A conformational restriction approach to the development of dual inhibitors of acetylcholinesterase and serotonin transporter as potential agents for Alzheimer's disease

Alzheimer's disease (AD) has been treated with acetylcholinesterase (AChE) inhibitors such as donepezil. However, the clinical usefulness of AChE inhibitors is limited mainly due to their adverse peripheral effects. Depression seen in AD patients has been treated with serotonin transporter (SERT) inhibitors. We considered that combining SERT and AChE inhibition could improve the clinical usefulness of AChE inhibitors. In a previous paper, we found a potential dual inhibitor, 1, of AChE (IC50=101 nM) and SERT (IC50=42 nM), but its AChE inhibition activity was less than donepezil (IC50=10 nM). Here, we report the conformationally restricted (R)-18a considerably enhanced inhibitory activity against AChE (IC50=14 nM) and SERT (IC50=6 nM).     

Effects of dimethoate on snail B-esterase and growth as a function of dose, time and exposure route in a laboratory bioassay.

The aim was to study the effects of dimethoate on enzymatic targets and on the growth of Helix aspersa for different times and modes of exposure under laboratory conditions. Young snails were exposed to increasing dimethoate concentrations in the food (D.exp) or in an artificial substrate (S.exp) for 1, 2, 7 and 14 days. Both acetylcholinesterase (AChE) and carboxylesterase (CaE) activities were measured in the foot of the snails for each concentration and exposure time tested. Growth was evaluated after 7 days of exposure. AChE inhibition, dose-dependent for all lengths of exposure, was stronger in S.exp. AChE was more sensitive than CaE for both modes of exposure. IC50(-7) days was 38.3 micrograms g-1 in D.exp and 11.7 micrograms g-1 in S.exp for AChE and was higher than 150 micrograms g-1 in two exposure modes for CaE. AChE activity decreased from the first day to reach maximum inhibition after 7 days of exposure. As noted for B-esterase activities, growth inhibition was stronger in S.exp and was only significant for AChE inhibition of > 90%. The present results show that AChE activity could be used to give early warning of toxic effects of dimethoate in terrestrial gastropods.     

Synthesis and biological evaluation of novel N,N'-bis-methylenedioxybenzyl-alkylenediamines as bivalent anti-Alzheimer disease ligands

A novel series of N,N'-bis-methylenedioxybenzyl-alkylenediamines 5a-5g have been designed, synthesized and evaluated as bivalent anti-Alzheimer's disease ligands. The enzyme inhibition assay results indicated that compounds 5e-5g inhibit both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the micromolar range (IC(50), 2.76-4.24 μM for AChE and 3.02-5.14 μM for BuChE), which was in the same potential as the reference compound rivastigmine (IC(50), 5.50 μM for AChE and 1.60 μM for BuChE). It was found that compounds could bind simultaneously to the peripheral and catalytic sites of AChE. β-Amyloid (Aβ) aggregation inhibition assay results showed that compound 5e exhibited highest self-mediated Aβ fibril aggregation inhibition activity (40.3%) with a similar potential as curcumin (41.6%). It was also found that 5e-5g did not affect neuroblastoma cell viability at the concentration of 50 μM.     

Acetylcholinesterase in the sea urchin Lytechinus variegatus: characterization and developmental expression in larvae

Acetylcholinesterase (AChE) in the echinoid Lytechinus variegatus has been characterized. Kinetic parameters V(max), K(m), K(ss), and b were 2594+/-1048 nmol ATCh hydrolyzed/min/mg tissue wet weight, 185+/-11 microM, 308+/-100 mM, and 0.2, respectively for the substrate ATCh and 17.8+/-6.87 nmol BTCh hydrolyzed/min/mg tissue wet weight, 654+/-424 microM, 36+/-31 mM, and 0.6, respectively for BTCh. Pharmacologic analyses were performed with four inhibitors of cholinesterases, physostigmine, BW284c51, ethopropazine, and iso-OMPA, and yielded IC(50) values of 106+/-4 nM, 718+/-118 nM, 2.57+/-0.6 mM, and >0.0300 M, respectively. Both kinetic and pharmacologic results confirmed the existence of AChE in larval L. variegatus. Dimeric and tetrameric globular forms (determined by velocity sedimentation on sucrose gradients) were present in L. variegatus larvae. Activity of AChE increased significantly as larvae progressed in development from embryos to eight-arm larvae. Acetylcholinesterase activity of F1 larvae derived from sea urchins collected from wild populations and of F1 larvae derived from sea urchins cultured in the laboratory and fed two different diets suggest that the nutritional and/or environmental history of the adult sea urchin affect the developmental progression of AChE activity in the F1 offspring.     

In vitro characterization of cholinesterases in the earthworm Eisenia andrei

Assessment of pollution impact in soil ecosystems has become a priority and interest has grown concerning the use of invertebrates as sentinel organisms. Inhibition of cholinesterase (ChE) activity has a great potential as a biomarker of pesticide exposure, and we evaluated the ChE kinetic parameters in the earthworm Eisenia andrei in the presence of acetylthiocholine (ASCh), proprionylthiocholine (PSCh) and butyrylthiocholine (BSCh). The highest ChE activity was found in the presence of ASCh and PSCh (42.45 and 49.82 nmol min(-1) mg protein(-1), respectively). BSCh was hydrolyzed at a rate of 4.04 nmol min(-1) mg protein(-1), but the time course did not reach a plateau under our experimental conditions. Km values were 0.142+/-0.006 and 0.183+/-0.053 mM for ASCh and PSCh, respectively. ASCh and PSCh hydrolysis were significantly inhibited by eserine (IC50 values were 1.44 x 10(-8) and 1.20 x 10(-8) M, respectively) and by carbaryl (IC50 values of 5.75 x 10(-9) and 4.79 x 10(-9) M). The presence of different ChEs in tissues from E. andrei was assessed by using selective inhibitors for AChE (BW284c51) and BChE (iso-OMPA). BW284c51 strongly reduced ASCh and PSCh hydrolysis and slightly affected that of BSCh, while iso-OMPA was without effect in all cases.     

Some new carbacylamidophosphates as inhibitors of acetylcholinesterase and butyrylcholinesterase

The differences in the inhibition activity of organophosphorus agents are a manifestation of different molecular properties of the inhibitors involved in the interaction with the active site of enzyme. We were interested in comparing the inhibition potency of four known synthesized carbacylamidophosphates with the general formula RC(O)NHP(O)Cl2, constituting organophosphorus compounds, where R = CCl3 (1), CHCl2 (2), CH2Cl (3) and CF3 (4), and four new ones with the general formula RC(O)NHP(O)(R')2, where R' = morpholine and R = CCl3 (5), CHCl2 (6), CH2Cl (7), CF3 (8), on AChE and BuChE activities. In addition, in vitro activities of all eight compounds on BuChE were determined. Besides, in vivo inhibition potency of compounds 2 and 6, which had the highest inhibition potency among the tested compounds, was studied. The data demonstrated that compound 2 from the compound series 1 to 4 and compound 6 from the compound series 5 to 8 are the most sensitive as AChE and BuChE inhibitors, respectively. Comparing the IC50 values of these compounds, it was clear that the inhibition potency of these compounds for AChE are 2- to 100-fold greater than for BuChE inhibition. Comparison of the kinetics (IC50, Ki, kp, KA and KD) of AChE and BuChE inactivation by these compounds resulted in no significant difference for the measured variables except for compounds 2 and 6, which appeared to be more sensitive to AChE and BuChE by significantly higher kp and Ki values and a lower IC50 value in comparison with the other compounds. The LD50 value of compounds 2 and 6, after oral administration, and the changes of erythrocyte AChE and plasma BuChE activities in albino mice were studied. The in vivo experiments, similar to the in vitro results, showed that compound 2 is a stronger AChE and BuChE inhibitor than the other synthesized carbacylamidophosphates. Furthermore, in this study, the importance of electropositivity of the phosphorus atom, steric hindrance and leaving group specificity were reinforced as important determinants of inhibition activity.     

Joint effects of dimethoate and heavy metals on metabolic responses in a grasshopper (Chorthippus brunneus) from a heavy metals pollution gradient

We studied how an exposure to an additional stressing factor-dimethoate, might affect detoxifying ability of grasshoppers collected at 5 meadow sites located along a heavy metal pollution gradient. Activities of esterases and enzymes linked with glutathione (GSH) metabolism were assayed 24 h after topical treatment with 0.32 microg dimethoate per insect. Inhibition of acetylcholinesterase (AChE) reaches nearly 50% of the value stated in untreated insects, without significant site-dependent differences. The pesticide also caused a significant decrease in activities of glutathione peroxidase (GPx) followed by a decrease in GSH levels in grasshoppers from all assayed groups, demonstrating high sensitivity of glutathione-dependent metabolism to the additional stressing factor. In the case of glutathione reductase (GR) and carboxylesterases (CarE) the fall of activity was shown especially in insects from less polluted meadows and the reference site. Glutathione reductase (GR) activity in individuals treated with dimethoate did not decrease only in insects from the most contaminated site I. This might suggest the trade-off mechanisms adapting grasshoppers to life in seriously polluted environments.     

Synthesis and biological evaluation of novel flavonoid derivatives as dual binding acetylcholinesterase inhibitors

A new series of flavonoid derivatives have been designed, synthesised and evaluated as acetylcholinesterase inhibitors that could bind simultaneously to the peripheral and catalytic sites of the enzyme. Among them, fifteen derivatives were found to inhibit the enzyme in the micromolar range and isoflavone derivatives possessed more potent inhibitory activity than other flavonoid derivatives. The best compound 9a had its inhibitory activity (IC(50) = 0.093 microM) in the same range as the reference compound, donepezil (IC(50) = 0.025 microM). Preliminary structure-activity relationships and a molecular modeling study for 9a have revealed that the isoflavone moiety plays a key role in the interaction of this series of derivatives with AChE by acting as an anchor in its peripheral anionic site.     

Identification of new allosteric sites and modulators of AChE through computational and experimental tools

Allosteric sites on proteins are targeted for designing more selective inhibitors of enzyme activity and to discover new functions. Acetylcholinesterase (AChE), which is most widely known for the hydrolysis of the neurotransmitter acetylcholine, has a peripheral allosteric subsite responsible for amyloidosis in Alzheimer’s disease through interaction with amyloid β-peptide. However, AChE plays other non-hydrolytic functions. Here, we identify and characterise using computational tools two new allosteric sites in AChE, which have allowed us to identify allosteric inhibitors by virtual screening guided by structure-based and fragment hotspot strategies. The identified compounds were also screened for in vitro inhibition of AChE and three were observed to be active. Further experimental (kinetic) and computational (molecular dynamics) studies have been performed to verify the allosteric activity. These new compounds may be valuable pharmacological tools in the study of non-cholinergic functions of AChE.     

The aryl acylamidase activity is much more sensitive to Alzheimer drugs than the esterase activity of acetylcholinesterase in chicken embryonic brain

The appearance of cholinergic trait often precedes synaptogenesis, indicating the involvement of cholinesterase proteins in nervous system development, particularly so acetylcholinesterase (AChE). In addition to AChE's acclaimed esterase activity, its lesser known non-cholinergic functions have gained much attention, because of AChE protein expression in areas other than cholinergic innervations; one such function could be exerted by its associated aryl acylamidase (AAA) activity. In this study, an attempt has been made in profiling esterase and AAA activities of AChE at different developmental stages of the chick embryo, e.g. at embryonic day 6 (E6), E9, E12, E15 and E18. AAA activity showed a correlated expression with esterase activity at all stages, but the relative ratios of AAA to esterase activity were higher at younger stages. The inhibition of AAA activity was shown to be more sensitive towards Huperzine, Donepezil whereas inhibition of esterase activity was sensitive to Tacrine and DFP. Remarkably, the major Alzheimer drugs- Huperzine and Donepezil, much more strongly inhibited AAA activity of AChE at younger developmental stages whose IC50 values are 0.01 μM and 0.1 μM respectively. In the case of BW284c51, inhibition was more pronounced at older stages and IC50 value was 0.1 μM. Since in Alzheimer's disease (AD), embryonic forms of AChE have been reported to reappear, a possible role of AAA activity in the pathogenesis of AD should be considered.     

Flow cytometric analysis of bacterial respiratory and enzymatic activity in the natural aquatic environment

The respiratory and enzymatic activity of bacteria in polluted and unpolluted river water was investigated by flow cytometry. Double staining with 6CFDA (6-carboxy fluorescein diacetate) and PI (propidium iodide) was used to examine bacterial esterase activity. CTC (5-cyano-2,3-ditolyl tetrazolium chloride) was employed as the indicator of bacterial respiration. The ratios of colony-forming units to total bacterial number were less than 2%, except highly polluted sites. The number of respiring bacteria determined by flow cytometry amounted to 10–15% of the total bacterial number at both unpolluted and polluted sampling stations, while it was 30% at the highly polluted station. Almost 50% of total bacteria demonstrated esterase activity in the unpolluted areas, whereas 70–90% of total bacteria retained this enzymatic activity in the polluted areas. Thus, many of the non-culturable bacteria in the natural aquatic environment have enzymatic activity regardless of the pollution level, and 6CFDA was more superior in the sensitivity for the evaluation of physiological activity of bacteria in the natural environment. The ratio of bacteria with esterase activity increased in direct proportion to the pollution level of river water and suggested that this ratio would be a useful indicator in evaluating the pollution levels in an aquatic environment.     

Design, synthesis, and evaluation of 2-phenoxy-indan-1-one derivatives as acetylcholinesterase inhibitors

A series of 2-phenoxy-indan-1-one derivatives have been designed, synthesized, and tested as acetylcholinesterase inhibitors. The most potent compound exhibited high AChE inhibitory activity (IC50 = 50 nM), and the molecular docking study indicated that it was nicely accommodated by AChE.     

A selective molecularly imprinted polymer for immobilization of acetylcholinesterase (AChE): an active enzyme targeted and efficient method

In the present study, we immobilized acetylcholinesterase (AChE) enzyme onto acetylcholine removed imprinted polymer and acetylcholine containing polymer. First, the polymers were produced with acetylcholine, substrate of AChE, by dispersion polymerization. Then, the enzyme was immobilized onto the polymers by using two different methods: In the first method (method A), acetylcholine was removed from the polymer, and then AChE was immobilized onto this polymer (acetylcholine removed imprinted polymer). In the second method (method B), AChE was immobilized onto acetylcholine containing polymer by affinity. In method A, enzyme‐specific species (binding sites) occurred by removing acetylcholine from the polymer. The immobilized AChE reached 240% relative specific activity comparison with free AChE because the active enzyme molecules bounded onto the polymer. Transmission electron microscopy results were taken before and after immobilization of AChE for the assessment of morphological structure of polymer. Also, the experiments, which include optimum temperature (25–65°C), optimum pH (3–10), thermal stability (4–70°C), kinetic parameters, operational stability and reusability, were performed to determine the characteristic of the immobilized AChE. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of two acetylcholinesterases in Pardosa pseudoannulata and the sensitivity to insecticides

Pardosa pseudoannulata is an important predatory enemy against insect pests, such as rice planthoppers and leafhoppers. In order to understand the insecticide selectivity between P. pseudoannulata and insect pests, two acetylcholinesterase genes, Pp-ace1 and Pp-ace2, were cloned from this natural enemy. The putative proteins encoded by Pp-ace1 and Pp-ace2 showed high similarities to insect AChE1 (63% to Liposcelis entomophila AChE1) and AChE2 (36% to Culex quinquefasciatus AChE2) with specific functional motifs, which indicated that two genes might encode AChE1 and AChE2 proteins respectively. The recombinant proteins by expressing Pp-ace1 and Pp-ace2 genes in insect sf9 cells showed high AChE activities. The kinetic parameters, V_max and K_m, of two recombinant AChE proteins were significantly different. The sensitivities to six insecticides were determined in two recombinant AChEs. Pp-AChE1 was more sensitive to all tested insecticides than Pp-AChE2, such as fenobucarb (54 times in K_i ratios), isoprocarb (31 times), carbaryl (13 times) and omethoate (6 times). These results indicated that Pp-AChE1 might be the major synaptic enzyme in the spider. By sequence comparison of P. pseudoannulata and insect AChEs, the key amino acid differences at or close to the functional sites were found. The locations of some key amino acid differences were consistent with the point mutation sites in insect AChEs that were associated with insecticide resistance, such as Phe331 in Pp-AChE2 corresponding to Ser331Phe mutation in Myzus persicae and Aphis gossypii AChE2, which might play important roles in insecticide selectivity between P. pseudoannulata and insect pests. Of course, the direct evidences are needed through further studies.     

Homo- and hetero-bivalent edrophonium-like ammonium salts as highly potent, dual binding site AChE inhibitors

A number of mono- and bis-quaternary ammonium salts, containing edrophonium-like and coumarin moieties tethered by an appropriate linker, proved to be highly potent and selective dual binding site acetylcholinesterase inhibitors with good selectivity over butyrylcholinesterase. Homobivalent bis-quaternary inhibitors 11 and 12, differing by only one methylene unit in the linker, were the most potent and selective inhibitors exhibiting a sub-nanomolar affinity (IC(50)=0.49 and 0.17 nM, respectively) and a high butyryl-/acetylcholinesterase affinity ratio (SI=1465 and 4165, respectively). The corresponding hetero-bivalent coumarinic inhibitors 13 and 14 were also endowed with excellent inhibitory potency but a lower AChE selectivity (IC(50)=2.1 and 1.0 nM, and SI=505 and 708, respectively). Docking simulations enabled clear interpretation of the structure-affinity relationships and detection of key binding interactions at the primary and peripheral AChE binding sites.     

Synthesis and evaluation of novel 4-[(3H,3aH,6aH)-3-phenyl)-4,6-dioxo-2-phenyldihydro-2H-pyrrolo[3,4-d]isoxazol-5(3H,6H,6aH)-yl]benzoic acid derivatives as potent acetylcholinesterase inhibitors and anti-amnestic agents

The present study was designed to synthesize and evaluate pyrrolo-isoxazole benzoic acid derivatives as potential acetylcholinesterase (AChE) inhibitors for the management of Alzheimer's disease. The synthesis of pyrrolo-isoxazole benzoic acid derivatives involved ring opening cyclization of p-aminobenzoic acid with maleic anhydride to yield maleanilic acid, which in turn afforded N-arylmaleimide via ring closed cyclization. Azomethine-N-oxides were obtained by condensation of N-arylhydroxylamine with differently substituted benzaldehydes followed by refluxing of N-arylmaleimide with differently substituted azomethine-N-oxides to pyrrolo-isoxazole benzoic acid derivatives as cis- and trans-stereoisomers. The synthesized compounds were evaluated in vitro for AChE inhibitory activity in rat brain homogenate with donepezil as standard AChE inhibitor. Thereafter, the most potent test compound was evaluated for in vitro butyrylcholinesterase inhibitory activity and in vivo memory evaluation in scopolamine (0.4mg/kg)-induced amnesia in mice by employing Morris water maze test. All pyrrolo-isoxazole benzoic acid derivatives demonstrated potent AChE inhibitory activity. Most of compounds exhibited similar activity to donepezil and four of them (7h, 7i, 8i, and 8h, IC(50)=19.1±1.9-17.5±1.5nM) displayed higher inhibitory activity as compared to donepezil (21.5±3.2nM) with compound 8ia (IC(50)=17.5±1.5nM) being the most active one. The test compound 8ia also ameliorated scopolamine-induced amnesia in mice in terms of restoration of time spent in target quadrant (TSTQ) and escape latency time (ELT). It may be concluded that pyrrolo-isoxazole benzoic acid derivatives may be employed as potential AChE inhibitors.     

Kinetic analysis of the inhibition of human butyrylcholinesterase with cymserine

Accompanying the gradual rise in the average age of the population of most industrialized countries is a regrettable progressive rise in the number of individuals afflicted with age-related neurodegenerative disorders, epitomized by Alzheimer's disease (AD) but, additionally, including Parkinson's disease (PD) and stroke. The primary therapeutic strategy, to date, involves the use of cholinesterases inhibitors (ChEIs) to amplify residual cholinergic activity. The enzyme, acetylcholinesterase (AChE), along with other elements of the cholinergic system is depleted in the AD brain. In contrast, however, its sister enzyme, butyrylcholinesterase (BuChE), that likewise cleaves acetylcholine (ACh), is elevated and both AChE and BuChE co-localize in high amounts with the classical pathological hallmarks of AD. The mismatch between increased brain BuChE and depleted levels of both ACh and AChE, particularly late in the disease, has supported the design and development of new ChEIs with a preference for BuChE; exemplified by the novel agent, cymserine, whose binding kinetics are characterized for the first time. Specifically, as assessed by the Ellman method, cymserine demonstrated potent concentration-dependent binding with human BuChE. The IC50 was determined as 63 to 100 nM at the substrate concentration range of 25 to 800 microM BuSCh. In addition, the following new binding constants were investigated for human BuChE inhibition by cymserine: T(FPnubeta), K(nubeta), K(Bs), K(MIBA), M(IC50), D(Sc), R(f), (O)K(m), OIC100, K(sl), theta(max) and R(i). These new kinetic constants may open new avenues for the kinetic study of the inhibition of a broad array of other enzymes by a wide variety of inhibitors. In synopsis, cymserine proved to be a potent inhibitor of human BuChE in comparison to its structural analogue, phenserine.     

Dual substrate model for novel approach towards a kinetic study of acetylcholinesterase inhibition by diazinon

Limited reports as compared to other insecticides appear in the literature for acetylcholinesterase (AChE) inhibition by diazinon. In the current study, new kinetic parameters of AChE inhibition by diazinon have been investigated. The assay was done with bovine retinal AChE using two different substrate (ASCh) concentrations in the absence and presence of diazinon (0.08-1.28 mM). The optical density was monitored up to 25 min (reaction time) for the assay. New kinetic parameters k'(oms), K'(sms), k(oms), K(sms), K'(asms) and K(asms) ) were calculated from these experimental data.     

Monoclonal Antibodies to Rabbit Brain Acetylcholinesterase: Selective Enzyme Inhibition, Differential Affinity for Enzyme Forms, and Cross-Reactivity with Other Mammalian Cholinesterases

Eleven unique monoclonal IgG antibodies were raised against rabbit brain acetylcholinesterase (AChE, EC 3.1.1.7), purified to electrophoretic homogeneity by a two-step procedure involving immunoaffinity chromatography. The apparent dissociation constants of these antibodies for rabbit AChE ranged from about 10 nM to more than 100 nM (assuming one binding site per catalytic subunit). Species cross-reactivity was investigated with crude brain extracts from rabbit, rat, mouse cat, guinea pig, and human. One antibody bound rabbit AChE exclusively; most bound AChE from three or four species; two bound enzyme from all species tested. Identical, moderate affinity for rat and mouse brain AChE was displayed by two antibodies; two others were able to distinguish between these similar antigens. Nine of the antibodies had lowered affinity for AChE in the presence of 1 M NaCl, but two were salt resistant. Analysis of mutual interferences in AChE binding suggested that certain of the antibodies were competing for nearby epitopes on the AChE surface. One antibody was a potent AChE inhibitor (IC50 = 10(-8) M), blocking up to 90% of the enzyme activity. Most of the antibodies were less able to bind the readily soluble AChE of detergent-free brain extracts than the AChE which required detergent for solubilization. The extreme case, an antibody that was unable to recognize nearly half of the "soluble" AChE, was suspected of lacking affinity for the hydrophilic enzyme form.     

Ester derivatives of annulated tetrahydroazocines: a new class of selective acetylcholinesterase inhibitors

A series of ester derivatives of annulated tetrahydroazocines, namely 2,3,6,11-tetrahydro-1H-azocino[4,5-b]indoles (5-10), 2,3,6,7-tetrahydro-1H-azocino[5,4-b]indoles (11-14), and 4,7,8,9-tetrahydro-1H-pyrrolo[2,3-d]azocines (15-18), synthesized through an efficient 6-->8 membered ring expansion procedure, were investigated for their acetylcholinesterase (AChE) inhibitory activities. Most of the compounds acted as AChE inhibitors in vitro, with IC(50) values ranging from 5 to 40 microM. The most potent compounds 11 and 15, both as racemic mixtures, proved selective toward AChE, exhibiting selectivity ratios versus butyrylcholinesterase (BuChE) of ca. 15 and more than 20, respectively. Structure-activity studies highlighted, among other factors, lipophilicity as a property modulating the AChE inhibition potency, as shown by a reasonable parabolic correlation between pIC(50) and experimental 1-octanol/water partition coefficient (logP), which described the prevailing behavior of the examined compounds (r(2)=0.665). Molecular docking simulations using the X-ray crystal structure of AChE from Torpedo californica suggested possible binding modes of the tetrahydroazocine ester derivatives 11 and 15.