Synthetic peptides mimicking the binding site of human acetylcholinesterase for its inhibitor fasciculin 2

Molecules capable of mimicking protein binding and/or functional sites present useful tools for a range of biomedical applications, including the inhibition of protein–ligand interactions. Such mimics of protein binding sites can currently be generated through structure‐based design and chemical synthesis. Computational protein design could be further used to optimize protein binding site mimetics through rationally designed mutations that improve intermolecular interactions or peptide stability. Here, as a model for the study, we chose an interaction between human acetylcholinesterase (hAChE) and its inhibitor fasciculin‐2 (Fas) because the structure and function of this complex is well understood. Structure‐based design of mimics of the hAChE binding site for Fas yielded a peptide that binds to Fas at micromolar concentrations. Replacement of hAChE residues known to be essential for its interaction with Fas with alanine, in this peptide, resulted in almost complete loss of binding to Fas. Computational optimization of the hAChE mimetic peptide yielded a variant with slightly improved affinity to Fas, indicating that more rounds of computational optimization will be required to obtain peptide variants with greatly improved affinity for Fas. CD spectra in the absence and presence of Fas point to conformational changes in the peptide upon binding to Fas. Furthermore, binding of the optimized hAChE mimetic peptide to Fas could be inhibited by hAChE, providing evidence for a hAChE‐specific peptide–Fas interaction. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Increased acetylcholinesterase inhibitor sensitivity as an intergenerational response to short-term acetylcholinesterase inhibitor exposure in Scapholeberis kingi

Limnology - To determine the potential long-term risks associated with continual use of pesticides, we investigated (1) whether short-term exposure (48 h) of first generation (1G)...     

Synthetic bioantioxidants--inhibitors of acetylcholinesterase activity]

The inhibitory effects of synthetic antioxidants (3-oxypyridine, pyrimidine and hindered phenols) on the enzymic activity of membrane-bound acetylcholinesterase (AChE) was studied. In terms of estimated kinetic characteristics of AChE-reaction (KM, Vmax, KI), the pattern of enzyme inhibition by the hindered phenol compounds was found to be of non-competitive or mixed type depending on the inhibitor structure or on the substrate acetylcholine or acetylthiocholine used. The comparative study of the inhibitory action of water-soluble derivatives of hindered phenols and fatty-soluble ionol made it possible to reveal possible contributions to the inhibition of both direct and mediated (by the membrane microsurroundings) effects on the membrane-bound AChE by the studied synthetic bioantioxidants.     

Energetics of ligand and inhibitor interactions with acetylcholinesterase

Acetylcholinesterase (AChE, EC 3.1.1.7) from Electrophorus electricus, purified by affinity chromatography to a specific activity of 7000-10,000 U/mg protein, was studied at 27 degrees C in conduction-type microcalorimeters for the heats of reaction, with the subsite-specific cationic ligands edrophonium and propidium and with the irreversible inhibitor diisopropylfluorophosphate (DFP), in an ion-free aqueous medium. Edrophonium and propidium, each at 0.5 x 10(-5) M, yielded reaction heats of +3.2 and -1.5 kcal/mol (1 kcal = 4.184 J) respectively, with 1.3 x 10(-5) M AChE active sites. DFP (1.3 x 10(-5) M) reacted exothermically yielding -0.5 kcal/mol at stoichiometric level with AchE active sites. Circular dichroic spectra showed that a ternary complex of AChE (6.5 x 10(-7) M active sites) and the two ligands (each at 1 x 10(-3) M) in 1 mM Tris-HCl buffer (pH 8.0) had a positive Cotton effect at 235 nm. Neither DFP nor phosphoric acid 2,2-dichloroethenyl dimethyl ester (DDVP) caused any appreciable change. DFP-AChE, however, behaved like a normal enzyme in showing a positive Cotton effect in association with the two ligands. DDVP-AChE showed an increase in negative ellipticity at 287 nm in the presence of the two ligands. Another cationic ligand, d-tubocurarine, when present together with edrophonium, increased negative ellipticity at 302 nm and blue-shifted a 265-nm peak of the normal AChE. DFP interactions with AChE appear to be energetically different from those of edrophonium, the latter of which is believed to associate with the acetylcholine-binding subsite.     

合成微生物群落用于木质纤维素生物质转化的研究进展

木质纤维素在自然界中的储量可观,是生物燃料生产的重要来源。联合生物加工(consolidated bioprocessing)指在不添加酶的情况下,将木质纤维素“一步”转化为生物燃料的过程,在能源危机日益严重的今天具有重要的应用价值。合成微生物群落(synthetic microbial consortia)是由两种或多种纯培养微生物(野生菌株或工程菌株)共同培养而形成的菌群,具有复杂性低、稳定性高等优点,通过协调微生物之间的相互作用以及整个生态系统的稳定,从而实现特定的功能。近年来,合成生物学的快速发展有利于开发新的方法和工具用于合成微生物群落的构建及优化,促进其在联合生物加工方面的应用。本文聚焦于木质纤维素的联合生物加工,综述了合成微生物群落在该领域的研究进展。简单介绍了系统生物学为合成微生物群落的设计提供指导,详细介绍了合成微生物群落的设计原则、优化工具和在实际生产中的应用与挑战,为木质纤维素的联合生物加工提供借鉴意义。     

ACAT inhibitor F-1394 prevents intimal hyperplasia induced by balloon injury in rabbits

Acyl-CoA:cholesterol acyltransferase (ACAT) is thought to contribute significantly to lipid deposition in macrophages, which subsequently leads to the initiation and progression of atherosclerosis. The aim of the present study was to examine the influence of hypercholesterolemia on arterial hyperplasia induced by endothelial denudation and the direct effect of ACAT inhibition on lesion formation. Rabbits were fed either a cholesterol diet or a regular diet for 4 weeks, and then the left common carotid arteries were denuded of endothelium. After the operation, all rabbits were kept on the regular diet for 2;-6 weeks. Two weeks after the denudation, the degree of intimal thickening and the number of proliferating cells (which were immunohistologically identified to be smooth muscle cells) were similar in hypercholesterolemic and normolipidemic rabbits. After that, both parameters progressively increased in hypercholesterolemic rabbits but declined in normolipidemic rabbits. Macrophages were apparent in the lesions only in hypercholesterolemic rabbits. Next, the effect of the ACAT inhibitor, (1S,2S)-2-[3-(2,2-dimethylpropyl)-3-nonylureido] cyclohexane-1-yl 3-[(4R)-N-(2,2,5,5-tetramethyl-1,3-dioxane-4-carbonyl)amino]propionate (F-1394), on neointimal formation in hypercholesterolemic rabbits was examined. Oral administration of F-1394 significantly reduced neointimal thickening and the extent of macrophages in lesions without affecting serum cholesterol levels. These results suggest that hypercholesterolemia causes macrophage-derived foam cell accumulation in lesions, and that the progression of lesions is accelerated by the presence of macrophages. Moreover, the study shows that F-1394 prevents neointimal formation even in the presence of hypercholesterolemia, indicating that F-1394 may be useful for treating restenosis after percutaneous translumenal coronary angioplasty in hyperlipidemic patients.     

Reagent-less detection of a competitive inhibitor of immobilized acetylcholinesterase

The interaction of monosulfonate tetraphenyl porphine (TPPS(1)) with immobilized acetylcholinesterase (AChE) yields a characteristic absorbance peak at 446 nm. Addition of acetylcholine iodide or the competitive inhibitor tetracaine to the immobilized TPPS(1)-AChE complex results in a decrease in absorbance intensity at 446 nm due to displacement of the porphyrin from the active site. The loss in intensity at 446 nm is linearly dependent on tetracaine concentration at levels below 100 ppb. Tetracaine concentrations as low as 300 ppt have been detected.     

Overexpression of SR-BI in hamsters treated with a novel ACAT inhibitor (F12511)

The effect of a novel acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor on cholesterol metabolism was studied in hamsters. Oral administration of F12511 (10 mg/kg/d) for 4 weeks produced a decrease in dietary cholesterol absorption (-18%) and in the liver concentration of esterified cholesterol (-75%), as compared with control values in untreated hamsters. While the hepatic expression of LDLr was unchanged by the treatment, that of SR-BI was increased (+142%), which suggests that the hepatic expression of SR-BI could be upregulated by a depletion of the cholesterol stores, due to ACAT inhibition. This SR-BI overexpression, however, did not induce a fall in plasma HDL-cholesterol concentration, in contrast with previous reports in transgenic mice overexpressing SR-BI at a higher extent.     

Differential response of dog and pig tracheal smooth muscle to the acetylcholinesterase inhibitor soman

The effect of the acetylcholinesterase inhibitor soman on tracheal smooth muscle (TSM) from the dog and pig was studied. In response to soman, tracheal ring preparations contract more and the resting tension for TSM preparations is higher for the dog compared with the pig. Tension induced by electrical field stimulation (EFS) and the half-time of EFS-train induced contractions have a similar dependence on soman exposure in both dog and pig TSM. These results suggest that the basal acetylcholine secretion or leakage within the TSM nerve terminal is probably higher for the dog compared with the pig.     

Paecilomide,a new acetylcholinesterase inhibitor from Paecilomyces lilacinus

Fungi are some of the most important organisms in the production of bioactive secondary metabolites. This success is related to the advances in biotechnology and also to the possibility of working with techniques such as the “OSMAC” (one strain-many compounds) to achieve different fungal secondary metabolites profiles upon modifying the culturing conditions. Using this approach, the fungal species Paecilomyces lilacinus was cultivated in potato dextrose broth under 14 different fermentative conditions by adding the bacterium Salmonella typhimurium to the growing medium in order to provide biotic stress. S. typhimurium was added alive or after inactivation by autoclave or microwave irradiation in different stages of fungal growth. Extracts were prepared by liquid–liquid extraction using ethyl acetate, a medium polarity solvent in order to avoid extracting culturing media components. Production of fatty acids of relevance for the pharmaceutical and food industries was enhanced by the modified fermentative conditions and they were identified and quantified. The extracts were evaluated for acetylcholinesterase inhibition and the more active extract (91 ± 2.91% inhibition) was prepared in large scale. From this active P. lilacinus extract, a novel pyridone alkaloid, named Paecilomide, was isolated and its structure was elucidated by modern nuclear magnetic resonance techniques and mass spectrometric analyses. Paecilomide (1) was also evaluated for acetylcholinesterase inhibition, presenting 57.5 ± 5.50% of acetylcholinesterase inhibition.     

The ACAT inhibitor CP-113,818 markedly reduces amyloid pathology in a mouse model of Alzheimer's disease

Amyloid beta-peptide (Abeta) accumulation in specific brain regions is a pathological hallmark of Alzheimer's disease (AD). We have previously reported that a well-characterized acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor, CP-113,818, inhibits Abeta production in cell-based experiments. Here, we assessed the efficacy of CP-113,818 in reducing AD-like pathology in the brains of transgenic mice expressing human APP(751) containing the London (V717I) and Swedish (K670M/N671L) mutations. Two months of treatment with CP-113,818 reduced the accumulation of amyloid plaques by 88%-99% and membrane/insoluble Abeta levels by 83%-96%, while also decreasing brain cholesteryl-esters by 86%. Additionally, soluble Abeta(42) was reduced by 34% in brain homogenates. Spatial learning was slightly improved and correlated with decreased Abeta levels. In nontransgenic littermates, CP-113,818 also reduced ectodomain shedding of endogenous APP in the brain. Our results suggest that ACAT inhibition may be effective in the prevention and treatment of AD by inhibiting generation of the Abeta peptide.     

Towards a species-selective acetylcholinesterase inhibitor to control the mosquito vector of malaria, Anopheles gambiae

Anopheles gambiae is the major mosquito vector of malaria in sub-Saharan Africa. At present, insecticide-treated nets (ITNs) impregnated with pyrethroid insecticides are widely used in malaria-endemic regions to reduce infection; however the emergence of pyrethroid-resistant mosquitoes has significantly reduced the effectiveness of the pyrethroid ITNs. An acetylcholinesterase (AChE) inhibitor that is potent for An. gambiae but weakly potent for the human enzyme could potentially be safely deployed on a new class of ITNs. In this paper we provide a preliminary pharmacological characterization of An. gambiae AChE, discuss structural features of An. gambiae and human AChE that could lead to selective inhibition, and describe compounds with 130-fold selectivity for inhibition of An. gambiae AChE relative to human AChE.     

Identification of ACAT1- and ACAT2-specific inhibitors using a novel, cell-based fluorescence assay: individual ACAT uniqueness

Acyl CoA:cholesterol acyltransferase 1 (ACAT1) and ACAT2 are enzymes responsible for the formation of cholesteryl esters in tissues. While both ACAT1 and ACAT2 are present in the liver and intestine, the cells containing either enzyme within these tissues are distinct, suggesting that ACAT1 and ACAT2 have separate functions. In this study, NBD-cholesterol was used to screen for specific inhibitors of ACAT1 and ACAT2. Incubation of AC29 cells, which do not contain ACAT activity, with NBD-cholesterol showed weak fluorescence when the compound was localized in the membrane. When AC29 cells stably transfected with either ACAT1 or ACAT2 were incubated with NBD-cholesterol, the fluorescent signal localized to the nonpolar core of cytoplasmic lipid droplets was strongly fluorescent and was correlated with two independent measures of ACAT activity. Several compounds were found to have greater inhibitory activity toward ACAT1 than ACAT2, and one compound was identified that specifically inhibits ACAT2. The demonstration of selective inhibition of ACAT1 and ACAT2 provides evidence for uniqueness in structure and function of these two enzymes. To the extent that ACAT2 is confined to hepatocytes and enterocytes, the only two cell types that secrete lipoproteins, selective inhibition of ACAT2 may prove to be most beneficial in the reduction of plasma lipoprotein cholesterol concentrations.     

Mechanisms of conversion of plasminogen activator inhibitor 1 from a suicide inhibitor to a substrate by monoclonal antibodies

We have delineated two different reaction mechanisms of monoclonal antibodies (mAbs), MA-8H9D4 and either MA-55F4C12 or MA-33H1F7, that convert plasminogen activator inhibitor 1 (PAI-1) to a substrate for tissue (tPA)- and urokinase plasminogen activators. MA-8H9D4 almost completely (98-99%) shifts the reaction to the substrate pathway by preventing disordering of the proteinase active site. MA-8H9D4 does not affect the rate-limiting constants (k(lim)) for the insertion of the reactive center loop cleaved by tPA (3.5 s(-1)) but decreases k(lim) for urokinase plasminogen activator from 25 to 4.0 s(-1). MA-8H9D4 does not cause deacylation of preformed PAI-1/proteinase complexes and probably acts prior to the formation of the final inhibitory complex, interfering with displacement of the acylated serine from the proteinase active site. MA-55F4C12 and MA-33H1F7 (50-80% substrate reaction) do not interfere with initial PAI-1/proteinase complex formation but retard the inhibitory pathway by decreasing k(lim) (>10-fold for tPA). Interaction of two mAbs with the same molecule of PAI-1 has been directly demonstrated for pairs MA-8H9D4/MA-55F4C12 and MA-8H9D4/MA-33H1F7 but not for MA-55F4C12/MA-33H1F7. The strong functional additivity observed for MA-8H9D4 and MA-55F4C12 demonstrates that these mAbs interact independently and affect different steps of the PAI-1 reaction mechanism.     

The ACAT inhibitor avasimibe increases the fractional clearance rate of postprandial triglyceride-rich lipoproteins in miniature pigs

Previously, we have shown, in vivo, that the acyl coenzyme A: cholesterol acyltransferase (ACAT) inhibitor avasimibe decreases hepatic apolipoprotein (apo) B secretion into plasma. To test the hypothesis that avasimibe modulates postprandial triglyceride-rich lipoprotein (TRL) metabolism in vivo, an oral fat load (2 g fat/kg) containing retinol was given to 9 control miniature pigs and to 9 animals after 28 days treatment with avasimibe (10 mg/kg/day, n=5; 25 mg/kg/day, n=4). The kinetic parameters for plasma retinyl palmitate (RP) metabolism were determined by multi-compartmental modeling using SAAM II. Avasimibe decreased the 2-h TRL (d<1.006 g/mL; S(f)>20) triglyceride concentrations by 34%. The TRL triglyceride 0-12 h area under the curve (AUC) was decreased by 21%. In contrast, avasimibe had no effect on peak TRL RP concentrations, time to peak, or its rate of appearance into plasma, however, the TRL RP 0-12 h AUC was decreased by 17%. Analysis of the RP kinetic parameters revealed that the TRL fractional clearance rate (FCR) was increased 1.4-fold with avasimibe. The TRL RP FCR was negatively correlated with very low density lipoprotein (VLDL) apoB production rate measured in the fasting state (r=-0.504). No significant changes in total intestinal lipid concentrations were observed. Thus, although avasimibe had no effect on intestinal TRL secretion, plasma TRL clearance was significantly increased; an effect that may relate to a decreased competition with hepatic VLDL for removal processes.     

Donepezil, an acetylcholinesterase inhibitor, enhances adult hippocampal neurogenesis

Donepezil hydrochloride is a potent and selective acetylcholinesterase inhibitor and has been treated for Alzheimer's disease, in which the cholinergic dysfunction is observed. Recently, the degeneration of medial septal cholinergic nuclei in adult rat suppressed the neurogenesis in hippocampal dentate gyrus (DG) was reported. Then, we determined whether donepezil which activated the brain cholinergic system could modulate hippocampal neurogenesis in normal rats. After the injection of 5'-bromo-2'-deoxyuridine (BrdU) to label dividing cells, we orally treated with donepezil (0.5 or 2mg/kg) once a day for 4 weeks. In the other group, we performed 4-week subcutaneous infusion of scopolamine (0.75 or 3mg/day), a muscarinic acetylcholine receptor blocker. The doses of donepezil and scopolamine we used in this study were reported to activate and inhibit cholinergic activity in rats, respectively. One day after the completion of drug treatment, the animals were sacrificed, and immunohistochemical analysis was performed. Donepezil increased, but scopolamine decreased, the number of BrdU-positive cells in the DG as compared with the vehicle-treated control. Neither drug had any effects on the percentage of BrdU-positive cells that were also positive for a neuronal marker NeuN, nor the number of proliferating cell nuclear antigen-positive cells in the DG. These results indicate that donepezil enhances and scopolamine suppresses the survival of newborn neurons in the DG without affecting the proliferation of neural progenitor cell and the neuronal differentiation. We also found that chronic treatment of donepezil enhanced, and scopolamine suppressed phosphorylation of cAMP response element binding protein (CREB), which was involved in cell survival, in the DG. These results suggest that donepezil activates the central cholinergic transmission and enhances the survival of newborn neurons in the DG via CREB signaling.     

The cyanobacterial alkaloid nostocarboline: an inhibitor of acetylcholinesterase and trypsin

Preselected cyanobacterial strains (available from culture collections and our own isolates), belonging primarily to the heterocystous cluster, were screened for inhibitors against butyrylcholinesterase. About one-half of the extracts exhibited inhibitory activity. Nostocarboline, the responsible metabolite in Nostoc 78–12A, was studied in more detail as an acetylcholinesterase (AChE) inhibitor. The compound showed potent activity against this enzyme (IC₅₀ = 5.3 μM), and the Michaelis-Menten kinetics indicated a non-competitive component in the inhibitory mechanism. In addition, nostocarboline turned out to be a potent inhibitor of trypsin (IC₅₀ = 2.8 μM), and thus is the first described cyanobacterial serine protease inhibitor of an alkaloid structure. The function of nostocarboline in aquatic ecosystems and its potential as a lead compound for the development of useful therapeutic AChE inhibitors is discussed.     

Discovery of a novel acetylcholinesterase inhibitor by structure-based virtual screening techniques

Acetylcholinesterase (AChE) is considered to be one of the most important targets for the treatment of Alzheimer's disease (AD). Previously our group has reported a series of tacrine-based hybrids as potent AChE inhibitors (AChEI). To discover more novel scaffolds, molecular docking and dynamics stimulation were applied to acquire the binding models of AChE with the most prominent compounds from our work. A structure-based pharmacophore model plus shape constraints was generated from the binding models and it was then employed to virtually screen commercial databases, giving a focused hit list of candidates. Subsequently, we scored the hit compounds by their molecular binding energies, which were calculated by MM/PBSA method. Fifteen compounds were selected and purchased for testing their anti-AChE effects, while seven of them showed inhibitory effects with IC(50) values ranging from 1.5 to 9.8 μM. The drug-like properties of these compounds, including LogD, AlogP, molecular volume and Lipinski rule of five, were also calculated. Compounds 12 and 16 (IC(50)=2.5 and 1.5 μM, respectively) exhibited potent activity and acceptable drug-like properties, thus might serve as leads for further modification. The data suggest that the presented model might be a valid approach for identification and development of new AChEIs.     

Synthesis of more potent analogues of the acetylcholinesterase inhibitor,huperzine B

The synthesis and acetylcholinesterase inhibition activity of analogues of huperzine B are reported. These new racemic analogues show a better AChE inhibitory activity than the natural product huperzine B.