. Stigmasterol and 1-hexacosanol were identified to be the chief orchestrators of larvicidal activity and their mode of action has been observed to be neurotoxicity. At a molecular level both stigmasterol and 1-hexacosanol were found to be inhibiting acetylcholinesterase activity in C. quinquefasciatus & A. aegypti. The acetylcholinesterase inhibitory effect was validated in vitro using recombinant acetylcholinesterase and ex vivo in larval homogenates of Culex and Aedes. Electrophysiological studies using electroantennography have shown enhanced neural response to these compounds. ConclusionsNeurotoxic effect of C. odorata derived stigmasterol and 1-hexacosanol, exerted through acetylcholinesterase inhibition was responsible for the mortality of C. quinquefasciatus, A. aegypti &Chironomus riparius. EAG studies pointed out hyper-excitability of the olfactory system by these compounds. General significanceThese compounds are natural agents for mosquito control that can be used in vector control as larvicidal compounds, pending further investigations.

A highly stable minimally processed plant-derived recombinant acetylcholinesterase for nerve agent detection in adverse conditions.

Although recent innovations in transient plant systems have enabled gram quantities of proteins in 1–2 weeks, very few have been translated into applications due to technical challenges and high downstream processing costs. Here we report high-level production, using a Nicotiana benthamiana/p19 system, of an engineered recombinant human acetylcholinesterase (rAChE) that is highly stable in a minimally processed leaf extract. Lyophylized clarified extracts withstand prolonged storage at 70 °C and, upon reconstitution, can be used in several devices to detect organophosphate (OP) nerve agents and pesticides on surfaces ranging from 0 °C to 50 °C. The recent use of sarin in Syria highlights the urgent need for nerve agent detection and countermeasures necessary for preparedness and emergency responses. Bypassing cumbersome and expensive downstream processes has enabled us to fully exploit the speed, low cost and scalability of transient production systems resulting in the first successful implementation of plant-produced rAChE into a commercial biotechnology product.

Preparation, in vitro screening and molecular modelling of monoquaternary compounds related to the selective acetylcholinesterase inhibitor BW284c51.

This paper describes preparation and in vitro evaluation of 19 compounds related to the selective experimental cholinesterase inhibitor BW284c51. The novel compounds were prepared as fragments of parent molecule BW284c51 and evaluated on the model of human recombinant acetylcholinesterase and human plasmatic butyrylcholinesterase. The IC₅₀ values of the prepared compounds were compared to the parent molecule BW284c51. None of the compounds was superior to the parent drug, but two BW284c51 fragments showed promising hAChE inhibition in µM scale and improved selectivity. These two fragments were further subjected to the molecular modelling study and their enzyme interactions were rationalized. The structure-activity relationship of the prepared series was stated.