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.

Structural and Biochemical Insights into the Inhibition of Human Acetylcholinesterase by G-Series Nerve Agents and Subsequent Reactivation by HI-6.

The recent use of organophosphate nerve agents in Syria, Malaysia, Russia, and the United Kingdom has reinforced the potential threat of their intentional release. These agents act through their ability to inhibit human acetylcholinesterase (hAChE; E.C. 3.1.1.7), an enzyme vital for survival. The toxicity of hAChE inhibition via G-series nerve agents has been demonstrated to vary widely depending on the G-agent used. To gain insight into this issue, the structures of hAChE inhibited by tabun, sarin, cyclosarin, soman, and GP were obtained along with the inhibition kinetics for these agents. Through this information, the role of hAChE active site plasticity in agent selectivity is revealed. With reports indicating that the efficacy of reactivators can vary based on the nerve agent inhibiting hAChE, human recombinatorially expressed hAChE was utilized to define these variations for HI-6 among various G-agents. To identify the structural underpinnings of this phenomenon, the structures of tabun, sarin, and soman-inhibited hAChE in complex with HI-6 were determined. This revealed how the presence of G-agent adducts impacts reactivator access and placement within the active site. These insights will contribute toward a path of next-generation reactivators and an improved understanding of the innate issues with the current reactivators.

[1,5]-Hydride Shift-Cyclization versus C(sp2)-H Functionalization in the Knoevenagel-Cyclization Domino Reactions of 1,4- and 1,5-Benzoxazepines.

Domino cyclization reactions of N-aryl-1,4- and 1,5-benzoxazepine derivatives involving [1,5]-hydride shift or C(sp2)-H functionalization were investigated. Neuroprotective and acetylcholinesterase activities of the products were studied. Domino Knoevenagel-[1,5]-hydride shift-cyclization reaction of N-aryl-1,4-benzoxazepine derivatives with 1,3-dicarbonyl reagents having active methylene group afforded the 1,2,8,9-tetrahydro-7bH-quinolino [1,2-d][1,4]benzoxazepine scaffold with different substitution pattern. The C(sp3)-H activation step of the tertiary amine moiety occurred with complete regioselectivity and the 6-endo cyclization took place in a complete diastereoselective manner. In two cases, the enantiomers of the chiral condensed new 1,4-benzoxazepine systems were separated by chiral HPLC, HPLC-ECD spectra were recorded, and absolute configurations were determined by time-dependent density functional theory- electronic circular dichroism (TDDFT-ECD) calculations. In contrast, the analogue reaction of the regioisomeric N-aryl-1,5-benzoxazepine derivative did not follow the above mechanism but instead the Knoevenagel intermediate reacted in an SEAr reaction [C(sp2)-H functionalization] resulting in a condensed acridane derivative. The AChE inhibitory assays of the new derivatives revealed that the acridane derivative had a 6.98 µM IC50 value.

Brachychiton populneus as a novel source of bioactive ingredients with therapeutic effects: antioxidant, enzyme inhibitory, anti-inflammatory properties and LC-ESI-MS profile.

Brachychiton populneus is one of the unexploited Tunisian plants, traditionally eaten as food and used for medicinal purposes. The present study aimed to investigate the phytochemical components of the seeds, leaves and flowers from B. populneus using three different solvents and to explore their antioxidant, anti-inflammatory and neuroprotective effects. Further, this study was focused on the identification of phenolic compounds from the most active extract. In vitro, all extracts showed strong antioxidant property by DPPH, ferrous ion chelating and lipid peroxidation-inhibiting assays, noticeable anti-inflammatory activity by protein denaturation and membrane stabilization methods and important neuroprotective effects by acetylcholinesterase inhibitory test. In vivo, B. populneus (50, 100 and 200 mg/kg, i.p.) showed significant dose-response anti-inflammatory effects against carrageenan-induced paw edema. With respect to the phenolic profile, the leaf methanol extract presented eight phenolic acids, one flavone and four flavonoids, with salvianolic acid B (820.3 mg/kg), caffeic acid (224.03 mg/kg), syringic acid (100.2 mg/kg) and trans-ferulic acid (60.02 mg/kg) as the major compounds. The results of the current study suggested that B. populneus could be a precious source of health-benefitting biomolecules and may be developed as new antioxidant, anti-inflammatory and AChE inhibitors.

Purification and characterization of acetylcholinesterase in Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae).

Red palm weevil (RPW), Rhynchophorus ferrugineus, is one of the most destructive pests of cultivated palm trees. The application of synthetic insecticides is currently a main strategy for RPW control. In this study we estimated the distribution of acetylcholinesterase (AChE), as a detoxifying enzyme and the target site of inhibition by insecticides, using ASChI as substrate in different organs of the pest including whole gut, cuticle, fat body, head and haemolymph. The activity ranged from 314.9 to 3868 U in individual organs while the specific activity ranged from 99 to 340.8 U/mg proteins; the cuticle had the highest enzyme level. During larval development, the 11th instar larvae had the highest enzyme content with 5630 U in the cuticle, with a specific activity of 140 U/mg protein. The two major AChE isoenzymes were purified by chromatography on gel filtration and ion exchange columns. They had specific activities of 3504.3 and 2979 U/mg protein, molecular weights of 33 and 54 kDa and activation energies of 8.3 and 4.4 kcal/mol, respectively. Both isoenzymes had monomeric forms, optimum activity at pH 8.0 and 40 °C, were completely inhibited by Hg2+ and Cu2 and showed similar trends towards the inhibitors eserine, BW284C51 and iso-OMPA. The catalytic properties were compared with those previously recorded for different insect species. This work will pave the way for more studies for improving the understanding of insecticide resistance and developing the field application of synthetic insecticides for controlling R. ferrugineus to ensure successful application.

Chemical characterization of the main bioactive polyphenols from the roots of Morus australis (mulberry).

Morus species, commonly known as mulberry, is widely distributed in China. The mulberry tree is a high-value plant in agriculture. Morus australis is one of the major Morus species growing in Northern China. However, the biological properties of the main constituents of M. australis roots were not well studied. In the present study, through extensive chromatographic and spectral analysis, 12 phenolic compounds were isolated and identified from the M. australis roots. Compounds 1, 2, 8, 9 and 12 were isolated from M. australis roots for the first time. Antitumor activities of these polyphenols were studied on the A549 cell line. Compounds 1, 5 and 6 exhibited cytotoxicity on A549 cells and induced apoptosis in A549 cells via the intrinsic mitochondrial pathway. They also mediated inhibition of autophagic flux contributed cell death via the PI3k/Akt/mTOR pathway. In order to explore more potential bioactivities of these isolates, α-glucosidase, acetylcholinesterase and tyrosinase inhibitory activities were studied, and the results demonstrated that the inhibitory activity of these polyphenols on enzymes was not defined by their basic structural skeletons, but by the substituted position.

Identification of Carboxylesterase, Butyrylcholinesterase, Acetylcholinesterase, Paraoxonase, and Albumin Pseudoesterase in Guinea Pig Plasma through Nondenaturing Gel Electrophoresis.

Drugs to protect against nerve agent toxicity are tested in animals. The current preferred small animal model is guinea pigs because their plasma bioscavenging capacity resembles that of NHP. We stained nondenaturing polyacrylamide slab gels with a variety of substrates, inhibitors, and antibodies to identify the esterases in heparinized guinea pig plasma. An intense band of carboxylesterase activity migrated behind albumin. Minor carboxylesterase bands were revealed after background activity from paraoxonase was inhibited by using EDTA. The major butyrylcholinesterase band was a disulfide-linked dimer. Incubation with the antihuman butyrylcholinesterase antibody B2 18-5 shifted the butyrylcholinesterase dimer band to slower migrating complexes. Carboxylesterases were distinguished from butyrylcholinesterase by their sensitivity to inhibition by bis-p-nitrophenyl phosphate. Acetylcholinesterase tetramers formed a complex with the antihuman acetylcholinesterase antibody HR2. Organophosphorus toxicants including cresyl saligenin phosphate, dichlorvos, and chlorpyrifos oxon irreversibly inhibited the serine esterases but not paraoxonase. Albumin pseudoesterase activity was seen in gels stained with α- or ß-naphthyl acetate and fast blue RR. We conclude that guinea pig plasma has 2 types of carboxylesterase, butyrylcholinesterase dimers and 5 minor butyrylcholinesterase forms, a small amount of acetylcholinesterase tetramers, paraoxonase, and albumin pseudoesterase activity. A knockout mouse with no carboxylesterase activity in plasma is available and may prove to be a better model for studies of nerve agent toxicology than guinea pigs.

Chemical Composition, Characteristics Profiles and Bioactivities of Tunisian Phalaris canariensis Seeds: a Potential Source of ω-6 and ω-9 Fatty Acids.

Seeds oils of Phalaris canariensis extracted by ultrasonication and cold maceration were evaluated for their physical characteristics, total phenol contents, fatty acid and sterol compositions as well as for their antioxidant, antibacterial and acetylcholinesterase activities. The physicochemical properties of ultrasonication and cold maceration oils respectively were: acid values (4.00 and 3.25) mg KOH/g, peroxide values (5.53 and 4.41) meq O2 Kg-1, iodine values (88.83 and 95.17) g/100 g of oil, saponification values (119.21 and 98.17) mg KOH/g, phenolic content (36.40 and 53.00) mg GAE/g extract, chlorophylls (0.52 and 0.60) mg/kg oil and carotenoids contents (1.92 and 1.88) mg/kg oil. Gas chromatography analysis revealed that linoleic (52.03 and 52.2%), oleic (31.75 and 31.84%) and palmitic (11.09 and 11.34 %) acids were the major fatty acids in the two oils. Specific extinctions at 232 nm (K232) and 270 nm (K270) were (0.58 and 0.44) and (0.42 and 0.33), respectively. The DSC melting curve showed that their melting points and melting enthalpies were (-28.05°C and 76.8 J/g) and (-27.47°C and 62.3 J/g), respectively. On the other hand, the evaluation of their DPPH radical scavenging, total antioxidant capacity, antibacterial and acetylcholinesterase activities showed interesting results. Thus, Phalaris canariensis seeds oils could deserve further consideration and investigation as a potentially new multi-purpose product for agro-food, medicinal and cosmetic uses.

Acetylcholinesterase Inhibitory Alkaloids from the Whole Plants of Zephyranthes carinata.

Eleven new alkaloids (1-11), classified as the 12-acetylplicamine (1), N-deformyl-seco-plicamine (2), plicamine (3-6), 4a-epi-plicamine (7), seco-plicamine (8), and lycorine (9-11) framework types, along with 15 known alkaloids (12-26) were isolated from the whole plants of Zephyranthes carinata. The structures of the new alkaloids 1-11 were established by extensive spectroscopic data interpretation. The absolute configurations of 9 and 10 were defined by single-crystal X-ray diffraction analysis. Zephycarinatines A (1), B (2), and G (7) represent the first examples of 12-acetylplicamine, N-deformyl-seco-plicamine, and 4a-epi-plicamine alkaloids, respectively. Alkaloids 6, 11, 17, and 20-23 exhibited AChE inhibitory activities with IC50 values ranging from 1.21 to 184.05 µM, and a preliminary structure-activity relationship is discussed.

Immunopurification of Acetylcholinesterase from Red Blood Cells for Detection of Nerve Agent Exposure.

Nerve agents and organophosphorus pesticides make a covalent bond with the active site serine of acetylcholinesterase (AChE), resulting in inhibition of AChE activity and toxic symptoms. AChE in red blood cells (RBCs) serves as a surrogate for AChE in the nervous system. Mass spectrometry analysis of adducts on RBC AChE could provide evidence of exposure. Our goal was to develop a method of immunopurifying human RBC AChE in quantities adequate for detecting exposure by mass spectrometry. For this purpose, we immobilized 3 commercially available anti-human acetylcholinesterase monoclonal antibodies (AE-1, AE-2, and HR2) plus 3 new monoclonal antibodies. The monoclonal antibodies were characterized for binding affinity, epitope mapping by pairing analysis, and nucleotide and amino acid sequences. AChE was solubilized from frozen RBCs with 1% (v/v) Triton X-100. A 16 mL sample containing 5.8 µg of RBC AChE was treated with a quantity of soman model compound that inhibited 50% of the AChE activity. Native and soman-inhibited RBC AChE samples were immunopurified on antibody-Sepharose beads. The immunopurified RBC AChE was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. The aged soman-modified PheGlyGluSerAlaGlyAlaAlaSer (FGESAGAAS) peptide was detected using a targeted analysis method. It was concluded that all 6 monoclonal antibodies could be used to immunopurify RBC AChE and that exposure to nerve agents could be detected as adducts on the active site serine of RBC AChE.

Recombinant Acetylcholinesterase purification and its interaction with silver nanoparticle.

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

Characterization of acetylcholinesterase from elm left beetle, Xanthogaleruca luteola and QSAR of temephos derivatives against its activity.

Insect acetylcholinesterase (AChE) is the principal target for organophosphate (OP) and carbamate (CB) insecticides. In this research, an AChE from third instar larvae of elm left beetle, Xanthogaleruca luteola was purified by affinity chromatography. The enzyme was purified 75.29-fold with a total yield of 8.51%. As shown on denaturing SDS-PAGE, the molecular mass of purified AChE was 70kDa. The enzyme demonstrated maximum activity at pH7 and 35°C. Furthermore, a series of temephos (Tem) derivatives with the general structure of P(O)XP(O) (1-44) were prepared, synthesized and characterized by 31P, 13C, 1H NMR and FT-IR spectral techniques. The toxicity of 36 new Tem derivatives was screened on the third instar larvae and the compound compound 1,2 cyclohexane-N,N'-bis(N,N'-piperidine phosphoramidate) exhibited the highest insecticidal potential. The method of kinetic analysis is applied in order to obtain the maximum velocity (Vmax), the Michaelis constant (Km) and the parameters characterizing the inhibition type for inhibitors with >75% mortality in preliminary bioassay. The inhibition mechanism was mixed and inhibitory constant (Ki) was calculated as 4.70µM-1min-1 for this compound. Quantitative structure-activity relationship (QSAR) equations of these compounds indicated that the electron orbital energy has major effect on insecticidal properties.

Chemical composition and in vitro evaluation of antioxidant, antimicrobial, cytotoxicity and anti-acetylcholinesterase properties of Tunisian Origanum majorana L. essential oil.

This study investigated the chemical composition and evaluated the antioxidant, antimicrobial, cytotoxic and anti-acetylcholinesterase properties of Tunisian Origanum majorana essential oil. The findings showed that the oil exhibited high activity, particularly in terms of reducing power and ß-Carotene bleaching, inducing higher IC50 values than BHT. The oil showed an important antimicrobial activity against 25 bacterial and fungal strains. In fact, the IZ, MIC and MBC values recorded for the bacterial strains were in the range of 8 ± 0-18.33 ± 0.57 mm, 0.097-3.125 and 0.39-6.25 mg/mL, respectively. The IZ, MIC and MFC values of the fungal strains varied between 11±0-28 ± 0 mm, 0.058-0.468 mg/mL and 0.234-1.875 mg/mL, respectively. A low cytotoxic effect was observed against cancer (Hep-2 and HT29) and continuous cell lineage (Vero), with CC50 values ranging from 13.73 to 85.63 mg/mL. The oil was also evaluated for anti-acetylcholinesterase effects, which showed that it exhibited significant activity with IC50 values reaching 150.33 ± 2.02 µg/mL.

Biphasic photoelectrochemical sensing strategy based on in situ formation of CdS quantum dots for highly sensitive detection of acetylcholinesterase activity and inhibition.

Herein, we reported a facile and highly sensitive biphasic photoelectrochemical (PEC) sensing strategy based on enzymatic product-mediated in situ formation of CdS quantum dots (QDs), and assayed the activity and inhibition of acetylcholinesterase (AChE) in its optimal state. Upon the hydrolysis of acetylthiocholine catalyzed by AChE, the product thiocholine stabilizes the in situ formation of CdS QDs in homogenous solution. Due to the electrostatic attraction, the resulting tertiary amino group-functionalized CdS QDs are attached to the surface of the negatively charged indium tin oxide (ITO) electrode, generating significant PEC response upon illumination in the presence of electron donors. By taking full advantage of the in situ formation of CdS QDs in homogenous solution, this strategy is capable of detecting AChE activity and inhibition in its optimal state. A directly measured detection limit of 0.01mU/mL for AChE activity is obtained, which is superior to those obtained by some fluorescence methods. The inhibition of AChE activity by aldicarb is successfully detected, and the corresponding IC50 is determined to be 13µg/L. In addition to high sensitivity and good selectivity, this strategy also exhibits additional advantages of simplicity, low cost and easy operation. To the best of our knowledge, the as-proposed strategy is the first example demonstrating the application of CdS QDs formed in situ for biphasic PEC detection of enzyme activity and inhibition. More significantly, it opens up a new horizon for the development of homogenous PEC sensing platforms, and has great potential in probing many other analytes.

Acetylcholinesterase and carbonic anhydrase isoenzymes I and II inhibition profiles of taxifolin.

Taxifolin, also known as dihydroquercetin, is a flavonoid commonly found in plants. Carbonic anhydrase (CA, EC 4.2.1.1) plays an important role in many critical physiological events including carbon dioxide (CO2)/bicarbonate (HCO3(-)) respiration and pH regulation. There are 16 known CA isoforms in humans, of which human hCA isoenzymes I and II (hCA I and II) are ubiquitous cytosolic isoforms. In this study, the inhibition properties of taxifolin against the slow cytosolic isoenzyme hCA I, and the ubiquitous and dominant rapid cytosolic isoenzyme hCA II were studied. Taxifolin, as a naturally bioactive flavonoid, has a K(i) of 29.2 nM against hCA I, and 24.2 nM against hCA II. For acetylcholinesterase enzyme (AChE) inhibition, K(i) parameter of taxifolin was determined to be 16.7 nM. These results clearly show that taxifolin inhibited both CA isoenzymes and AChE at the nM levels.

Comparative chemical profiling, cholinesterase inhibitions and anti-radicals properties of essential oils from Polygonum hydropiper L: a preliminary anti- Alzheimer's study.

BACKGROUND: Cholinesterase inhibition is a vital target for the development of novel and mechanism based inhibitors, owing to their role in the breakdown of acetylcholine (ACh) neurotransmitter to treat various neurological disorders including Alzheimer's disease (AD). Similarly, free radicals are implicated in the progression of various diseases like neurodegenerative disorders. Due to lipid solubility and potential to easily cross blood brain barrier, this study was designed to investigate the anticholinesterase and antioxidant potentials of the standardized essential oils from the leaves and flowers of Polygonum hydropiper. METHODS: Essential oils from the leaves (Ph.LO) and flowers (Ph.FO) of P. hdropiper were isolated using Clevenger apparatus. Oil samples were analyzed by GC-MS to identify major components and to attribute the antioxidant and anticholinesterase activity to specific components. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory potentials of the samples were determined following Ellman's assay. Antioxidant assays were performed using 1,1-diphenyl,2-picrylhydrazyl (DPPH), 2,2-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid (ABTS) and hydrogen peroxide (H2O2) free radical scavenging assays. RESULTS: In the GC-MS analysis 141 and 122 compounds were indentified in Ph.LO and Ph.FO respectively. Caryophylene oxide (41.42 %) was the major component in Ph.FO while decahydronaphthalene (38.29 %) was prominent in Ph.LO. In AChE inhibition, Ph.LO and Ph.FO exhibited 87.00** and 79.66***% inhibitions at 1000 µg/ml with IC50 of 120 and 220 µg/ml respectively. The IC50 value for galanthamine was 15 µg/ml. In BChE inhibitory assay, Ph.LO and Ph.FO caused 82.66*** (IC50 130 µg/ml) and 77.50***% (IC50 225 µg/ml) inhibitions respectively at 1000 µg/ml concentration. In DPPH free radical scavenging assay, Ph.LO and Ph.FO exhibited IC50 of 20 and 200 µg/ml respectively. The calculated IC50s were 180 & 60 µg/ml for Ph.LO, and 45 & 50 µg/ml for Ph.FO in scavenging of ABTS and H2O2 free radicals respectively. CONCLUSIONS: In the current study, essential oils from leaves and flowers of P. hydropiper exhibited dose dependent anticholinesterase and antioxidant activities. Leaves essential oil were more effective and can be subjected to further in-vitro and in-vivo anti-Alzheimer's studies.

In situ induced metal-enhanced fluorescence: a new strategy for biosensing the total acetylcholinesterase activity in sub-microliter human whole blood.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities (i.e., total AChE) in human blood are biomarkers for theranostic monitoring of organophosphate neurotoxin-poisoned patients. We developed an ultra-sensitive method to detect the total AChE activity in sub-microliter human whole blood based on in situ induced metal-enhanced fluorescence (MEF). Both AChE and BChE can catalyze the hydrolysis of the acetylthiocholine (ATCh) substrate and produce positively-charged thiocholine (TCh). TCh can reverse the negatively-charged surface of core-shell Ag@SiO2 nanoparticles (NPs). The negatively-charged fluorescent dye (8-hydroxypyrene-1,3,6-trisulfonic acid, HPTS) is then confined to the surface of Ag@SiO2 NPs and generates an enhanced fluorescence signal in situ. Changes in the surface charge of Ag@SiO2 NPs are monitored by Zeta potential, and the MEF effect is confirmed by the measurements of fluorescence time decay. AChE activity has a dynamic range of 0 U/mL to 0.005 U/mL and a detection limit of 0.05 mU/mL. The total AChE activity in the sub-microliter human whole blood could be determined; the results were further validated. Therefore, combining the AChE catalytic reaction with MEF provides a simple, ultra-sensitive, and cost-effective "in situ MEF" approach to determine the total AChE activity in human whole blood sample down to sub-microliters without matrix interferences. The strategy also allows potential usage in other tissues and other fields.

Discovery of butyrylcholinesterase inhibitors among derivatives of azaphenothiazines.

The study presents the discovery of novel butyrylcholinesterase (BuChE) inhibitors among derivatives of azaphenothiazines by application of in silico and in vitro screening methods. From an in-house library of compounds, 143 heterocyclic molecules derived from the azaphenothiazine scaffold were chosen for virtual screening. Based on results of the docking procedure, 15 compounds were identified as exhibiting the best fit for the two screening complexes (ligand - AChE and ligand - BuChE). Five compounds displayed moderate AChE and good BuChE inhibitory activity at screening concentrations of 10 µM. The IC50 values for active BuChE inhibitors were in the 11.8-122.2 nM range. Three of the most active inhibitors are tetra- or pentacyclic derivatives of azaphenothiazines with the same N-methyl-2-piperidinethyl substituent.

Novel tacrine analogs as potential cholinesterase inhibitors in Alzheimer's disease.

Acetylcholinesterase inhibitors (AChEIs) are used for the treatment of Alzheimer's disease (AD). The increase in ACh levels ameliorates the symptoms of the disease. Tacrine is the first clinically approved drug as AChEI used in the treatment of AD. In this paper, we synthesized new tacrine analogs to act on catalytic and peripheral sites of AChE. Their inhibitory activity was evaluated. All novel compounds except 7a showed promising results toward AChE. Two compounds, 10b and 11b, are more potent than tacrine. Furthermore, molecular-modeling studies were performed for these two compounds to rationalize the obtained pharmacological activity. Moreover, various drug-likeness properties of the new compounds were predicted.

Comparative studies on acetylcholinesterase characteristics between the aphids, Sitobion avenae and Rhopalosiphum padi.

The aphids Sitobion avenae (Fabricius) and Rhopalosiphum padi (Linnaeus) (Hemiptera: Aphidiae) are serious pests on grain crops and usually coexist on late period of wheat growth in China. Bioassays showed that R. padi was more susceptible than S. avenae to pirimicarb that is used for wheat aphid control, and the determination of acetylcholinesterase (AChE, EC 3.1.1.7) sensitivity showed that the sensitivity of AChE to pirimicarb was significantly higher in R. padi than in S. avenae ( Lu and Gao 2009 ). AChE is the target enzyme of the carbamates, including pirimicarb, hence, to understand the mechanism responsible for the tolerance difference to carbamate insecticides of S. avenae and R. padi, we purified AChE from both aphid species using procainamide affinity column and characterized the AChE. The purification factor and yield from S. avenae (234.7-fold and 92.9%) were far higher than that from R. padi 17.3-fold and 13.9%. The results of substrate and inhibitor specificities of purified enzyme from both S. avenae and R. padi indicated that the purified enzyme was a typical AChE. The crude AChE extract from S. avenae was 5.4-, 4.3- and 8.1-fold less sensitive to inhibition by pirimicarb, methomyl and thiodicarb, respectively, than that from R. padi, whereas for the purified AChE, S. avenae was only 1.6-, 1.3- and 1.7-fold less sensitive to inhibition by pirimicarb, methomyl and thiodicarb, respectively, than R. padi. This suggests that eserine and BW284C51 may bind with other proteins, such as carboxylesterase, in the crude extract to reduce their inhibition against AChE. These results are useful for planning the chemical control of aphids on wheat.