Active Site Of Acetylcholinesterase Research Articles

Molecular modeling and in vitro reactivation study between the oxime BI-6 and acetylcholinesterase inhibited by different nerve agents

Nerve agents are organophosphates acting as potent inhibitors of acetylcholinesterase (AChE), the enzyme responsible for the hydrolysis of acetylcholine and, consequently, the termination of the transmission of nerve impulses. The inhibition of AChE by an organophosphate can be reversed by a nucleophilic agent able to dephosphorylate a serine residue in the active site of AChE. In this sense, the oximes are compounds capable of removing the nerve agent and reactivate the enzyme. Here, we have applied a methodology involving theoretical docking and Quantum Mechanics/Molecular Mechanics, using the softwares Molegro® and Spartan®, to evaluate the kinetic constants of reactivation and the interactions of the oxime BI-6 with AChE inhibited by different organophosphorus compounds in comparison to in vitro data. Results confirm that this method is suitable for the prediction of kinetic and thermodynamic parameters of oximes, which may be useful in the design and selection of new and more effective oximes.

Insights and Ideas Garnered from Marine Metabolites for Development of Dual-Function Acetylcholinesterase and Amyloid-β Aggregation Inhibitors

Due to the diversity of biological activities that can be found in aquatic ecosystems, marine metabolites have been an active area of drug discovery for the last 30 years. Marine metabolites have been found to inhibit a number of enzymes important in the treatment of human disease. Here, we focus on marine metabolites that inhibit the enzyme acetylcholinesterase, which is the cellular target for treatment of early-stage Alzheimer’s disease. Currently, development of anticholinesterase drugs with improved potency, and drugs that act as dual acetylcholinesterase and amyloid-β aggregation inhibitors, are being sought to treat Alzheimer’s disease. Seven classes of marine metabolites are reported to possess anti-cholinesterase activity. We compared these metabolites to clinically-used acetylcholinesterase inhibitors having known mechanisms of inhibition. We performed a docking simulation and compared them to published experimental data for each metabolite to determine the most likely mechanism of inhibition for each class of marine inhibitor. Our results indicate that several marine metabolites bind to regions of the acetylcholinesterase active site that are not bound by the clinically-used drugs rivastigmine, galanthamine, donepezil, or tacrine. We use the novel poses adopted for computational drug design of tighter binding anticholinesterase drugs likely to act as inhibitors of both acetylcholinesterase activity and amyloid-β aggregation inhibition.

Novel structural hybrids of pyrazolobenzothiazines with benzimidazoles as cholinesterase inhibitors

Two series of novel pyrazolobenzothiazine-based hybrid compounds were efficiently synthesized starting from saccharin sodium salt. Pyrazolo[4,3-c][1,2]benzothiazine scaffolds were N-arylated by using p-fluorobenzaldehyde, followed by the incorporation of a benzimidazole or similar ring systems by treatment with arylenediamines. These phenylene-connected hybrid compounds were investigated as potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Compounds 12d and 12k were the most potent AChE inhibitors with IC50 values of 11 and 13 nM, respectively, while 6j (IC50 = 17 nM) proved to be the most active inhibitor against BuChE with remarkable selectivity for BuChE over AChE. Molecular docking studies were also performed on human AChE and BuChE to suggest possible binding modes in which the inhibitor's extended structure is accommodated along the active site gorge of both enzymes.

ZnII/pyridyloxime complexes as potential reactivators of OP-inhibited acetylcholinesterase: In vitro and docking simulation studies

In order to investigate the ability of metal complexes to act as reactivators of organophosphorus compounds (OP)-inhibited acetylcholinesterase (AChE), we have synthesized and crystallographically characterized three novel mononuclear ZnII complexes formulated as [ZnCl2{(4-py)CHNOH}2] (1), [ZnBr2{(4-py)CHNOH}2] (2) and [Zn(O2CMe)2{(4-py)CHNOH}2]∙2MeCN (3∙2MeCN), where (4-py)CHNOH is 4-pyridinealdoxime. Their reactivation potency was tested in vitro with a slight modification of the Ellman's method using Electric eel acetylcholinesterase and the insecticide paraoxon (diethyl 4-nitrophenyl phosphate) as inhibitor. The activity of the already reported complex [Zn2(O2CPh)2{(4-py)CHNOH}2]·2MeCN (4·2MeCN) and of the clinically used drug obidoxime 1,1′-[oxybis(methylene)]bis{4-[(E)- (hydroxyimino)methyl]pyridinium} was also examined. The results of the in vitro experiments demonstrate moderate reactivation of the metal complexes compared to the drug obidoxime. On the other hand, it is clearly shown that the metal complex is the responsible molecular entity for the observed activity, as the reactivation efficacy of the organic ligand (4-pyridinealdoxime) is found to be inconsequential. Docking simulation studies were performed in the light of predicted complex-enzyme interactions using the paraoxon-inhibited enzyme along with the four ZnII complexes and obidoxime as a reference reactivator. The results showed that the three mononuclear metal complexes possess the required characteristics to be accommodated into the active site of AChE, while the entrance of the dinuclear ZnII compound is unsuccessful. An interesting outcome of docking simulations is the fact that the mononuclear compounds accommodate into the active site of AChE in a similar mode as obidoxime.

O- and N-Substituted Derivatives of Planetol as Valuable Bioactive Compounds

The compounds bearing sulfamoyl and acetamoyl groups have been found to show various biological activities. In the present research work, a series of O- and N-substituted derivatives were synthesized, starting with planetol (1). First N-methyl-4-hydroxyanilinium sulfate (1; planetol or metol) was treated with different aryl sulfonyl chlorides (2a-i) using aq. sodium carbonate solution as reaction medium to yield N-substituted derivatives 3a-i. The electrophile, N-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-bromoacetamide (5) was prepared by the reaction of 2,3-dihydro-1,4-benzodioxin-6-amine (4) and 2-bromoacetylbromide in a weak basic aqueous medium. The target O-substituted molecules 6a-i, were synthesized by gearing up the electrophile 5, with the molecules 3a-i, in a polar aprotic solvent using LiH as an activator. The proposed structures of all the synthesized molecules were corroborated by IR, H-1 NMR and EIMS spectral data. The in vitro enzyme inhibition and antibacterial studies rendered the synthesized molecules as better cholinesterase inhibitors and moderately better antibacterial agents. To explore the binding modes of the synthesized compounds, all of them were computationally docked against the active sites of acetyl cholinesterase (AChE), butyryl cholinesterase (BChE) and lipoxygenase (LOX). The compounds showed significant interactions and good correlation with the experimental data.

Synthesis of Highly Functionalised Dispiropyrrolidine Derivatives as Novel Acetylcholinesterase Inhibitors

In the effort of finding novel acetyl cholinesterase (AChE) inhibitors to improve the efficacy of Alzheimer's disease (AD) treatment, series of substituted aryl-1′-methyldispiro[indan-2,2′ pyrrolidine-3′, 2-indan]-1,3,1′-trione and substituted 7′-aryl-5′, 6′,7′,7a′-tetrahydrodispiro-[indane-2,5′-pyrrolo[1,2-c] [1,3]thiazole-6′,2-indan]-1,3,1-trione analogues were synthesized using [3+2]-cycloaddition reactions. These newly synthesized pyrrolidine compounds were assayed for their biological activity using Ellman's method. The structural elucidation of the compounds was performed by using 1-NMR, 13-NMR, ESI-MS spectra and elemental analyses. Eight out of twenty synthesized compounds showed more than 50% inhibition at concentration of 10 μM. Compound 2e, 2i and 3e were among the most active one, giving IC value as 3.3 μM for 2e, 3.7 μM for 2i and 5.5 μM for 3e, respectively. Lineweaver-Burk plot indicated that 2i inhibits AChE in a competitive manner. Molecular modelling study was performed to disclose the binding interaction of these compounds with the active site of AChE.

Synthesis, Biological Activity Evaluation and Molecular Modeling Study on the New Isoconessimine Derivatives as Acetylcholinesterase Inhibitors

AbstractNew isoconessimine derivatives were synthesized from conessine (1) and evaluated as acetylcholinesterase (AChE) inhibitors. The derivatives were prepared via two reaction steps, N‐demethylation and nucleophilic substitution. All of the synthesized derivatives exhibited more potential anti‐acetylcholinesterase activities than conessine (1) (IC50=16 µmol·L−1) and isoconessimine (2) (IC50>300 µmol·L−1). Compound 7b (3β‐[methyl‐[2‐(4‐nitrophenoxy)ethyl]amino]con‐5‐enine) showed the most potent inhibitory activity with an IC50 of 110 nmol/L which is close to that of reference compound huperzine A (IC50=70 nmol/L). The mode of AChE inhibition by 7b was reversible and non‐competitive. In addition, molecular modeling was performed to explore the binding mode of inhibitor 7b at the active site of AChE and the results showed that 7b could be docked into the acetylcholinesterase active site and compound 7b had hydrophobic interactions with Trp279 and Leu282.

Identification of potential bivalent inhibitors from natural compounds for acetylcholinesterase through in silico screening using multiple pharmacophores

The symptomatic cure observed in the treatment of Alzheimer's disease (AD) by FDA approved drugs could possibly be due to their specificity against the active site of acetylcholinesterase (AChE) and not by targeting its pathogenicity. The AD pathogenicity involved in AChE protein is mainly due to amyloid beta peptide aggregation, which is triggered specifically by peripheral anionic site (PAS) of AChE. In the present study, a workflow has been developed for the identification and prioritization of potential compounds that could interact not only with the catalytic site but also with the PAS of AChE. To elucidate the essential structural elements of such inhibitors, pharmacophore models were constructed using PHASE, based on a set of fifteen best known AChE inhibitors. All these models on validation were further restricted to the best seven. These were transferred to PHASE database screening platform for screening 89,425 molecules deposited at the “ZINC natural product database”. Novel lead molecules retrieved were subsequently subjected to molecular docking and ADME profiling. A set of 12 compounds were identified with high pharmacophore fit values and good predicted biological activity scores. These compounds not only showed higher affinity for catalytic residues, but also for Trp86 and Trp286, which are important, at PAS of AChE. The knowledge gained from this study, could lead to the discovery of potential AChE inhibitors that are highly specific for AD treatment as they are bivalent lead molecules endowed with dual binding ability for both catalytic site and PAS of AChE.

Targeted oxidation of Torpedo californica acetylcholinesterase by singlet oxygen: identification of N-formylkynurenine tryptophan derivatives within the active-site gorge of its complex with the photosensitizer Methylene Blue

The principal role of AChE (acetylcholinesterase) is termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine. The active site of AChE is near the bottom of a long and narrow gorge lined with aromatic residues. It contains a CAS (catalytic 'anionic' subsite) and a second PAS (peripheral 'anionic' site), the gorge mouth, both of which bind acetylcholine via π-cation interactions, primarily with two conserved tryptophan residues. It was shown previously that generation of (1)O(2) by illumination of MB (Methylene Blue) causes irreversible inactivation of TcAChE (Torpedo californica AChE), and suggested that photo-oxidation of tryptophan residues might be responsible. In the present study, structural modification of the TcAChE tryptophan residues induced by MB-sensitized oxidation was investigated using anti-N-formylkynurenine antibodies and MS. From these analyses, we determined that N-formylkynurenine derivatives were specifically produced from Trp(84) and Trp(279), present at the CAS and PAS respectively. Peptides containing these two oxidized tryptophan residues were not detected when the competitive inhibitors, edrophonium and propidium (which should displace MB from the gorge) were present during illumination, in agreement with their efficient protection against the MB-induced photo-inactivation. Thus the bound MB elicited selective action of (1)O(2) on the tryptophan residues facing on to the water-filled active-site gorge. The findings of the present study thus demonstrate the localized action and high specificity of MB-sensitized photo-oxidation of TcAChE, as well as the value of this enzyme as a model system for studying the mechanism of action and specificity of photosensitizing agents.

Acetylcholinesterase-Inhibiting Activity of Salicylanilide N-Alkylcarbamates and Their Molecular Docking

A series of twenty-five novel salicylanilide N-alkylcarbamates were investigated as potential acetylcholinesterase inhibitors. The compounds were tested for their ability to inhibit acetylcholinesterase (AChE) from electric eel (Electrophorus electricus L.). Experimental lipophilicity was determined, and the structure-activity relationships are discussed. The mode of binding in the active site of AChE was investigated by molecular docking. All the discussed compounds expressed significantly higher AChE inhibitory activity than rivastigmine and slightly lower than galanthamine. Disubstitution by chlorine in C'(3,4) of the aniline ring and the optimal length of hexyl-undecyl alkyl chains in the carbamate moiety provided the most active AChE inhibitors. Monochlorination in C'(4) exhibited slightly more effective AChE inhibitors than in C'(3). Generally it can be stated that compounds with higher lipophilicity showed higher inhibition, and the activity of the compounds is strongly dependent on the length of the N-alkyl chain.

SYNTHESIS AND INVESTIGATION OF ANTI-ACETYLCHOLINESTERASE ACTIVITY IN SILICO AND IN VITRO OF SOME CHALCONES

Background: Alzheimer is a major cause of dementia in the elderly and acetylcholinesterase inhibitors are used to treat the symptoms of this disease. Recently, chalcones have been reported as potential acetylcholinesterase inhibitors. Materials and methods: In this study, Claisen-Schmidt condensation reaction was applied to synthesize chalcones. Anti-acetylcholinesterase activity of these chalcones was determined by Ellman method. Molecular docking studies on acetylcholinesterase were performed to explain the interaction between these chalcone analogues and acetylcholinesterase active site at molecular level. Results: A total of twenty chalcones were synthesized and determined for in vitro anti-acetylcholinesterase activity. The results indicated that six compounds having IC50 value below 100 µM, three compounds having IC50 value in the range of 100 µM and 300 µM, the rest having IC50 value above 300 µM. Chalcone S17 (4’-amino-2-chlorochalcone) shows the strongest anti-acetylcholinesterase activity in the investigated group with IC50 value of 36.10 µM. In combination with the results of the in vitro anti-acetylcholinesterase activity, molecular docking study is used to explain the interaction between chalcone molecules and their active site, and the structure-activity relationship is abstracted. Conclusions: Our study indicated that the 2’-hydroxychalcones with halogen functional groups on B ring are strong acetylcholinesterase inhibitors. Chalcone S17 (4’-amino-2-chlorochalcone) could be considered as a potential lead compound for the development of new acetylcholinesterase inhibitors. Keywords: acetylcholinesterase, AChE, Alzheimer, chalcon, docking. Key words: A cetylcholinesterase, AChE, Alzheimer, chalcon, docking

Effect of Different Oximes on Rat and Human Cholinesterases Inhibited by Methamidophos: A ComparativeIn VitroandIn SilicoStudy

Methamidophos is one of the most toxic organophosphorus (OP) compounds. It acts via phosphorylation of a serine residue in the active site of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), leading to enzyme inactivation. Different oximes have been developed to reverse this inhibition. Thus, our work aimed to test the protective or reactivation capability of pralidoxime and obidoxime, as well as two new oximes synthesised in our laboratory, on human and rat cholinesterases inhibited by methamidophos. In addition, we performed molecular docking studies in non-aged methamidophos-inhibited AChE to understand the mechanisms involved. Our results suggested that pralidoxime protected and reactivated methamidophos-inhibited rat brain AChE. Regarding human erythrocyte AChE, all oximes tested protected and reactivated the enzyme, with the best reactivation index observed at the concentration of 50 μM. Concerning BChE, butane-2,3-dionethiosemicarbazone oxime (oxime 1) was able to protect and reactivate the methamidophos-inhibited BChE by 45% at 50 μM, whereas 2(3-(phenylhydrazono)butan-2-one oxime (oxime 2) reactivated 28% of BChE activity at 100 μM. The two classical oximes failed to reactivate BChE. The molecular docking study demonstrated that pralidoxime appears to be better positioned in the active site to attack the O-P moiety of the inhibited enzyme, being near the oxyanion hole, whereas our new oximes were stably positioned in the active site in a manner similar to that of obidoxime. In conclusion, our work demonstrated that the newly synthesised oximes were able to reactivate not only human erythrocyte AChE but also human plasma BChE, which could represent an advantage in the treatment of OP compounds poisoning.

Steroidal alkaloids from Holarrhena antidysenterica as acetylcholinesterase inhibitors and the investigation for structure–activity relationships

AimsInhibition of acetylcholinesterase (AChE) is still considered as a strategy for the treatment of neurological disorders such as Alzheimer's disease (AD). Many plant derived alkaloids (such as huperzine A, galanthamine and rivastigmine) are known for their AChE inhibitory activity. The aim of the present work was to isolate and identify new AChE inhibitors from Holarrhen antidysenterica. Main methodsThese compounds were tested for AChE inhibiting activity by the Ellman's method in 96-well microplates. In addition, molecular modeling was performed to explore the binding mode of inhibitors 1–5 at the active site of AChE, and the preliminary structure–activity relationships (SARs) were discussed. Key findingsIn the course of searching for AChE inhibitors from herb medicines, the total alkaloidal extract from the seeds of H. antidysenterica was found having potent AChE inhibitory activity with an IC50 value of 6.1μg/mL. Further bioactivity-guided chromatographic fractionation afforded five steroidal alkaloids, conessine 1, isoconessimine 2, conessimin 3, conarrhimin 4 and conimin 5. All the isolated compounds, except for 2, showed strong AChE inhibiting activity with IC50 values ranging from 4 to 28μM. The most active inhibitor is compound 3 with an IC50 value of 4μM. The mode of AChE inhibition by 3 was reversible and non-competitive. SignificanceThe results suggest that these alkaloids could be potential candidates for further development of new drugs against AD.

Novel and Viable Acetylcholinesterase Target Site for Developing Effective and Environmentally Safe Insecticides

Insect pests are responsible for human suffering and financial losses worldwide. New and environmentally safe insecticides are urgently needed to cope with these serious problems. Resistance to current insecticides has resulted in a resurgence of insect pests, and growing concerns about insecticide toxicity to humans discourage the use of insecticides for pest control. The small market for insecticides has hampered insecticide development; however, advances in genomics and structural genomics offer new opportunities to develop insecticides that are less dependent on the insecticide market. This review summarizes the literature data that support the hypothesis that an insect-specific cysteine residue located at the opening of the acetylcholinesterase active site is a promising target site for developing new insecticides with reduced off-target toxicity and low propensity for insect resistance. These data are used to discuss the differences between targeting the insect-specific cysteine residue and targeting the ubiquitous catalytic serine residue of acetylcholinesterase from the perspective of reducing off-target toxicity and insect resistance. Also discussed is the prospect of developing cysteine-targeting anticholinesterases as effective and environmentally safe insecticides for control of disease vectors, crop damage, and residential insect pests within the financial confines of the present insecticide market.

Acetylcholinesterase based assay of eleven organophosphorus pesticides: finding of assay limitations

The study includes findings about limitations of acetylcholinesterase (AChE) based assay. Eleven organophosphorus pesticides: chlorpyrifos ethyl, chlorpyrifos methyl, DFP, dichlorvos, dimethoate, fenthion, paraoxon ethyl, paraoxon methyl, phosalone, pirimiphos methyl and pirimiphos ethyl were photometrically assayed using AChE as a recognition element. The study was carried out in order to find approachability of AChE based assay. In the first round, common organic solvents were tested for interfering in assay, since samples collection and extraction is a necessary part in samples processing. Isopropanol was found as the most convenient due to minimal inhibition not exceeding 5%. Though all analysed pesticides inhibit AChE in vivo, some of them are toxic after metabolisation. We found AChE based assay approachable for assay of DFP, paraoxons, and dichlorvos. These are oxoforms of organophosphorus pesticides. From thioforms of assayed pesticides, only fenthion was able significantly inhibit AChE in vitro. Electrochemical biosensor with AChE attached on platinum electrode was used for confirmation of interaction pesticide – AChE and complex stability estimation. DFP, paraoxons and dichlorvos were allowed to interact with AChE in biosensor. These pesticides were settled firmly in AChE active site as no spontaneous recovery of AChE activity was observed.

Quantum mechanical/molecular mechanical analysis of mechanisms of enzyme action. Human acetylcholinesterase

The influence of the system separation into the QM and MM parts on the result of quantum mechanical/molecular mechanical (QM/MM) modeling of acetylcholine hydrolysis in the acetylcholinesterase active site was considered. The minimum acceptable quantum subsystem that provides an adequate description of the enzymatic reaction energy profile was identified. The computed energy profiles were analyzed and possible inaccuracies in QM/MM calculations were estimated. The fairly high tetrahedral intermediate stability was demonstrated, which is in a good agreement with the newest experimental observations.

Benzofurans from Styrax agrestis As Acetylcholinesterase Inhibitors: Structure–Activity Relationships and Molecular Modeling Studies

An extract of Styrax agrestis fruits, collected in Vietnam, significantly inhibited acetylcholinesterase (AChE) in vitro. Bioassay-guided fractionation revealed three new egonol-type benzofurans: egonol-9(Z),12(Z) linoleate (1), 7-demethoxyegonol-9(Z),12(Z) linoleate (2), and 7-demethoxyegonol oleate (4). Ten known egonol-type benzofurans were also isolated (3, 5, 6-13). In order to better understand structure-activity relationships in this series, egonol derivatives 14-19 were prepared by chemical modifications and evaluated for their inhibition of AChE, butyrylcholinesterase (BChE), and AChE-induced Aβ aggregation. Compounds 1-4 were the most potent inhibitors of the series, which exhibited inhibitory activity against AChE (IC50 1.4-3.1 μM) and, for 1, Aβ aggregation (77.6%). Molecular docking studies were also performed to investigate interaction of these compounds with the active site of AChE.

An aporphine alkaloid from Nelumbo nucifera as an acetylcholinesterase inhibitor and the primary investigation for structure–activity correlations

N-methylasimilobine (1), a new-found strong acetylcholinesterase (AChE) inhibitor, along with two weakly active aporphine alkaloids, nuciferine (2) and nornuciferine (3) were separated from Nelumbo nucifera. N-methylasimilobine (1) inhibited 50% of AChE activity at the concentrations of 1.5 ± 0.2 µg mL−1 when the standard IC50 value of Physostigmine was 0.013 ± 0.002 µg mL−1. The mode of AChE inhibition by 1 was reversible and non-competitive. In addition, molecular modelling was performed to explore the binding mode of inhibitor 1 at the active site of AChE.

Geissoschizine methyl ether, a corynanthean-type indole alkaloid from Uncaria rhynchophylla as a potential acetylcholinesterase inhibitor

Geissoschizine methyl ether (1), a newly discovered strong acetylcholinesterase (AChE) inhibitor, along with six weakly active alkaloids, vallesiachotamine (2), hisuteine (3), hirsutine (4), isorhynchophylline (5), cisocorynoxeine (6) and corynoxeine (7) have been isolated from Uncaria rhynchophylla. Geissoschizine methyl ether (1) inhibited 50% of AChE activity at concentrations of 3.7 ± 0.3 µg mL−1 while the IC50 value of physostigmine as a standard was 0.013 ± 0.002 µg mL−1. The mode of AChE inhibition by 1 was reversible and non-competitive. In addition, molecular modelling was performed to explore the binding mode of inhibitor 1 at the active site of AChE.

Influence of the Acetylcholinesterase Active Site Protonation on Omega Loop and Active Site Dynamics

Existence of alternative entrances in acetylcholinesterase (AChE) could explain the contrast between the very high AChE catalytic efficiency and the narrow and long access path to the active site revealed by X-ray crystallography. Alternative entrances could facilitate diffusion of the reaction products or at least water and ions from the active site. Previous molecular dynamics simulations identified side door and back door as the most probable alternative entrances. The simulations of non-inhibited AChE suggested that the back door opening events occur only rarely (0.8% of the time in the 10ns trajectory). Here we present a molecular dynamics simulation of non-inhibited AChE, where the back door opening appears much more often (14% of the time in the 12ns trajectory) and where the side door opening was observed quite frequently (78% of trajectory time). We also present molecular dynamics, where the back door does not open at all, or where large conformational changes of the AChE omega loop occur together with alternative passage opening events. All these differences in AChE dynamical behavior are caused by different protonation states of two glutamate residues located on bottom of the active site gorge (Glu202 and G450 in Mus Musculus AChE). Our results confirm the results of previous molecular dynamics simulations, expand the view and suggest the probable reasons for the overall conformational behavior of AChE omega loop.