Reversible Inhibition Of Acetylcholinesterase Research Articles

Development of a Transdermal Delivery System for Tacrine

Tacrine has several mechanisms of action. The putative primary mechanism of action of tacrine for Alzheimer's disease is reversible inhibition of acetylcholinesterase (AChE), which thus slows the breakdown of the chemical messenger acetylcholine (ACh) in the brain. Tacrine also inhibits butyrylcholinesterase activity. In accumulation, tacrine blocks sodium and potassium channels. Tacrine also acts as a histamine N-methyltransferase inhibitor. This study was carried out to develop matrix based transdermal patches containing Tacrine to overcome the first pass metabolism and to reduce frequency of dosing compared to oral route. . Matrix type of transdermal patches was developed by using Methocel K4M, Methocel K15M, Methocel K100M and Xanthan gum polymers. Transdermal patches were prepared by employing solvent casting method. Drug excipients compatibility studies were carried out by using FTIR, and it was observed that there were no interactions. Formulations were prepared with the varying concentrations polymers ranging from F1-F16, and all the formulations were evaluated for various physical parameters Physical appearance, Flatness, Weight variation, Thickness, Folding endurance, Drug content, Moisture uptake, Moisture content and Swelling study and all the results were found to be within the pharmacopeial limits, invitro drug release studies by using dialysis membrane. Among all the 16 Tacrine transdermal patches formulations F5 formulations which contain Methocel K15M 100 mg had shown 97.61% % cumulative drug release within 12 hours.

Amiridine-piperazine hybrids as cholinesterase inhibitors and potential multitarget agents for Alzheimer's disease treatment

We synthesized eleven new amiridine-piperazine hybrids 5a-j and 7 as potential multifunctional agents for Alzheimer's disease (AD) treatment by reacting N-chloroacetylamiridine with piperazines. The compounds displayed mixed-type reversible inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Conjugates were moderate inhibitors of equine and human BChE with negligible fluctuation in anti-BChE activity, whereas anti-AChE activity was substantially dependent on N4-substitution of the piperazine ring. Compounds with para-substituted aromatic moieties (5g, 5h, and bis-amiridine 7) had the highest anti-AChE activity in the low micromolar range. Top-ranked compound 5h, N-(2,3,5,6,7,8-hexahydro-1H-cyclopenta[b]quinolin-9-yl)-2-[4-(4-nitro-phenyl)-piperazin-1-yl]-acetamide, had an IC50 for AChE = 1.83 ± 0.03 μM (Ki = 1.50 ± 0.12 and αKi = 2.58 ± 0.23 μM). The conjugates possessed low activity against carboxylesterase, indicating a likely absence of unwanted drug-drug interactions in clinical use. In agreement with analysis of inhibition kinetics and molecular modeling studies, the lead compounds were found to bind effectively to the peripheral anionic site of AChE and displace propidium, indicating their potential to block AChE-induced β-amyloid aggregation. Similar propidium displacement activity was first shown for amiridine. Two compounds, 5c (R = cyclohexyl) and 5e (R = 2-MeO-Ph), exhibited appreciable antioxidant capability with Trolox equivalent antioxidant capacity values of 0.47 ± 0.03 and 0.39 ± 0.02, respectively. Molecular docking and molecular dynamics simulations provided insights into the structure-activity relationships for AChE and BChE inhibition, including the observation that inhibitory potencies and computed pKa values of hybrids were generally lower than those of the parent molecules. Predicted ADMET and physicochemical properties of conjugates indicated good CNS bioavailability and safety parameters comparable to those of amiridine and therefore acceptable for potential lead compounds at the early stages of anti-AD drug development.

Experimental and Computational Evaluation of Piperonylic Acid Derived Hydrazones Bearing Isatin Moieties as Dual Inhibitors of Cholinesterases and Monoamine Oxidases.

A set of piperonylic acid derived hydrazones with variable isatin moieties was synthesized and evaluated for their inhibitory activity against the enzymes acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and monoamine oxidases A and B (MAO-A/B). The results of in vitro studies revealed IC50 values in the micromolar range, with the majority of the compounds showing selectivity for the MAO-B isoform. N-[2-Oxo-1-(prop-2-ynyl)indolin-3-ylidene]benzo[d][1,3]dioxole-5-carbohydrazide (3) was identified as a lead AChE inhibitor with IC50 =0.052±0.006 μm. N-[(3E)-5-chloro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-2H-1,3-benzodioxole-5-carbohydrazide (2) was the lead MAO-B inhibitor with IC50 =0.034±0.007 μm, and showed 50 times greater selectivity for MAO-B over MAO-A. The kinetic studies revealed that compounds 2 and 3 displayed competitive and reversible inhibition of AChE and MAO-B, respectively. The molecular docking studies revealed the significance of hydrophobic interactions in the active site pocket of the enzymes under investigation. Further optimization studies might lead to the development of potential neurotherapeutic agents.

The in vitro protective effects of the three novel nanomolar reversible inhibitors of human cholinesterases against irreversible inhibition by organophosphorous chemical warfare agents

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.

Potentiometric enzyme biosensor for aflatoxin B1 detection – Kinetic simulation

We have investigated the operation of a potentiometric biosensor based on reversible acetylcholinesterase inhibition for determination of aflatoxin B1. The biochemical reactions between enzyme, substrate, and inhibitor are described using rate equations. A sensitivity study of the biosensor response to the biochemical reaction rates was done by varying each rate constant. The response to substrate and inhibitor was used as a fitting target for evaluation of the biochemical reaction rates. Initial concentrations of the enzyme (2.0 × 10−5 M acetylcholinesterase), the substrate (4.0 × 10−3 M acetylcholine chloride), and the inhibitor (9.6 × 10−6 M aflatoxin B1) in the biosensor membrane act as the boundary conditions for the rate equations. This kinetic model allowed us to establish the effect of reaction rates on the biosensor response and reproduce the response to different inhibitor concentrations. The results may be used for biosensor design and optimization.

In vitro antioxidant assessment and a rapid HPTLC bioautographic method for the detection of anticholinesterase inhibitory activity of Geophila repens

ObjectiveGeophila repens (L.) I. M. Johnst. (Rubiaceae), a small, creeping, perennial herb, is claimed to have memory-enhancing property. The goal of this study was to assess its antioxidant and anticholinesterase activity and conduct a rapid bioautographic enzyme assay for screening acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition of G. repens extracts. MethodsAntioxidant activity of G. repens extracts was assessed by performing 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide (NO), superoxide (SOD), hydroxyl (OH) and total antioxidant capacity (TAC) assays. Anticholinesterase activity was investigated by quantifying the AChE and BChE inhibitory activities of chloroform (CGR), ethyl acetate (EGR) and methanol (MGR) extract fractions from G. repens leaves. A rapid high-performance thin-layer chromatography (HPTLC) bioautographic method for the detection of AChE and BChE inhibition was performed. ResultsAmong all extract fractions, EGR exhibited the highest half maximal inhibitory concentration (IC50) in DPPH, SOD, NO, OH and TAC assays, with IC50 of (38.33 ± 3.21), (45.14 ± 1.78), (59.81 ± 1.32), (39.45 ± 0.79) and (43.76 ± 0.81) µg/mL respectively. EGR displayed competitive, reversible inhibition of AChE and BChE activities with IC50 of (68.63 ± 0.45) and (59.45 ± 0.45) µg/mL, respectively. Total phenolic and flavonoids contents of EGR were found to be 360.42 mg gallic acid equivalents and 257.31 mg quercetin equivalents per gram of extract. Phytoconstituents of the EGR extract that were inhibitors of cholinesterase produced white spots on the yellow background of HPTLC plates in the bioautographic test. ConclusionThe results of this study revealed that phenols and flavonoids could be responsible for the antioxidant, anticholinesterase activities of G. repens.

Persistence and Comparative Pesticidal Potentials of Some Constituents of Lippia adoensis (Hochst. ex Walp.) (Lamiales: Verbenaceae) Essential Oil against Three Life Stages of Callosobruchus maculatus (Fab.) (Coleoptera: Bruchidae)

Over the past decades, the development of an alternative and eco-friendly pest control strategies has become a public concern for the sake of mankind. Plant essential oils are complex mixtures of volatile organic compounds, which play indispensable roles in the environment, for the plant itself, as well as for humans. Aims: The objectives of this study were (i) to identify and report the volatile organic compounds of Original Research Article Akami et al.; BBJ, 13(4): 1-16, 2016; Article no.BBJ.26087 2 Lippia adoensis (Hochst. Ex Walp.) (Lamiales: verbenaceae) essential oil (EO, herein after) and to compare the bioactivity of its four major compounds with the crude EO at relatively low dosages on some fitness parameters of Callosobruchus maculatus (F.) (Coleptera: Bruchidae), (ii) to test the persistency of each compounds over time, and (iii) to evaluate the type of interaction of the major constituents of the EO (whether antagonistic, synergistic or additive) in order to project the biological interaction of the whole compounds. Study Design: Young leaves of L. adoensis were randomly collected in Mbe locality (NgaoundereCameroon) for EO extraction which was then analysed by the means of Gas Chromatography (GCFID) coupled with Mass Spectrometry (GC-MS). Bioassays consisted of three shelves treatments for adult mortality, eggs laying, larval development and progeny production. A complete randomized design (CRD) with five replications was set for each treatment. Places and Duration of Study: Laboratory of Biology of the Faculty of Science, University of Ngaoundere (Cameroon) and Pest Control laboratory of Professor Niu Changying HZAU, Wuhan (China) from February 2014 to January 2016. Methodology: Essential oil was extracted with a Clevencher-type apparatus, hydrodistilled and airdried. Gas Chromatography-Flame Ionization Detector (GC-FID) and Gas Chromatography coupled with Mass Spectrometry (GC-MS) were carried out to analyze the constituents. The toxicity of crude EO and its four major constituents was evaluated at 0.5, 5, 10, 20 and 40 μL/g. Ten glass jars (volume 800mL) containing 50 g of cowpea seeds were prepared. After treatment, ten couples of C. maculatus aged 1-7days were randomly selected and separately introduced in each glass jar and kept at 22.72±1.06°C, 83.73±1.28% RH. Control ja rs were treated only with pure acetone. Each treatment was replicated five times. The exposure lasted for six days post treatment. Data on adult’s mortality, eggs laying, larvae and progeny production were assessed and monitored. Results: The GC–MS analysis allowed the identification of 43 volatile components representing 93.54% of total oil. The major components were Thymol (22.01%), Thymol-acetate (15.21%), paracymene (13.85%) and Triacetin (9.131%). The crude EO suppressed adults, completely inhibited eggs laying and adult emergence at 5 and 10 L/g after 24 h, respectively. Complete adults suppression was observed with thymol and the mixture at 20 L/g. Para-cymene and Triacetin caused complete adult mortality at 40 L/g but did not inhibited eggs laying and progeny production. EO and the mixture have been the most potent and persistent with a higher persistency throughout the experimentation. The synergism ratios (SR) were all higher than 1, thereby suggesting a significant (P = .05) synergistic interaction of major constituents although lower than that of the crude EO. Data on behaviours at death suggests that this EO may be neurotoxic probably through the blocking of the cholinergic receptors through reversible inhibition of acetylcholinesterase. Conclusion: The results obtained from this study revealed a significant (P = .05) insecticidal bioefficacy of EO extracted from young leaves of L. adoensis from Cameroon. This insecticidal properties are due to its richness in chemical constituents (43 in total) that sustained its persistency during the treatment. L. adoensis could therefore be a suitable topical agent to control C. maculatus infestations and could be of value for commercial formulations.

Development of a Larvicidal Nanoemulsion with Pterodon emarginatus Vogel Oil.

Pterodon emarginatus Vogel is a Brazilian species that belongs to the family Fabaceae, popularly known as sucupira. Its oil has several biological activities, including potent larvicidal property against Aedes aegypti. This insect is the vector of dengue, a tropical disease that has been considered a critical health problem in developing countries, such as Brazil. Most of dengue control methods involve larvicidal agents suspended or diluted in water and making active lipophilic natural products available is therefore considered a technological challenge. In this context, nanoemulsions appear as viable alternatives to solve this major problem. The present study describes the development of a novel nanoemulsion with larvicidal activity against A. aegypti along with the required Hydrophile Lipophile Balance determination of this oil. It was suggested that the mechanism of action might involve reversible inhibition of acetylcholinesterase and our results also suggest that the P. emarginatus nanoemulsion is not toxic for mammals. Thus, it contributes significantly to alternative integrative practices of dengue control, as well as to develop sucupira based nanoproducts for application in aqueous media.

Inhibitory Effect of Ebselen on Cerebral Acetylcholinesterase Activity In Vitro: Kinetics and Reversibility of Inhibition

Ebselen is a synthetic organoselenium compound that has been considered a potential pharmacological agent with low toxicity, showing antioxidant, anti-inflammatory and neuroprotective effects. It is bioavailable, blood-brain barrier permeant and safe based on cellular toxicity and Phase I-III clinical trials. There is evidence that ebselen inhibits acetylcholinesterase (AChE) activity, an enzyme that plays a key role in the cholinergic system by hydrolyzing acetylcholine (ACh), in vitro and ex vivo. This system has a well-known relationship with cognitive process, and AChE inhibitors, such as donepezil and galantamine, have been used to treat cognitive deficits, mainly in the Alzheimer's Disease (AD). However, these drugs have poor bioavailability and a number of side effects, including gastrointestinal upsets and hepatotoxicity. In this way, this study aimed to evaluate the effect of ebselen on cerebral AChE activity in vitro and to determine the kinetic profile and the reversibility of inhibition by dialysis. Ebselen inhibited the cerebral AChE activity with an IC50 of 29 µM, similar to IC50 found with pure AChE from electric eel, demonstrating a mixed and reversible inhibition of AChE, since it increased Km and decreased Vmax. The AChE activity was recovered within 60 min of dialysis. Therefore, the use of ebselen as a therapeutic agent for treatment of AD should be considered, although memory behavior tasks are needed to support such hypothesis.

Charge Density Studies on Anti-Alzheimer Agents

The average age of people is increasing continuously thanks to the progress in the medicinal sciences and further social advances. As a consequence, however, diseases which affect people more likely at a higher age also increase. In this course Alzheimer's disease (AD) and related brain disorders distribute rapidly and have to be taken more serious. One of the most frequently applied drugs against AD is donepezil®. Its function is a reversible inhibition of acetylcholinesterase (AChE), thereby reducing the deficit of acetylcholine associated with the occurrence of AD. As one result from the charge density (CD) of the small-molecule structure containing the donepezilium cation comparable electronic interactions were identified as in the macromolecular TcAChE-donepezil complex which were made visible by electrostatic potential and Hirshfeld surfaces.[1] Two newer developments of Alzheimer agents are bexarotene and methylene blue. For the first one a therapeutic effect on AD in a mouse model was recently reported. From a comparative CD study on bexarotene and its disila analogue differences in the electrostatic potentials were identified, while the spherical structures showed no significant differences. The second one, methylene blue, targets the abnormal tangle type tau protein aggregation inside the nerve cells in the brain and stops its spread. The molecule is positively charged with various counterions. From the CD an answer to the not yet understood question is expected whether the formal positive charge is localized or delocalized.

An In Vivo Zebrafish Screen Identifies Organophosphate Antidotes with Diverse Mechanisms of Action

Organophosphates are a class of highly toxic chemicals that includes many pesticides and chemical weapons. Exposure to organophosphates, either through accidents or acts of terrorism, poses a significant risk to human health and safety. Existing antidotes, in use for over 50 years, have modest efficacy and undesirable toxicities. Therefore, discovering new organophosphate antidotes is a high priority. Early life stage zebrafish exposed to organophosphates exhibit several phenotypes that parallel the human response to organophosphates, including behavioral deficits, paralysis, and eventual death. Here, we have developed a high-throughput zebrafish screen in a 96-well plate format to find new antidotes that counteract organophosphate-induced lethality. In a pilot screen of 1200 known drugs, we identified 16 compounds that suppress organophosphate toxicity in zebrafish. Several in vitro assays coupled with liquid chromatography/tandem mass spectrometry–based metabolite profiling enabled determination of mechanisms of action for several of the antidotes, including reversible acetylcholinesterase inhibition, cholinergic receptor antagonism, and inhibition of bioactivation. Therefore, the in vivo screen is capable of discovering organophosphate antidotes that intervene in distinct pathways. These findings suggest that zebrafish screens might be a broadly applicable approach for discovering compounds that counteract the toxic effects of accidental or malicious poisonous exposures.

Molecular docking and receptor-specific 3D-QSAR studies of acetylcholinesterase inhibitors

The reversible inhibition of acetylcholinesterase (AChE) has become a promising target for the treatment of Alzheimer's disease (AD) which is mainly associated with low in vivo levels of acetylcholine (ACh). The availability of AChE crystal structures with and without a ligand triggered the effort to find a structure-based design of acetylcholinesterase inhibitors (AChEIs) for AD. The major problem observed with the structure-based design was the feeble robustness of the scoring functions toward the correlation of docking scores with inhibitory potencies of known ligands. This prompted us to develop new prediction models using the stepwise regression analysis based on consensus of different docking and their scoring methods (GOLD, LigandFit, and GLIDE). In the present investigation, a dataset of 91 molecules belonging to 9 different structural classes of heterocyclic compounds with an activity range of 0.008 to 281,000 nM was considered for docking studies and development of AChE-specific 3D-QSAR models. The model (M1) developed using consensus of docking scores of scoring functions viz. Glide score, Gold score, Chem score, ASP score, PMF score, and DOCK score was found to be the best (R(2) = 0.938, Q(2) = 0.925, R(pred)(2) = 0.919, R(2)m((overall)) = 0.936) compared to other consensus models. Docking studies revealed that the molecules with proper alignment in the active site gorge and the ability to interact with all the crucial amino acid residues, in particular by forming π-π stacking interactions with Trp84 at the catalytic anionic site (CAS) and Trp279 at peripheral anionic site (PAS), showed augmented potencies with consequent improvement in patient cognition and reduced the formation of senile plaques associated with AD. Further, the descriptors that signify the association of the ligands with the receptor as well as ADME properties of the ligands were also analyzed by means of the set of ligands that have been pre-positioned with respect to a receptor after docking analysis and considered as independent variables to generate a linear model (M3 and M4) using a stepwise multiple linear regression method to get additional insight into the physicochemical requirements for effective binding of ligands with AChE as well as for prediction of AChE inhibition. The developed AChE-specific prediction models (M1-M4) satisfactorily reflect the structure-activity relationship of the existing AChEIs and have all the potential to facilitate the process of design and development of new potent AChEIs.

Lethal Poisoning with Ethiofencarb and Ethanol

Ethiofencarb is one of the carbamate compounds, which are, in general, less toxic than organophosphorus insecticides. This is due to their reversible acetylcholinesterase inhibition and relative inability to cross the blood-brain barrier. Generally, ethiofencarb is regarded to be of low toxicity (LD(50) > 200 mg/kg); however, severe poisoning and death are not uncommon. To our knowledge, no measurements of ethiofencarb and its metabolites in human postmortem whole blood have been published. We present here a case report of fatal ethiofencarb intoxication with quantitative analysis of ethiofencarb and its metabolites in ante- and postmortem blood. In addition, postmortem urine was collected and analyzed. A 56-year-old man, who worked as a gardener, was found in poor condition, sitting in his car seat. He had been vomiting. The man was admitted to the local hospital about 1 h later. At admission, he was conscious, but unable to speak clearly. His condition deteriorated, and he developed severe pulmonary edema. Resuscitation with atropine and adrenaline were attempted, but he died approximately 3 h after admission. The analysis of postmortem peripheral blood revealed 0.12 g/100 mL ethanol, 26.4 mg/L ethiofencarb, 37.9 mg/L ethiofencarbsulfoxide, and 0.9 mg/L ethiofencarbsulfone. Ethanol (0.26 g/100 mL), ethiofencarb, ethiofencarbsulfoxide, and ethiofencarbsulfone were also detected in urine.

Active site directed docking studies: Synthesis and pharmacological evaluation of cis-2,6-dimethyl piperidine sulfonamides as inhibitors of acetylcholinesterase

Hypocholinergic function associated with Alzheimer's disease (AD) is well-accepted hypothesis, in this regard, many research attempts have been made to elevate the reduced cholinergic neurotransmission, among them two main treatment strategies were widely explored, namely stimulation of muscarinic receptor 1 and/or reversible inhibition of acetylcholinesterase (AChE) enzyme. In an attempt to improve the efficacy and to minimize general side effects of these AChE inhibitors, many lead molecules are developed in research; one among them is piperidine derivative. Donazepil is a widely prescribed AChE inhibitor which displays a piperidine ring in its structure. In the present study, we have docked cis-2,6-dimethyl piperidine sulfonamides ( 3a– i) on AChE enzyme and synthesized by nucleophilic substitution reaction between cis-2,6-dimethyl piperidine and alkyl/aryl sulfonyl chlorides in the presence of triethylamine. These piperidine sulfonamides were subjected to in vitro AChE enzyme inhibition studies and in vivo antiamnesic study to reverse scopolamine induced memory loss in rats. Two derivatives ( 3a and f) in this class of piperidines ( 3a– i) showed considerable inhibition against different sources of AChE in vitro and reduced average number of mistakes done by wistar rats as compared to scopolamine treated group in vivo (rodent memory evaluation).

Mechanisms of Organophosphate Toxicity and Detoxication with Emphasis on Studies in Croatia

This review comprises studies on the mechanisms of toxicity and detoxication of organophosphorus (OP) compounds done in Croatia in different research areas. One area is the synthesis of antidotes against OP poisoning and their in vivo testing in experimental animals. In vitro studies included in this review focus on the mechanisms of reversible inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), protection of cholinesterases from inhibition by OPs, and reactivation of phosphylated cholinesterases. The third area comprises distribution profiles of BChE and paraoxonase (PON) phenotypes in selected population groups and the detection of OPs and metabolites in humans. Finally, methods are described for the detection of OP compounds in human blood and other media by means of cholinesterase inhibition.

The effect of ionic strength on the reversible inhibition of acetylcholinesterase under the influence of thionephosphonates of different hydrophobicity

280 A study of the anticholinesterase efficacy of a series of esters of thionephosphonic acids was a premise for obtaining a new group of reversible organophosphorous inhibitors (OPIs) of cholinesterases characterized by a competitive type of action [1‐3]. To this end, it was of interest to study the characteristics of the mechanism of action of these OPIs, such as the possibility of contribution of the uncompetitive component to the overall pattern of reversible inhibition, the effect of the structural characteristics of a reversible OPI molecule on the esterase catalysis upon change in ionic strength, and the role of hydrophobic‐hydrophilic interactions in the sorption of the ligand on the catalytic surface of the enzyme. Clarification of these issues will help to determine the advantages of one or another structure of the inhibitor’s molecule taking into account the ion‐ion and hydrophobic interactions taking place during their reaction with cholinesterase. The presence of the trimethylammonium group in the molecule of acetylcholine, the natural substrate of cholinesterase, determines the substantial effect of both ionic strength and the nature of ions on the enzymatic hydrolysis as such, which was studied in sufficient detail earlier [1, 3]. In the line with these studies, the effect of chlorides of alkali metals and alkali-earth metals on various kinetic characteristics, including the parameters of nonproductive substrate binding [4, 5], of enzymatic hydrolysis by serum cholinesterase of acetylcholine and its analogue, N -acetoxyethylene- N -ethylpiperidinium iodide [6]; the molecule of this analogue contains the N -ethylpiperidinium group instead of the trimethylammonium group. That study showed that the introduction of salts to the reaction mixture differentially influenced the catalytic site activity ( a c ) and the Michaelis constant ( K M ) depending on the nature of cation and the structure of substrate. However, the a c / K M ratio, which to a certain extent characterizes the affinity of the substrate for the enzyme [3], calculated for the two substrates of different structure, was similar irrespective of the presence of cations and their nature [6].

Reversible inhibition of acetylcholinesterase by carbamates or huperzine A increases residual activity of the enzyme upon soman challenge

The treatment options in soman poisoning are very limited due to rapid aging of the inhibited acetylcholinesterase, which makes the enzyme essentially intractable. Hence, oxime treatment probably comes too late in realistic scenarios. As an alternative, protecting part of the enzyme by reversible inhibition prior to soman exposure has been proposed. This strategy was successfully tested in animal experiments, but its efficacy still awaits complete understanding. In particular, it is unclear whether survival is improved by a higher residual activity of acetylcholinesterase during the acute phase, when the reversible and irreversible inhibitors are present together. In previous experiments with carbamate pre-treatment and paraoxon challenge we noticed an increased residual activity of erythrocyte acetylcholinesterase compared to non-pre-treatment. This result was encouraging to also test for comparable effects when using soman. Immobilized human erythrocytes were continuously perfused for real-time measurement of acetylcholinesterase activity by a modified Ellman method using 0.45 mM acetylthiocholine. After having established the inhibition rate constant of soman, we tested the prophylactic potential of physostigmine, pyridostigmine and huperzine A. Pre-treatment with the reversible inhibitors inhibited the enzyme by 20–95%. Additional perfusion with 10 nM soman for 30 min resulted in a residual activity of 1–5%, at low and high pre-inhibition, respectively. The residual activity was markedly higher than in the absence of reversibly blocking agents (0.1%). After discontinuation of soman and the reversible inhibitors, enzyme activity recovered up to 30% following pre-inhibition by 50%. The experimental data agreed with computer simulations when feeding the kinetic-based model with the established rate constants. The results with soman essentially agreed with those obtained previously with paraoxon.

Huperzine A.

Huperzine A, an alkaloid isolated from Huperzia serrata, is a putative nootropic agent developed by the Chinese Academy of Sciences. Huperzine A is currently in phase III trials in China for the treatment of patients with Alzheimer's disease. The mechanism of action of huperzine A is suggested to be facilitated through the slow reversible inhibition of acetylcholinesterase. Marco Hi-Tech Joint Venture has exclusive worldwide marketing and distribution rights to huperzine A. Marco Hi-Tech Joint Venture is a corporation principally owned by Hi-Tech Pharmacal and Marco International, a global trading and finance firm formed to import huperzine A from China. Marco Hi-Tech Joint Venture also has rights to synthetic analogues of huperzine A. In July 2003, Savient Pharmaceuticals acquired the exclusive rights from Marco Hi-Tech to market huperzine A in Europe and the US. Clinical trials of huperzine A in elderly patients with age-associated memory loss are underway in the US, and a phase II study funded by an NCI grant is being planned.

The Effect of Continuous Pyridostigmine Administration on Functional (A12) Acetylcholinesterase Activity in Guinea-Pig Muscles

Pyridostigmine which causes a reversible inhibition of acetylcholinesterase (AChE), was administered continuously for 6 days to guinea-pigs, via a subcutaneously implanted osmotic pump. This produced 40–50% inhibition of red cell acetylcholinesterase (AChE). Controls were animals treated with saline via pumps, and untreated animals. The activities of the functional A12 molecular form of AChE were compared in diaphragm, extensor digitorum longus (EDL) and soleus muscles in the three animal groups at 6 days. The pumps were removed at 6 days and the A12 AChE activities were determined at various times thereafter. As the enzyme separation procedure was lengthy, drug-induced inhibition was no longer present when the enzyme activity was measured. At 6 days, the activity was significantly higher in EDL (over 50% higher) and soleus (over two-fold higher) in pyridostigmine-treated animals than saline-treated animals. In the diaphragm, the activities in pyridostigmine and saline-treated animals were similar but both were significantly (over two-fold) higher than in untreated animals. At 1 day after pump removal (day 7) the activity had declined in all three muscles of the pyridostigmine-treated animals and in the diaphragm of saline-treated animals. Thereafter, in the diaphragm (but not the EDL or soleus) in pyridostigmine-treated animals, there were marked variations in the enzyme activity up to day 20. In saline-treated animals there was a marked transient increase in activity at day 13 in all muscles. The results indicate that the homeostatic control of functional AChE had been affected in both the pyridostigmine and saline treatment groups.

Accurate prediction of the bound conformation of galanthamine in the active site of torpedo californica acetylcholinesterase using molecular docking

The alkaloid (−)-galanthamine is known to produce significant improvement of cognitive performances in patients with the Alzheimer’s disease. Its mechanism of action involves competitive and reversible inhibition of acetylcholinesterase (AChE). Herein, we correctly predict the orientation and conformation of the galanthamine molecule in the active site of AChE from Torpedo californica (TcAChE) using a combination of rigid docking and flexible geometry optimization with a molecular mechanics force field. The quality of the predicted model is remarkable, as indicated by the value of the RMS deviation of ∼0.5Å when compared with the crystal structure of the TcAChE-galanthamine complex. A molecular model of the complex between TcAChE and a galanthamine derivative, SPH1107, with a long chain substituent on the nitrogen has been generated as well. The side chain of this ligand is predicted to extend along the enzyme active site gorge from the anionic subsite, at the bottom, to the peripheral anionic site, at the top. The docking procedure described in this paper can be applied to produce models of ligand-receptor complexes for AChE and other macromolecular targets of drug design.