Abstract Acetylcholinesterase Research Articles

Nematicidal activity of tirotundin and parthenolide isolated from Tithonia diversifolia and Chrysanthemum parthenium

Acetylcholinesterase (AChE) is an enzyme that catalyzes acetylcholine into choline and acetic acid. Conventional pesticides, including organophosphates and carbamates target and inhibit the activity of AChE. To obtain more pesticide precursors that meet the safety requirements, more than 200 compounds were screened. Tirotundin and parthenolide identified as potential neurotoxins to nematodes were isolated from Tithonia diversifolia and Chrysanthemum parthenium, respectively. Their IC50 values were 6.89 ± 0.30 and 5.51 ± 0.23 μg/mL, respectively against the AChE isolated from Caenorhabditis elegans. AChE was inhibited in a dose-dependent manner using the two compounds. And the Lineweaver-Burk and Dixon plots indicated that tirotundin and parthenolide were reversible inhibitors against AChE, both inhibiting AChE in a mixed-type competitive manner and demonstrating these compounds may possess dual binding site AChE inhibitors. LC50 values of tirotundin and parthenolide against C. elegans were 9.16 ± 0.21 and 7.23 ± 0.48 μg/mL, respectively. These results provide a certain theoretical basis for the development and utilization of novel pesticides.

Association of Salivary Cholinesterase With Arthropod Vectors of Disease

Acetylcholinesterase (AChE) was previously reported to be present in saliva of the southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), with proposed potential functions to 1) reduce acetylcholine toxicity during rapid engorgement, 2) modulate host immune responses, and 3) to influence pathogen transmission and establishment in the host. Potential modulation of host immune responses might include participation in salivary-assisted transmission and establishment of pathogens in the host as has been reported for a number of arthropod vector-borne diseases. If the hypothesis that tick salivary AChE may alter host immune responses is correct, we reasoned that similar cholinesterase activities might be present in saliva of additional arthropod vectors. Here, we report the presence of AChE-like activity in the saliva of southern cattle ticks, Rhipicephalus (Boophilus) microplus; the lone star tick, Amblyomma americanum (Linnaeus); Asian tiger mosquitoes, Aedes albopictus (Skuse); sand flies, Phlebotomus papatasi (Scopoli); and biting midges, Culicoides sonorensis Wirth and Jones. Salivary AChE-like activity was not detected for horn flies Haematobia irritans (L.), stable flies Stomoxys calcitrans (L.), and house flies Musca domestica L. Salivary cholinesterase (ChE) activities of arthropod vectors of disease-causing agents exhibited various Michaelis-Menten KM values that were each lower than the KM value of bovine serum AChE. A lower KM value is indicative of higher affinity for substrate and is consistent with a hypothesized role in localized depletion of host tissue acetylcholine potentially modulating host immune responses at the arthropod bite site that may favor ectoparasite blood-feeding and alter host defensive responses against pathogen transmission and establishment.

Structural basis of fullerene derivatives as novel potent inhibitors of protein acetylcholinesterase without catalytic active site interaction: insight into the inhibitory mechanism through molecular modeling studies

Acetylcholinesterase (AChE) is an important kind of esterase that plays a key biological role in the central and peripheral nervous systems. Recent research has demonstrated that some fullerene derivatives serve as a new nanoscale class of potent inhibitors of AChE, but the specific inhibition mechanism remains unclear. In the present article, several molecular modeling methods, such as molecular docking, molecular dynamics simulations and molecular mechanics/generalized Born surface area calculations, were used for the investigation of the binding mode and inhibition mechanism of fullerene inhibitions for AChE. Results revealed that fullerene inhibitors block the entrance of substrates by binding with the peripheral anionic site (PAS) region. Thus, fullerene derivatives might mainly serve as competitive inhibitors. The interactions of a fullerene backbone with AChE are at the same level in different single side chain systems and seem to be related to the length or aromaticity of the side chain. The inhibitor with multihydroxyl side chains shows an obviously large electrostatic interaction as it forms additional hydrogen bonds with AChE. Moreover, fullerene derivatives might exhibit noncompetitive inhibition partly by affecting some secondary structures around them. Thus, the destructions of these secondary structures can lead to conformational changes in some important regions, such as the catalytic triad and acyl pocket. The investigation is of great importance to the discovery of good fullerene inhibitors.Communicated by Ramaswamy H. Sarma

Computational approach to suggest a new multi-target-directed ligand as a potential medication for Alzheimer’s disease

Acetylcholinesterase (AChE) enzyme and myeloid differentiation 2 protein (MD2) are two critical proteins involved in Alzheimer’s disease (AD). Since the nature of the active site of AChE and the binding pocket of MD2 are similar, some ligands can inhibit both of them appropriately. Oxidative stress has also been known as an important cause of AD. Designing an effective common inhibitor with antioxidant activity to inhibit AChE and MD2 proteins is the main goal of this work. In this regard, we used tacrine molecule with a high ligand efficiency (LE) and dehydrozingerone (DHZ) with anti-inflammatory, antioxidant and anti-Alzheimer activities. Some modifications on DHZ structure can increase its antioxidant activity. So, tacrine molecule was combined with modified DHZ to present a new multi-target-directed ligand (MTDL). The ability of the designed ligand to inhibit AChE and MD2 proteins was confirmed by molecular docking, molecular dynamics (MD) simulation, and binding-free energy calculations. Therefore, the designed ligand can target two proteins involved in AD. It can also act as a potent antioxidant. In general, three important causative agents of AD are targeted by the designed ligand. Moreover, the inhibition of MD2, as the main source of oxidative stress, significantly reduces the production of free radicals.

MOLECULAR DOCKING STUDY ON 1-(3-(4-BENZYLPIPERAZIN-1-YL)PROPYL)-3,7-DIMETHYL-1H-PURINE-2,6(3H,7H)-DIONE AS AN ACETYLCHOLINESTERASE INHIBITOR

Acetylcholinesterase (AChE) is a good target in the design of new drugs for the treatment of Alzheimer’s disease. The currently known drugs -donepezil, galantamine and rivastignime- act as moderate AChE inhibitors. In the present study, we docked a newly synthesized arylpiperazine derivative 1-(3-(4-benzylpiperazin-1-yl)propyl)-3,7-dimethyl-1H-purine-2,6(3H,7H)-dione (LA1) into rhAChE and identified its binding mode. The docking pose of the studied LA1 molecule depends of the protonated state of the nitrogen atom of the piperazine moiety where in the best scored poses, the xanthine moiety of LA1 is bound into the catalytic active site (CAS) of AChE, while the arylpiperazine fragment is placed into the peripheral binding site (PAS). The Ellman’s test confirmed the compound binding. LA1 has good permeability through the GIT and BBB assessed by PAMPA. LA1 is a prospective lead for AChE inhibition.

The Biochemical Response of Acetyl Cholinesterasein Prostate Cancer in relation to the Environment

Acetylcholinesterase (ACHE) is an enzyme. Its main extracellular function isto hydrolyze the Acetylcholine to choline and acetate,and maintain the correct levels of Acetylcholine in the body and according to the Environmental Factors. Another function for (ACHE) in intra cellular compartment is to stimulate cell proliferation and apoptosis. In this study, we try to investigate the (ACHE) and prostate cancer overlapping to get new diagnostic markerand/or treatment for prostate cancer. Results: There is no significant difference in serum (ACHE) of patients with prostate cancer (12.90 ± 4.17ng /ml), in comparison to control group (9.0 ± 1.78 ng /ml),(p>0.05). But there is significant increase in serum (ACHE) incases with high grade prostate cancer with Gleason grade more than 7,(11.90 ± 2.11ng /ml),in comparison to lower grade prostate cancerwith Gleason grade less than 7, and control group (10.20 ± 2.20and 9.0 ± 1.78ng /ml respectively),(p<0.05). Conclusion: Because of thevariation inthe level of serum (ACHE) in patients with prostate cancer, this marker can't consider as prognostic protein for diagnosis ofprostate cancer. However further prospective studies are recommended to demonstrate its role in the diagnosis, grading and elimination of the tumor.

Acetylcholinesterase plays a non-neuronal, non-esterase role in organogenesis

Acetylcholinesterase (AChE) is crucial for degrading acetylcholine at cholinergic synapses. In vitro studies suggest that, in addition to its role in nervous system signaling, AChE can also modulate non-neuronal cell properties, although it remains controversial whether AChE functions in this capacity in vivo . Here, we show that AChE plays an essential non-classical role in vertebrate gut morphogenesis. Exposure of Xenopus embryos to AChE-inhibiting chemicals results in severe defects in intestinal development. Tissue-targeted loss-of-function assays (via microinjection of antisense morpholino or CRISPR-Cas9) confirm that AChE is specifically required in the gut endoderm tissue, a non-neuronal cell population, where it mediates adhesion to fibronectin and regulates cell rearrangement events that drive gut lengthening and digestive epithelial morphogenesis. Notably, the classical esterase activity of AChE is dispensable for this activity. As AChE is deeply conserved, widely expressed outside of the nervous system, and the target of many environmental chemicals, these results have wide-reaching implications for development and toxicology.

Understanding the conformational flexibility and electrostatic properties of curcumin in the active site of rhAChE via molecular docking, molecular dynamics, and charge density analysis

Acetylcholinesterase (AChE) is an important enzyme responsible for Alzheimer’s disease, as per report, keto-enol form of curcumin inhibits this enzyme. The present study aims to understand the binding mechanism of keto-enol curcumin with the recombinant human Acetylcholinesterase (rhAChE) from its conformational flexibility, intermolecular interactions, charge density distribution, and the electrostatic properties at the active site of rhAChE. To accomplish this, a molecular docking analysis of curcumin with the rhAChE was performed, which gives the structure and conformation of curcumin in the active site of rhAChE. Further, the charge density distribution and the electrostatic properties of curcumin molecule (lifted from the active site of rhAChE) were determined from the high level density functional theory (DFT) calculations coupled with the charge density analysis. On the other hand, the curcumin molecule was optimized (gas phase) using DFT method and further, the structure and charge density analysis were also carried out. On comparing the conformation, charge density distribution and the electrostatic potential of the active site form of curcumin with the corresponding gas phase form reveals that the above said properties are significantly altered when curcumin is present in the active site of rhAChE. The conformational stability and the interaction of curcumin in the active site are also studied using molecular dynamics simulation, which shows a large variation in the conformational geometry of curcumin as well as the intermolecular interactions.

Assay for heavy metals using an inhibitive assay based on the acetylcholinesterase from Clarias batrachus

Acetylcholinesterase (AChE) is usually used as an inhibitive assay for insecticides. A lesser-known property of AChE is its inhibition by heavy metals. In this work, we evaluate an AChE from brains of Clarias batrachus (catfish) exposed to wastes from aquaculture industry as an inhibitive assay for heavy metals. We discovered that the AChE was inhibited completely by Hg2+, Ag2+, Pb2+, Cu2+, Cd2+, Cr6+ and Zn2+ during initial screening. When tested at various concentrations, the heavy metals exhibited exponential decay type inhibition curves. The calculated IC50 (mg/L) for the heavy metals Ag2+, Cu2+, Hg2+, Cr6+ and Cd2+ were 0.088, 0.078, 0.071, 0.87 and 0.913, respectively. The IC50 for these heavy metals are comparable, and some are lower than the IC50 values from the cholinesterases from previously studied fish. The assay can be carried out in less than 30 minutes at ambient temperature.

Inhibitory activity of Acetylcholinesterase (AChE) and Antioxidant activity of methanolic extract of Desmodium gangeticum (L.)

Acetylcholinesterase (AChE) inhibition and antioxidant activity are considered to be highly correlated with Alzheimer’s disease treatment. The present study was designed to investigate the antioxidant and acetylcholinesterase inhibitory activity of Desmodium gangeticum L. Properly identified powdered plant material was extracted successively using methanol as a solvent. Acetylcholinesterase inhibitory activity was measured with modified Ellman method at 405 nm and antioxidant activity measured based on 1, 1- diphenyl-2-picrylhydrazil (DPPH) free radical scavenging test at 517 nm. Percentage inhibition for AChE ranged from 28.78±0.12 to 40.83±0.05 whereas DPPH radical scavenging percentage ranged from 24.68±0.72 to 42.22±0.67.

Next Effort in Cholinesterases Biosensors

Acetyl cholinesterase (AChE) and butyryl cholinesterase (BChE) are two known enzymes in the body with ability to hydrolyze acetylcholine. While AChE plays crucial role in acetylcholine mediated neurotransmission, BChE is secreted from livers to plasma and biological role of the enzyme is not fully understood [1]. The both cholinesterases are very sensitive to inhibition by nerve agents such as sarin, soman, tabun, VX, and some carbamate inhibitors such as pesticide carbofuran or malaoxon. Pharmacologically important compounds such as galantamine, donepezil, rivastigmine, huperzine, pyridostigmine and physostigmine used for e.g. Alzheimer disease, vascular dementia, myasthenia gravis, glaucoma, and delayed gastric emptying are inhibitors of either AChE or the both cholinesterases [2].

Effect of very Low Frequency Electromagnetic Radiation on Acetylcholine Esterase Activity

Acetylcholinesterase (AChE) is the enzyme that controls the acetylcholine (ACh) concentrations in cholinergic synaptic clefts by hydrolyzing ACh to choline and acetate. In this paper, we investigate the effect of very low frequency (VLF) electromagnetic field (EMF) radiation on AChE activity. AChE was exposed to VLF-EMFs with electric field intensity ranged from 0 to 200V/m and exposure time ranged from 0 to 120min. The results demonstrated that the VLF-EMF increased enzyme activity when the intensity ranged from 80V/m to 100V/m as well as exposure time ranged from 60min to 80min. And the AChE activity decreased when the intensity ranged from 120V/m to 140V/m and exposure time ranged from 20min to 120min. Thus, those results are significant in clinical treatments.

Acetylcholinesterase Complexes with the Natural Product Inhibitors Dihydrotanshinone I and Territrem B: Binding Site Assignment from Inhibitor Competition and Validation Through Crystal Structure Determination

Acetylcholinesterase (AChE) is a critical enzyme that regulates neurotransmission by degrading the neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for both therapeutic drugs that treat Alzheimer's disease and organophosphate (OP) chemical warfare agents that cripple the nervous system and cause death through paralysis. We are exploring a strategy to design compounds that bind tightly at or near a peripheral or P-site near the mouth of the AChE active site gorge and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. Here, we review our recent reports on two uncharged, natural product inhibitors of AChE, dihydrotanshinone I and territrem B, that have relatively high affinities for the enzyme. We describe an inhibitor competition assay and comment on the structures of these inhibitors in complex with recombinant human acetylcholinesterase as determined by X-ray crystallography. Our results reveal that dihydrotanshinone I binding is specific to only the P-site, while territrem B binding spans the P-site and extends into the acylation or A-site at the base of the gorge.

To Measure the Absorbance at 328nm by Ultraviolet-Visible Spectrophotometer - an Alternative Pathway to Measure the Activity of Acetylcholinesterase

Acetylcholinesterase (AChE) is an important enzyme in mammalian nervous systems. The Ellman assay is usually used to measure AChE activity and calculate the inhibition rate by measure the absorbance at 412nm by UV-Vis spectrophotometer. However, an alternative absorbance at 329nm which assigned as S-S of 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) was found, and it negatively correlated with the absorbance at 412nm. Therefore, the absorbance at 329nm was also possible used to measure AChE activity and calculate the inhibition rate. In addition, the reaction time should be considered in the process to transfer the relationship of these two absorbance values, because Cu (II) would combine with 5'-mercapto-2'-nitrobenzoic acid (5-MNBA).

Quantification of the Transcripts Encoding Different Forms of AChE in Various Cell Types: Real-Time PCR Coupled with Standards in Revealing the Copy Number

Acetylcholinesterase (AChE) is encoded by a single gene, and the alternative splicing at the 3' end produces different isoforms, including tailed (AChET), read-through (AChER), and hydrophobic (AChEH). Different forms of this enzyme exist in different cell types. Each AChE form has been proposed to have unique function, and all of them could be found in same cell type. Thus, the splicing process of different AChE forms remains unclear. Here, we aimed to establish a quantification method in measuring the absolute amount of each AChE splicing variants within a cell type. By using real-time PCR coupled with standard curves of defined copy of AChE variants, the copies of AChET transcript per 100ng of total RNA were 5.7 × 10(4) in PC12 (rat neuronal cell), 1.3 × 10(4) in Caco-2 (human intestinal cell), 0.67 × 10(4) in TF-1 (human erythropoietic precursor), 133.3 in SH-SY5Y (human neuronal cell), and 56.7 in human umbilical vein endothelial cells (human endothelial cells). The copies of AChEH in these cell types were 0.3 × 10(4), 3.3 × 10(4), 2.7 × 10(4), 133.3, and 46.7, respectively, and AChER were 0.07 × 10(4), 0.13 × 10(4), 890, 3.3, and 2.7, respectively. Furthermore, PC12 and TF-1 cells were chosen for the analysis of AChE splicing pattern during differentiation. The results demonstrated a selective increase in AChET mRNA but not AChER or AChEH mRNAs in PC12 upon nerve growth factor-induced neuronal differentiation. PC12 cells could therefore act as a good cell model for the study on alternative splicing mechanism and regulation of AChET.

Collagen Q is a Key Player for Developing Rational Therapy for Congenital Myasthenia and for Dissecting the Mechanisms of Anti-MuSK Myasthenia Gravis

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ) in the form of asymmetric AChE (AChE/ColQ). We exploited the proprietary NMJ-targeting signals of ColQ to treat congenital myasthenia and to explore the mechanisms of autoimmune myasthenia gravis (MG). Mutations in COLQ cause congenital endplate AChE deficiency (CEAD). First, a single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq−/− mice normalized motor functions, synaptic transmission, and partly the NMJ ultrastructure. Additionally, injection of purified recombinant AChE/ColQ protein complex into gluteus maximus accumulated AChE in non-injected forelimbs. Second, MuSK antibody-positive MG accounts for 5-15 % of MG. In vitro overlay of AChE/ColQ to muscle sections of Colq−/− mice, as well as in vitro plate-binding of MuSK to ColQ, revealed thatMuSK-IgG blocks binding of ColQ to MuSK in a dose-dependent manner. Passive transfer of MuSK-IgG to wild-type mice markedly reduced the size and intensity of ColQ signals at NMJs. MuSK-IgG thus interferes with binding of ColQ to MuSK. Elucidation of molecular mechanisms of specific binding of ColQ to NMJ enabled us to ameliorate devastating myasthenic symptoms of Colq−/− mice and also to reveal underlying mechanisms of anti-MuSK-MG.

In Vivo Acetylcholinesterase Reactivation in Male Guinea Pigs and Rhesus Macaques Following Cyclosarin Exposure and Treatment With 1,1′-Methylenebis{4-[(hydroxyimino)methyl] pyridinium} Dimethanesulfonate

Acetylcholinesterase (AChE) reactivation studies were conducted in guinea pigs (GPs) and nonhuman primates (NHPs) to determine the 1,1'-methylenebis{4-[(hydroxyimino)methyl] pyridinium} dimethanesulfonate (MMB4 DMS) dose that reactivated at least 20% of blood AChE within 15 minutes following cyclosarin (GF) dosing (used as the criterion for efficacy). Male GPs and male rhesus macaques (NHPs) were pretreated with atropine 15 minutes prior to GF administration (1 × median lethal dose [LD50]) and MMB4 DMS 15 minutes following GF administration. The GP survival was 5 of 8, 8 of 8, 8 of 8, and 6 of 8 for the 0.75, 3.0, 6.0, or 12.0 mg/kg MMB4 DMS treatment groups, respectively. In NHPs, survival was 6 of 6 at 0.5, 1.2, 3.0, or 9.3 mg/kg MMB4 DMS, respectively, 24 hours post-challenge, with the majority of animals noted as clinically normal by 24 hours. Pharmacokinetic/pharmacodynamic modeling revealed that 1.8 mg/kg in GPs or 0.013 mg/kg in NHPs would result in an average 20% reactivation; human equivalent doses were calculated as 0.39 mg/kg (based on GP data) and 0.004 mg/kg (based on NHP data). The model suggested that MMB4 plasma concentrations of 1000 ng/mL and AChE reactivation of 80% would be most effective. Although a 0.5 mg/kg MMB4 DMS dose in NHPs resulted in 100% survival and an average of 78% AChE reactivation, adverse effects associated with GF administration were still observed 24 hours post-challenge (tremors, mydriasis, and weakness were observed in 3 of 6 animals). In comparison, 6 of 6 animals treated with 1.2 mg/kg MMB4 DMS were observed as clinically normal 24 hours post-challenge.

Characterization of acetylcholinesterase in Hong Kong oyster (Crassostrea hongkongensis) from South China Sea

Acetylcholinesterase (AChE) activity has been used to evaluate the exposure of mollusk bivalves to organophosphates, carbamate pesticides, and heavy metals. Crassostrea hongkongensis is a Hong Kong endemic oyster, and has a high commercial value along the coastal area of South China. The use of this species as a bio-indicator of the marine environment, and the use of AChE activity measurements in tissues of C. hongkongensis require prior characterization of AChE in this species. Here, we report that gill tissue contains the highest AChE activity in C. hongkongensis, and that the molecular form of AChE is most likely to be a dimeric form. In addition, the effect of the pesticide acephate on AChE activity in the gill of C. hongkongensis was analyzed, and the mean inhibition concentration (IC50) value was determined. This study suggests that AChE activity in the gill tissue of C. hongkongensis might be used as a biomarker in monitoring organophosphate contamination in the marine fauna of South China.

Lycopodiaceae from Panama: A new source of acetylcholinesterase inhibitors

Acetylcholinesterase (AChE) inhibitors have been used for the symptomatic treatment of Alzheimer's disease. Eleven whole plants from Panama belonging to the Lycopodiaceae family have been screened for their anticholinesterase inhibitory and antioxidant activities by a thin-layer chromatography (TLC) bioautography method. Of these, only Lycopodium clavatum subsp. clavatum showed strong AChE inhibition. Seven plant extracts showed moderate inhibition, two of them, Huperzia cf chamaeleon and Huperzia reflexa, also possessed an antioxidant activity. This is the first report of anticholinesterase and antioxidant activities in these two native plants. Additionally, alkaloid extracts of the Lycopodiaceae plants were also analysed by TLC and LC-MS to identify the well-known AchE inhibitor, huperzine A. Two plants, H. cf chamaeleon and H. reflexa var. minor, showed the presence of huperzine.

Acetylcholinesterase inhibitory activity of Chinese sufu (fermented tofu) ethanol-extract

Acetylcholinesterase (AChE) inhibitory activities of commercial sufu and self-produced sufu were investigated in this experiment. The anti-AChE activities of commercial sufu samples of 15 brands, sourcing from various parts of China, and self-produced sufu, fermented with Actinomucor elegans 3.118, were measured. The results indicated that ethanol extract of Chinese sufu exhibited significant inhibitory activity against AChE in vitro. The inhibitory activity of No. 5 sufu was the strongest (IC₅₀, 0.191 mg/ml), while the pre-fermented sufu showed the highest inhibitory activity during sufu manufacturing. In addition, soybean extracts and potato extracts were used to culture A. elegans 3.118 in order to estimate which culture was preferable for the production of these AChE inhibitors. The soybean extracts, after fermentation by A. elegans 3.118, showed higher anti-AChE activity than did the potato extracts. The IC₅₀ of the soybean extracts was 1.29 μg/ml.