Human Erythrocyte Acetylcholinesterase Research Articles

Acetylcholinesterase purification from human erythrocytes using magnetic nanoparticles containing procainamide

This work presents a magnetic purification method of human erythrocyte Acetylcholinesterase (EC 3.1.1.7; AChE) based on affinity binding to procainamide (Proca) as ligand. Acetylcholinesterase is an acetylcholine-regulating enzyme found in different areas of the body and associated with various neurological disorders, such as Parkinson, Alzheymer and Amyotrophic Lateral Sclerosis. AChE from human erythrocyte purification has been attempted in recent years with low degree of purity. Here, magnetic nanoparticles (MNP) were synthesized and coated with polyaniline (PANI) and procainamide (PROCA) was covalently linked to the PANI. The extracted human erythrocyte AChE formed a complex with the MNP@PANI-PROCA and an external magnet separated it from the undesired proteins. Finally, the enzyme was collected by increasing the ionic strength. Experimental Box-Behnken design was developed to optimize this process of human erythrocyte AChE purification protocol. The enzyme was purified in all fifteen experiments. However, the best AChE purification result was achieved, about 2000 times purified, when 100 mg of MNP@PANI-PROCA was incubated for one hour with 4 ml hemolysate extract. The SDS-PAGE of this preparation presented a molecular weight of approximately 70 kDa, corroborating with few previous studies of AChE from erythrocyte purification.

Inhibitory Action of Omega-3 and Omega-6 Fatty Acids Alpha-Linolenic, Arachidonic and Linoleic acid on Human Erythrocyte Acetylcholinesterase.

Acetylcholinesterase (AChE, E.C. 3.1.1.7) termed as the true cholinesterase functions to end cholinergic transmission at synapses. Due to its diverse expression in non-neural tissues such as erythrocytes and bones along with its various molecular forms, researchers seek a non-classical role for this protein. Here, the inhibitory action of unsaturated 18 carbon fatty acids linoleic acid and alpha-linolenic acid and 20 carbon fatty acid arachidonic acid on AChE were investigated. Enzyme activity was measured in kinetic assay method according to Ellman assay utilizing acetylthiocholine. Analysis of the activity data revealed that among the fatty acids examined the IC50 values differed according to the length of the fatty acid and the number of the double bonds. Arachidonic acid, a 20-carbon fatty acid with 4 unsaturated bonds (20:4 n-6, cis 5,8,11,14) displayed an IC50 value of 2.78 µM and Ki value of 396.35 µM. Linoleic acid, an essential 18-carbon fatty acid (18:2 n-6, cis 9,12) had an IC50 value of 7.95 µM and Ki value of 8027.55 µM. The IC50 value of alpha-linolenic acid, 18-carbon fatty acid (18:3 n-3, cis-9,12,15) was found as 179.11 µM. Analysis of the data fit the inhibition mechanism for linoleic, alpha-linolenic and arachidonic acid as mixed-type; non-competitive. Molecular docking complied with these results yielding the best score for arachidonic acid. The alkenyl chain of the fatty acids predictably reached to the catalytic site while the carboxylate strongly interacted with the peripheric anionic site.

Non-covalent acetylcholinesterase inhibitors: In vitro screening and molecular modeling for novel selective insecticides

Insecticides represent the most crucial element in the integrated management approach to malaria and other vector-borne diseases. The evolution of insect resistance to long-used substances and the toxicity of organophosphates (OPs) and carbamates are the main factors contributing to the development of new, environmentally safe pesticides. In our work, fourteen compounds of 7-methoxytacrine-tacrine heterodimers were tested for their insecticidal effect. Compounds were evaluated in vitro on insect acetylcholinesterase from Anopheles gambiae (AgAChE) and Musca domestica (MdAChE). The evaluation was executed in parallel with testing on human erythrocyte acetylcholinesterase (HssAChE) and human butyrylcholinesterase (HssBChE) using a modified Ellman's method. Compound efficacy was determined as IC50 values for the respective enzymes and selectivity indexes were expressed to compare the interspecies selectivity. Docking studies were performed to predict the binding modes of selected compounds. K1328 and K1329 provided high HssAChE/AgAChE selectivity outperforming standard pesticides (carbofuran and bendiocarb), and thus can be considered as suitable lead structure for novel anticholinesterase insecticides.

Inhibition of cholinesterases by safranin O: Integration of inhibition kinetics with molecular docking simulations

In the present study, the inhibitory mechanisms and effects of a synthetic phenazine dye, safranin O (SO) on human plasma butyrylcholinesterase (BChE), human erythrocyte acetylcholinesterase (AChE) and recombinant BChE mutants were investigated. Kinetic studies showed the following information: SO leaded to linear competitive inhibition of human plasma BChE with Ki = 0.44 ± 0.085 μM; α = ∞. It acted as a hyperbolic noncompetitive inhibitor of human erythrocyte AChE with Ki = 0.69 ± 0.13; α = 1; β = 0.08 ± 0.02. On the other hand, the inhibitory effects of SO on two BChE mutants, where A328 was modified to either F or Y, revealed differences in terms of inhibitory patterns and Ki values, compared to the obtained results with recombinant wild type BChE. SO was found to act as a linear competitive inhibitor of A328F and A328Y BChE mutants. Compared to recombinant wild type BChE, A328Y and A328F BChE mutants caused a 4- and 10-fold decrease in Ki value for SO, respectively. These findings were supported by molecular modelling studies. In conclusion, SO is a potent inhibitor of human cholinesterases and may be useful in the design and development of new drugs for the treatment of AD.

Arachidonoylcholine and Other Unsaturated Long-Chain Acylcholines Are Endogenous Modulators of the Acetylcholine Signaling System.

Cholines acylated with unsaturated fatty acids are a recently discovered family of endogenous lipids. However, the data on the biological activity of acylcholines remain very limited. We hypothesized that acylcholines containing residues of arachidonic (AA-CHOL), oleic (Ol-CHOL), linoleic (Ln-CHOL), and docosahexaenoic (DHA-CHOL) acids act as modulators of the acetylcholine signaling system. In the radioligand binding assay, acylcholines showed inhibition in the micromolar range of both α7 neuronal nAChR overexpressed in GH4C1 cells and muscle type nAChR from Torpedo californica, as well as Lymnaea stagnalis acetylcholine binding protein. Functional response was checked in two cell lines endogenously expressing α7 nAChR. In SH-SY5Y cells, these compounds did not induce Ca2+ rise, but inhibited the acetylcholine-evoked Ca2+ rise with IC50 9 to 12 μM. In the A549 lung cancer cells, where α7 nAChR activation stimulates proliferation, Ol-CHOL, Ln-CHOL, and AA-CHOL dose-dependently decreased cell viability by up to 45%. AA-CHOL inhibited human erythrocyte acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) by a mixed type mechanism with Ki= 16.7 ± 1.5 μM and αKi= 51.4 ± 4.1 μM for AChE and Ki= 70.5 ± 6.3 μM and αKi= 214 ± 17 μM for BChE, being a weak substrate of the last enzyme only, agrees with molecular docking results. Thus, long-chain unsaturated acylcholines could be viewed as endogenous modulators of the acetylcholine signaling system.

Conjugates of methylene blue with \u03b3-carboline derivatives as new multifunctional agents for the treatment of neurodegenerative diseases

We studied the inhibitory activity of methylene blue (MB) γ-carbolines (gC) conjugates (MB-gCs) against human erythrocyte acetylcholinesterase (AChE), equine serum butyrylcholinesterase (BChE), and a structurally related enzyme, porcine liver carboxylesterase (CaE). In addition, we determined the ability of MB-gCs to bind to the peripheral anionic site (PAS) of Electrophorus electricus AChE (EeAChE) and competitively displace propidium iodide from this site. Moreover, we examined the ability of MB-gCs to scavenge free radicals as well as their influence on mitochondrial potential and iron-induced lipid peroxidation. We found that MB-gCs effectively inhibited AChE and BChE with IC50 values in the range 1.73–10.5 μM and exhibited low potencies against CaE (9.8–26% inhibition at 20 μM). Kinetic studies showed that MB-gCs were mixed-type reversible inhibitors of both cholinesterases. Molecular docking results showed that the MB-gCs could bind both to the catalytic active site and to the PAS of human AChE and BChE. Accordingly, MB-gCs effectively displaced propidium from the peripheral anionic site of EeAChE. In addition, MB-gCs were extremely active in both radical scavenging tests. Quantum mechanical DFT calculations suggested that free radical scavenging was likely mediated by the sulfur atom in the MB fragment. Furthermore, the MB-gCs, in like manner to MB, can restore mitochondrial membrane potential after depolarization with rotenone. Moreover, MB-gCs possess strong antioxidant properties, preventing iron-induced lipid peroxidation in mitochondria. Overall, the results indicate that MB-gCs are promising candidates for further optimization as multitarget therapeutic agents for neurodegenerative diseases.

Synthesis of new N-(pyridin-3-ylmethyl)-2-aminothiazoline derivatives possessing anticholinesterase and antiradical activity as potential multifunctional agents for the treatment of neurodegenerative diseases

New N-(pyridin-3-ylmethyl)-2-aminothiazolines containing various substituents at the 5 position of the thiazoline ring and the 4-tert-butylbenzyl, 4-isopropylbenzyl, or 4-fluorobenzyl moiety at the nitrogen atom of the amino group were synthesized. The inhibitory activity of the synthesized compounds against human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7), equine serum butyrylcholinesterase (BChE, EC 3.1.1.8), and porcine liver carboxylesterase (CaE, EC 3.1.1.1) was evaluated and their antioxidant properties were studied by ABTS assay. N-(Pyridin-3-ylmethyl)-2-aminothiazolines proveded to be very weak AChE inhibitors, while their inhibitory activity against BChE and CaE was structure-dependent. 2-Aminothiazolines containing the 4-tert-butylbenzyl moiety are more efficient BChE inhibitors compared to the derivatives containing the 4-isopropylbenzyl or 4-fluorobenzyl substituent. An analysis of the dependence of the esterase profile of N-(pyridin-3-ylmethyl)-2-aminothiazolines on the structure of the substituent at the 5 position of the thiazoline ring of these compounds demonstrated that the derivatives containing the iodomethyl substituent possess the highest anti-BChE activity, the compounds with R2 = H and R3 = CH2I have the optimal esterase profile. Regardless of the structure of the substituents in the benzyl moiety, all N-(pyridin-3-ylmethyl)-2-aminothiazolines containing the iodomethyl substituent at the 5 position of the thiazoline ring exhibited high radical scavenging activity comparable with that of the standard antioxidant Trolox. N-(Pyridin-3-ylmethyl)-2-aminothiazolines were shown to be a new promising class of compounds for the design of multifunctional agents for the treatment of neurodegenerative diseases.

Avaliação da atividade de aldeídos aromáticos e suas oximas frente à enzima acetilcolinesterase eritrocitária humana

Considerando a importância terapêutica das drogas inibidoras da acetilcolinesterase, bem como dos regeneradores da enzima, em casos de intoxicações por organofosforados. A atividade de aldeídos aromáticos e suas respectivas oximas foi avaliada frente à acetilcolinesterase eritrocitária humana. As oximas foram preparadas a partir da oximação do benzaldeído, do 4-hidroxibenzaldeído, do 4-hidroxi-3-metoxibenzaldeído e do 4-dimetialminobenzaldeído, e em seguida, o ensaio de Ellman foi empregado para avaliar o comportamento destes compostos frente à acetilcolinesterase ativa e inibida por diclorvós. Os aldeídos avaliados apresentaram atividade inibidora da acetilcolinesterase nas porcentagens de 21,33%, 53,00%, 44,00% e 62,67% respectivamente para benzaldeído, 4-hidroxibenzaldeído, 4-hidroxi-3-metoxibenzaldeído e 4- dimetilminobenzaldeído. Dentre as oximas avaliadas, apenas a dimetilaminobenzaldoxima apresentou atividade inibitória (36,67%). A benzaldoxima, a 4-hidroxibenzaldoxima e a 4-hidroxi-3-metoxibenzaldoxima mostraram-se capazes de regenerar modestamente a atividade enzimática em 9,21%, 32,83% e 8,90%, respectivamente, quando a enzima se encontrava inibida por diclorvós. Os aldeídos aromáticos testados apresentaram potencial atividade inibitória da acetilcolinesterase eritrocitária, na concentração de 100mM, sendo o maior potencial demonstrado pelo 4-dimetilamibenzaldeído, com 62,67% de inibição. As oximas, com exceção da dimetilaminobenzaldoxima, se comportaram como regeneradores fracos da enzima inibida por diclorvós, mostrando a relevância do grupo oxima para a regeneração da enzima inibida por organofosforados.

Human Erythrocyte Acetylcholinesterase in Health and Disease.

The biochemical properties of erythrocyte or human red blood cell (RBC) membrane acetylcholinesterase (AChE) and its applications on laboratory class and on research are reviewed. Evidence of the biochemical and the pathophysiological properties like the association between the RBC AChE enzyme activity and the clinical and biophysical parameters implicated in several diseases are overviewed, and the achievement of RBC AChE as a biomarker and as a prognostic factor are presented. Beyond its function as an enzyme, a special focus is highlighted in this review for a new function of the RBC AChE, namely a component of the signal transduction pathway of nitric oxide.

Activity of cholinesterases in a young and healthy middle-European population: Relevance for toxicology, pharmacology and clinical praxis

The activity of human cholinesterases, erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7) and plasma butyrylcholinesterase (BChE; EC 3.1.1.8) represents an important marker when monitoring exposure to pesticides/nerve agents, and may also be used in occupational medicine in diagnosis and prognosis of some diseases. In this study “normal/baseline” AChE and BChE activity has been investigated in a young and healthy population, with subsequent evaluation of several intra-population factors including sex, age (categories 18–25, 26–35 and 36–45 years old) and smoker status.The modified Ellman's method was used for enzyme activity assessment in 387 young and healthy individuals (201 males and 186 females aged 18–45). A significant inter-sexual difference in AChE and BChE activity was found (AChE: 351±67 for males and 377±65 for females, (μmol/min)/(μmol of hemoglobin), p<0.001; BChE: 140±33 for males and 109±29 for females, μkat/l, p<0.001; mean±SD). Despite the finding that mean AChE activity somewhat decreased whereas BChE activity grew within the age categories of the tested subjects, no significant effect of age on cholinesterase activity was found (p>0.05). Smoking influenced cholinesterase activity – AChE activity in smokers was elevated (approx. 3% in males; 8% in females) relative to that in non-smokers (p<0.05). Smoking was found not to have any effect on BChE activity.Reference values based on confidence intervals for AChE and BChE activity were established. The presented results might be useful in routine clinical practice where the monitoring of blood AChE and plasma BChE activity is crucial for prognosis and diagnosis of organophosphate poisoning, in occupational medicine and in relevant mass casualty scenarios.

Differential inhibition of human erythrocyte acetylcholinesterase by polyphenols epigallocatechin-3-gallate and resveratrol. Relevance of the membrane-bound form.

The activity of acetylcholinesterase (AChE) from human erythrocytes was tested in the presence of the phenolic compounds resveratrol and epigallocatechin-3-gallate (EGCG). Even though the stilbene barely changed this enzymatic activity, EGCG did inhibit AChE. Importantly, it preferentially acted on the membrane-bound enzyme rather than on its soluble form. Actually, it was shown that this flavonoid may bind to the red blood cell membrane surface, which may improve the interaction between EGCG and AChE. Therefore, caution should be taken when screening AChE inhibitors. In fact, testing compounds with the soluble form of the enzyme may underestimate the activity of some of these potential inhibitors, hence it would be advisable not to use them as a sole model system for screening. Moreover, erythrocyte AChE is proposed as a good model for these enzymatic assays. © 2016 BioFactors, 43(1):73-81, 2017.

Synthesis and studies of biological activity of N-(4-tert-butylbenzyl)-N-(pyridin-3-ylmethyl)-2-aminothiazolines as potential multifunctional agents for treatment of neurodegenerative diseases

A number of N-(4-tert-butylbenzyl)-N-(pyridin-3-ylmethyl)-2-aminothiazolines with different substituents at position 5 of thiazoline ring was synthesized and their inhibitory activity against human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7), horse serum butyrylcholinesterase (BChE, EC 3.1.1.8), and porcine liver carboxylesterase (CaE, EC 3.1.1.1) was studied, as well as their antioxidant properties in DPPH and ABTS tests (DPPH is the 2,2-diphenyl-1-picrylhydrazyl, ABTS is the 2,2´-azinobis(3-ethylbenzthiazolino-6-sulfonic acid)). All the studied compounds virtually do not inhibit AChE, whereas the inhibition degree of BChE and CaE depends on the substituent structure. The presence of radical-binding activity comparable with the standard antioxidant ascorbic acid was shown for compounds containing a iodomethyl substituent. The found lead compound (R1 = H, R2 = CH2I) combines the antioxidant properties and the ability to efficiently inhibit BChE, simultaneously possessing an optimal esterase profile. The performed studies showed that N-(4-tert-butylbenzyl)-N-(pyridin-3-ylmethyl)-2-aminothiazolines are a new promising class of compounds, which can be used for the development of multifunctional agents for treatment of neurodegenerative diseases.

Alkyl 2-arylhydrazinylidene-3-oxo-3-polyfluoroalkylpropionates as new effective and selective inhibitors of carboxylesterase.

A series of alkyl 2-Arylhydrazinylidene-3-oxo-3-polyfluoroalkylpropionates was synthesized and their inhibitory activity with respect to porcine liver carboxylesterase (CaE, EC 3.1.1.1), human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7), and horse serum butyrylcholinesterase (BChE, EC 3.1.1.8) was studied. The molecular docking method was used to study the binding mode of the compounds in the active site of CaE. It was found that compounds containing the trifluoromethyl group in the third position of carbonyl chain are highly effective and selective inhibitors of CaE with nanomolar IC50 values, which agrees well with the results of molecular docking.

Effects of bispyridinium non-oximes on nerve agent blocked neuromuscular transmission in rat diaphragm muscle and neuronal nicotinic receptors

Reactivation by bispyridinium mono-oximes (Hagedorn-oximes) and some classical oximes (0.03 or 1 mM) was studied in vitro of rat, bovine and human erythrocyte acetylcholinesterase and of electric eel acetylcholinesterase inhibited by soman. Relative reactivating potencies of the oximes are similar for the three inhibited erythrocyte enzymes. In general, Hagedorn-oximes are more potent than the classical oximes. Amont the Hagedorn-oximes, HI-6 is the most potent reactivator for the three inhibited enzymes. Relative reactivating potencies for the inhibited erythrocyte acetylcholinesterases and electric eel acetylcholinesterase, however, clearly differ.Since the reactivation experiments were carried out with racemic soman, a mixture of the two inhibited enzymes may be formed, which may cause additional problems in the comparison of various results. In order to get more detailed information on differences between human erythrocyte and electric eel acetylcholinesterase, reactivation of these enzymes inhibited with the P(−)-isomers of C(+)- and C(−)-soman were studied separately. Reactivation appeared to be dependent on the chirality of the α-carbon atom in the pinacolyl group. HI-6 is by far the most potent reactivator for the human enzyme inhibited by the two P(−)-isomers. It is suggested that electric eel acetylcholinesterase is not a reliable model for in vitro testing of therapeutic potencies of oximes against soman intoxication in mammals.Rate constants of aging of the four acetylcholinesterases inhibited with racemic soman and of the human and eel enzyme inhibited by the P(−)-isomers of C(+)- and C(−)-soman were also determined. The aging of the inhibited rat enzymes proceeds remarkably slowly (t12 = 21 min). The rate of aging is not affected by the chirality on the α-carbon atom in the pinacolyl group.Consequences of the present results are discussed in view of extrapolation of reactivation data of a series of reactivators to their relative therapeutic effect, ultimately in man. It is speculated that the more rapid aging of the human inhibited enzyme may hamper oxime-therapy in man more seriously than in rat.

Synthesis, Anticholinesterase, and Antibacterial Activity of N-Aroyl-a-Aminoacrylic Acid Dialkylaminoalkylamides

N-Aroyl-α-aminoacrylic acid 2-(dialkylamino)alkylamides and their quaternary ammonium salts were synthesized and their physicochemical properties were determined. Results obtained from studies of their interactions with human erythrocyte acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BChE) are presented. IC50 values (concentrations producing 50% inhibition of the rate of cholinesterase hydrolysis of 0.1 mM acetylthiocholine) were determined for all the compounds synthesized. The results show that all the compounds synthesized had anticholinesterase properties, mainly specific in relation to BChE. Data on the antibacterial properties of the synthesized compounds are also presented.

Synthesis and in vitro kinetic evaluation of N-thiazolylacetamido monoquaternary pyridinium oximes as reactivators of sarin, O-ethylsarin and VX inhibited human acetylcholinesterase (hAChE)

Presently available medications for treatment of organiphosphorus poisoning are not sufficiently effective due to various pharmacological and toxicological reasons. In this regard, herein we report the synthesis of a series of N-thiazolylacetamide monoquaternary pyridinium oximes and its analogs (1a–1b to 6a–6b) with diversely substituted thiazole ring and evaluation of their in vitro reactivation efficacies against nerve agent (sarin, O-ethylsarin and VX) inhibited human erythrocyte acetylcholinesterase (hAChE). Reactivation kinetics was performed to determine dissociation constant (KD), reactivity rate constant (kr) and the second order rate constant (kr2) for all the compounds and compared their efficacies with commercial antidotes viz. 2-PAM and obidoxime. All the newly synthesized oximes were evaluated for their physicochemical parameters (pKa) and correlated with their respective reactivation efficacies to assess the capability of the oxime reactivator. Three of these novel compounds showed promising reactivation efficacies toward OP inhibited hAChE. Molecular docking studies were performed in order to correlate the reactivation efficacies with their interactions in the active site of the AChE.

Синтез, антихолинэстеразная и антибактериальная активность диалкиламиноалкиламидов &lt;i&gt;N&lt;/i&gt;-ароил-α-аминоакриловой кислоты

Осуществлен синтез и описаны физико-химические характеристики 2-(диалкиламино)алкиламидов N-ароил-α-аминоакриловой кислоты и их четвертичных аммониевых солей. Приведены результаты изучения их взаимодействия с эритроцитарной ацетилхолинэстеразой (АХЭ) и плазменной бутирилхолинэстеразой (БуХЭ) человека. Для всех синтезированных соединений определены значения ИК50 (ингибирующая концентрация исследуемого соединения, при которой наблюдается 50 % торможение скорости холинэстеразного гидролиза 0,1 мМ ацетилтиохолина). Согласно полученным данным все синтезированные вещества проявляют антихолинэстеразные свойства и, в основном, специфичны по отношению к БуХЭ. Приведены также данные антибактериальных свойств синтезированных соединений.

The effect of glyphosate, its metabolites and impurities on erythrocyte acetylcholinesterase activity

Glyphosate [N-(phosphonomethyl)glycine] is used all over the world to protect agricultural and horticultural crops. According to initial reports, glyphosate has been considered to be safe for humans and animals; nevertheless, recent investigations had proven its toxicity. Extensive use of glyphosate and the conviction of its low toxicity leads to a situation in which it is used in excessive amounts in agriculture. That is why, we have investigated the effect of the most commonly used pesticide: glyphosate, its metabolites and impurities on acetylcholinesterase (AChE) activity (in vitro) in human erythrocytes, which is biochemically similar to acetylcholinesterase present in neural synapses.The analysis of noxious effects of metabolites and impurities of pesticides seems to be very important to evaluate toxicological risk that is associated with the effect of pesticide formulations (requirement of the EU regulations 1107/200/EC).The erythrocytes were incubated with xenobiotics at concentrations range from 0.01 to 5mM for 1 and 4h. Statistically significant decrease in AChE activity (about 20%) was observed only at high concentrations of the compounds (0.25–5mM), which enter body only as a result of acute poisoning. There were no statistically significant differences in the effect of the investigated compounds, while the changes caused by them were similar after 1 and 4h incubation. The investigated metabolites and impurities did not cause stronger changes in AChE activity than glyphosate itself.It may be concluded that the compounds studied (used in the concentrations that are usually determined in the environment) do not disturb function of human erythrocyte acetylcholinesterase.

Development of a method for extraction and assay of human erythrocyte acetylcholinesterase and pesticide inhibition

A method of extracting membranes from red blood cells (RBCs) is described, which were in turn used to assay acetylcholinesterase (AChE) activity. The evidence for the enzyme activity was established by selective inhibition using 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide, tetraisopropyl pyrophosphoramide and neostigmine. Blood samples were exposed to three organophosphorus (dichlorvos, chlorpyrifos and diazinon) and two carbamate (carbaryl and carbofuran) pesticides. Afterwards AChE activities in RBC membranes were determined. The concentrations capable to inhibit the enzyme activity by 50% (IC₅₀) for the pesticides were 10.66 µM (dichlorvos), 21.42 µM (chlorpyrifos), 109.98 µM (carbaryl) and 5.44 µM (carbofuran). The results related to 20% enzyme inhibition (level used in the estimation of threshold limits for anticholinesterase compounds) were below those acceptable daily intake values enacted by relevant national and international regulations. These results suggest that the proposed AChE extraction from RBC and assay could be a suitable method for monitoring occupational exposure to pesticides.

Biological Activity of sym-Triazines with Acetylcholine-like Substitutions as Multitarget Modulators of Alzheimer’s Disease

The bioactivities of two novel compounds (TAE-1 and TAE-2) that contain a sym-triazine scaffold with acetylcholine-like substitutions are examined as promising candidate agents against Alzheimer's disease. Inhibition of amyloid-β fibril formation in the presence of Aβ1-42, evaluated by Thioflavin T fluorescence, demonstrated comparable or improved activity to a previously reported pentapeptide-based fibrillogenesis inhibitor, iAβ5p. Destabilization of Aβ1-42 assemblies by TAE-1 and TAE-2 was confirmed by scanning electron microscopy imaging. sym-Triazine inhibition of acetylcholinesterase (AChE) activity was observed in cytosol extracted from differentiated human SH-SY5Y neuronal cells and also using human erythrocyte AChE. The sym-triazine derivatives were well tolerated by these cells and promoted beneficial effects on human neurons, upregulating expression of synaptophysin, a synaptic marker protein, and MAP2, a neuronal differentiation marker.