Rapid TLC/GC-MS identification of acetylcholinesterase inhibitors in alkaloid extracts

Alkaloid extracts from 12 plant species of the families Amaryllidaceae, Fumariacae and Papaveraceae were studied with respect to their acetylcholinesterase inhibitory activity and alkaloid patterns. Fifty-three alkaloids were identified by GC-MS, including known acetylcholinesterase (AChE) inhibitors such as galanthamine, epigalanthamine, sanguinine and epinorgalanthamine in extracts of Amaryllidaceae plants and protopine in extracts of Fumariaceae and Papaveraceae plants. The galanthamine-containing extracts of the amaryllidaceous plants were found to be the most active while the extract of Corydalis bulbosa was the most active among the extracts of the tested plants from the Fumariaceae and Papaveraceae plants. TLC bioautographic assay, preparative TLC and GC-MS analysis were combined to identify the active compounds in the studied extracts. Galanthamine was isolated from the known AChE inhibitors in the extracts of Amaryllidaceae plants. Corydaline, bulbocapnine and stylopine were found to be active in the extracts of plant species of the families Fumariaceae and Papaveraceae. Available standards of deshydrocorydaline--a precursor of corydaline, corydaline and stylopine--were tested for AChE inhibitory activity. Deshydrocorydaline and corydaline showed potent inhibitory activity comparable with that of the positive control galanthamine.     

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.     

Acetylcholinesterase in Mouse Neuroblastoma NB2A Cells: Analysis of Production, Secretion, and Molecular Forms

The mouse neuroblastoma cell line NB2A produces cellular and secreted acetylcholinesterase (AChE). After incubation of the cells for 4 days the ratio between AChE secreted into the medium and AChE in the cells was 1:1. The cell-associated enzyme could be subdivided into soluble AChE (25%) and detergent-soluble AChE (75%). Both extracts contained predominantly monomeric AChE (4.6S) and minor amounts of tetrameric AChE (10.6S), whereas the secreted AChE in the culture supernatant contained only the tetrameric form. All forms were partially purified by affinity chromatography. It could be demonstrated that the secretory and the intracellular soluble tetramers were hydrophilic, whereas the detergent-soluble tetramer was an amphiphilic protein. On the other hand the soluble and the detergent-soluble monomeric forms were amphiphilic and their activity depended on the presence of detergent. By digestion with proteinase K amphiphilic monomeric and tetrameric AChE could be converted to a hydrophilic form that no longer required detergent for catalytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]diisopropylfluorophosphate-labelled AChE gave one band at 64 kilodaltons (kD) under reducing conditions and two additional bands at 120 kD and 140 kD under nonreducing conditions.     

Acetylcholinesterase-inhibiting steroidal alkaloid from the sponge Corticium sp

A new stigmastane-type steroidal alkaloid, 4-acetoxy-plakinamine B (1), was isolated from the Thai sponge Corticium sp. The compound was subjected to the acetylcholinesterase inhibitory activity determination to reveal a high inhibitory activity (IC(50) 3.75+/-1.69 microM). The kinetics of enzyme inhibition showed a decrease in V(max), whereas K(m) was increased, thus suggesting an unusual mixed-competitive mode of inhibition. Compound 1 is the first steroidal alkaloid bearing a stigmastane skeleton ever been reported to exhibit such good potency in the acetylcholinesterase inhibition bioassay.     

The Brazilian Amaryllidaceae as a source of acetylcholinesterase inhibitory alkaloids

Nine Brazilian Amaryllidaceae species were studied for their alkaloid composition and acetylcholinesterase (AChE) inhibitory activity via GC–MS and a modified Ellman assay, respectively. A total of thirty-six alkaloids were identified in these plants, of which Hippeastrum papilio and H. glaucescens exhibited the highest galanthamine content and the best IC₅₀ values against AChE. Furthermore, Hippeastrum vittatum and Rhodophiala bifida also showed notable AChE inhibitory effects. X-ray crystallographic data for four galanthamine-type compounds revealed significant differences in the orientation of the N-methyl group, which are shown to be related to AChE inhibition.     

Acetylcholinesterase inhibitors from plants

Inhibition of acetylcholinesterase (AChE), the key enzyme in the breakdown of acetylcholine, is considered as a promising strategy for the treatment of neurological disorders such as Alzheimer's disease, senile dementia, ataxia and myasthenia gravis. A potential source of AChE inhibitors is certainly provided by the abundance of plants in nature. This article aims to provide a comprehensive literature survey of plants that have been tested for AChE inhibitory activity. Numerous phytoconstituents and promising plant species as AChE inhibitors are being reported in this communication.     

Amphiphilic and hydrophilic molecular forms of acetylcholinesterase in membranes derived from sarcoplasmic reticulum of skeletal muscle

Native molecular forms of acetylcholinesterase (AChE) present in a microsomal fraction enriched in SR of rabbit skeletal muscle were characterized by sedimentation analysis in sucrose gradients and by digestion with phospholipases and proteinases. The hydrophobic properties of AChE forms were studied by phase-partition of Triton X-114 and Triton X-100-solubilized enzyme and by comparing their migration in sucrose gradient containing either Triton X-100 or Brij 96. We found that in the microsomal preparation two hydrophilic 13.5 S and 10.5 S forms and an amphiphilic 4.5 S form exist. The 13.5 S is an asymmetric molecule which by incubation with collagenase and trypsin is converted into a 'lytic' 10.5 S form. The hydrophobic 4.5 S form is the predominant one in extracts prepared with Triton X-100. Proteolytic digestion of the membranes with trypsin brought into solution a significant portion of the total activity. Incubation of the membranes with phospholipase C failed to solubilize the enzyme. The sedimentation coefficient of the amphiphilic 4.5 S form remained unchanged after partial reduction, thus confirming its monomeric structure. Conversion of the monomeric amphiphilic form into a monomeric hydrophilic molecule was performed by incubating the 4.5 S AChE with trypsin. This conversion was not produced by phospholipase treatment.     

Alanine reverses the inhibitory effect of phenylalanine on acetylcholinesterase activity

The aim of this work was to evaluate, in vitro, the effect of L-alanine (Ala) on suckling rat brain acetylcholinesterase (AChE) and on eel Electrophorus electricus pure AChE inhibited by L-phenylalanine (Phe) as well as to investigate whether Phe or Ala is a competitive inhibitor or an effector of the enzyme. AChE activity was determined in brain homogenates and in the pure enzyme after 1 h preincubation with 1.2 mM of Phe or Ala as well as with Phe plus Ala. The activity of the pure AChE was also determined using as a substrate different amounts of acetylthiocholine. Ala reversed completely the inhibited AChE by Phe (18-20% in 500-600 microM substrate, p<0.01). Lineweaver-Burk plots showed that Vmax remained unchanged. However, Km was found increased with Phe (150%, p<0.001), decreased with Ala alone (50%, p<0.001) and unaltered with Phe plus Ala. It is suggested that: a) Phe presents a competitive inhibitory action with the substrate whereas Ala a competitive activation; b) Ala competition with Phe might unbind the latter from AChE molecule inducing the enzyme stimulation; c) Ala might reverse the inhibitory effect of Phe on brain AChE in phenylketonuric patients, if these results are extended into the in vivo reality.     

Assembly of monomeric acetylcholinesterase into tetrameric and asymmetric forms

A pulse-chase experiment was performed in embryonic rat myotube cultures to examine possible precursor-product relationships among the various molecular forms of acetylcholinesterase (AChE). AChE was labeled with paraoxon, a compound which diethylphosphorylates AChE at its active site. Diethylphosphorylated (labeled) AChE is inactive but can be reactivated by treatment with 1-methyl-2-hydroxyiminomethyl-pyridinium. Thus labeled enzyme could be followed as AChE that regained activity following treatment with 1-methyl-2-hydroxyiminomethylpyridium. To selectively label monomeric AChE (the hypothesized precursor form), cultures were treated with methanesulfonylfluoride which irreversibly inactivated more than 97% of total cellular AChE. Methylsulfonylfluoride was then washed from the cultures, and they were labeled with paraoxon during a 40-55-min recovery period. AChE appearing in the cultures during this recovery period is newly synthesized and consists almost entirely (92%) of the monomeric form. Immediately and 120-130 min after labeling, cultures were subjected to a sequential extraction procedure to separate globular from asymmetric forms. Individual forms were then separated by velocity sedimentation on sucrose gradients. In our first series of experiments, we observed a 55% decrease in labeled monomers during the chase, a 36% increase in labeled tetramers, and a 36% increase in labeled asymmetric forms. In a second series of experiments focused on individual asymmetric forms, we observed a 55% decrease in labeled monomers, a 58% increase in labeled tetramers, an overall increase of 81% in labeled asymmetric forms, and a 380% increase in labeled A12 AChE. These data provide the first uniequivocal proof that complex forms of AChE are assembled from active monomeric precursors.     

不同蕨类植物提取物乙酰胆碱酯酶抑制活性的评价

为对采自浙江天目山的40种蕨类植物乙醇提取物进行乙酰胆碱酯酶抑制活性研究。本文采用乙酰胆碱酯酶抑制活性筛选模型。结果表明：在供试质量浓度在1mg／mL时,40种植物的乙醇提取物均具有明显的乙酰胆碱酯酶抑制活性,其中密羽贯众、野雉尾金粉蕨和狗脊等13种蕨类植物表现出较强的抑制活性,抑制率均大于90％,其余大部分植物的抑制率均大于60％。而在供试质量浓度在0．025mg／mL时,只有少数植物表现出较高的乙酰胆碱酯酶抑制活性,其中狭顶鳞毛蕨的抑制率为62．63％&#177;3．72％,蜈松草的抑制率为48．01％&#177;2．87％,其余大部分植物的抑制活性均小于40％,甚至有些植物提取物不仅对乙酰胆碱酯酶没有抑制活性,反而表现出一定的增强作用。     

Interaction of 3-Alkylpyridinium Polymers from the Sea Sponge Reniera sarai with Insect Acetylcholinesterase

3-Alkylpyridinium polymers (poly-APS), composed of 29 or 99 N-butyl-3-butyl pyridinium units, were isolated from the marine sponge Reniera sarai. They act as potent cholinesterase inhibitors. The inhibition kinetics pattern reveals several successive phases ending in irreversible inhibition of the enzyme. To provide more information on mechanism of inhibition, interaction of poly-APS and N-butyl-3-butyl pyridinium iodide (NBPI) with soluble dimeric and monomeric insect acetylcholinesterase (AChE) was studied by using enzyme intrinsic fluorescence and light scattering, conformational probes ANS and trypsin, and SDS–PAGE. Poly-APS quenched tryptophan fluorescence emission of AChE more extensively than NBPI. Both inhibitors exhibited a pseudo-Lehrer type of quenching. Interaction of poly-APS with dimeric AChE did not induce significant changes of the enzyme conformation as assayed by using the hydrophobic probe ANS and trypsin digestion. In contrast to NBPI, titration of both monomeric and dimeric AChE with poly-APS resulted in the appearance of large complexes detected by measuring light scattering. An excess of poly-APS produced AChE precipitation as proved on SDS–PAGE. None of the effects were observed with trypsin as a control. It was concluded that AChE aggregation and precipitation rather than the enzyme conformational changes accounted for the observed irreversible component of poly-APS inhibition.     

Interaction of 3-Alkylpyridinium Polymers from the Sea Sponge Reniera sarai with Insect Acetylcholinesterase

3-Alkylpyridinium polymers (poly-APS), composed of 29 or 99 N-butyl-3-butyl pyridinium units, were isolated from the marine sponge Reniera sarai. They act as potent cholinesterase inhibitors. The inhibition kinetics pattern reveals several successive phases ending in irreversible inhibition of the enzyme. To provide more information on mechanism of inhibition, interaction of poly-APS and N-butyl-3-butyl pyridinium iodide (NBPI) with soluble dimeric and monomeric insect acetylcholinesterase (AChE) was studied by using enzyme intrinsic fluorescence and light scattering, conformational probes ANS and trypsin, and SDS–PAGE. Poly-APS quenched tryptophan fluorescence emission of AChE more extensively than NBPI. Both inhibitors exhibited a pseudo-Lehrer type of quenching. Interaction of poly-APS with dimeric AChE did not induce significant changes of the enzyme conformation as assayed by using the hydrophobic probe ANS and trypsin digestion. In contrast to NBPI, titration of both monomeric and dimeric AChE with poly-APS resulted in the appearance of large complexes detected by measuring light scattering. An excess of poly-APS produced AChE precipitation as proved on SDS–PAGE. None of the effects were observed with trypsin as a control. It was concluded that AChE aggregation and precipitation rather than the enzyme conformational changes accounted for the observed irreversible component of poly-APS inhibition.     

Thermal stability of acetylcholinesterase from Bungarus fasciatus venom as investigated by capillary electrophoresis

Previous studies on the conformation of the monomeric acetylcholinesterase (AChE) from the krait (Bungarus fasciatus) venom showed that the protein possesses a large permanent dipole moment. These studies predicted that thermal irreversible denaturation must occur via partially unfolded states. The thermal stability of Bungarus AChE was determined using capillary electrophoresis (CE) with optimized conditions. Runs performed at convenient temperature scanning rates provided evidence for an irreversible denaturation process according to the Lumry and Eyring model. The mid-transition temperature, T(m), and the effective enthalpy change, DeltaH(m) were determined at different pH. The temperature dependence of the free energy, DeltaG, of Bungarus AChE unfolding was drawn using values of T(m), DeltaH(m) and DeltaC(p) determined by CE. The thermodynamic parameters for the thermal denaturation of the monomeric snake enzyme were compared with those of different dimeric and tetrameric ChEs. It was shown that the changes in the ratio of DeltaH(cal/)DeltaH(vH) and DeltaC(p) reflect the oligomerization state of these proteins. All these results indicate that wild-type monomeric Bungarus AChE is a stable enzyme under standard conditions. However, designed mutants of this enzyme capable of degrading organophosphates have to be engineered to enhance their thermostability.     

Substituted spiro [2.3'] oxindolespiro [3.2″]-5,6-dimethoxy-indane-1″-one-pyrrolidine analogue as inhibitors of acetylcholinesterase

Series of pyrolidine analogues were synthesized and examined as acetylcholinesterase (AChE) inhibitors. Among the compounds, compounds 4k and 6k were the most potent inhibitors of the series. Compound 4k, showed potent inhibitory activity against acetyl cholinesterase enzyme with IC(50) 0.10 μmol/L. Pyrolidine analogues might be potential acetyl cholinesterase agents for AD.     

Axonal Transport Reversal of Acetylcholinesterase Molecular Forms in Transected Nerve

Reversal of anterograde rapid axonal transport of four molecular forms of acetylcholinesterase (AChE) was studied in chick sciatic nerve during the 24-h period following a nerve transection. Reversal of AChE activity started ～1 h after nerve transection, and all the forms of the enzyme, except the monomeric ones, showed reversal of transport. The quantity of enzyme activity reversed 24 h after transection was twofold greater than that normally conveyed by retrograde transport. We observed no leakage of the enzyme at the site of the nerve transection and no reversal of AChE activity transport in the distal segment of the severed nerve, a result indicating that the material carried by retrograde axonal transport cannot be reversed by axotomy. Thus, a nerve transection induces both quantitative and qualitative changes in the retrograde axonal transport, which could serve as a signal of distal injury to the cell body. The velocity of reverse transport, measured within 6 h after transection, was found to be 213 mm/day, a value close to that of retrograde transport (200 mm/day). This suggests that the reversal taking place in severed sciatic nerve is similar to the anterograde-to-retrograde conversion process normally occurring at the nerve endings.     

Nantenine as an acetylcholinesterase inhibitor: SAR,enzyme kinetics and molecular modeling investigations

Nantenine, as well as a number of flexible analogs, were evaluated for acetylcholinesterase (AChE) inhibitory activity in microplate spectrophotometric assays based on Ellman’s method. It was found that the rigid aporphine core of nantenine is an important structural requirement for its anticholinesterase activity. Nantenine showed mixed inhibition kinetics in enzyme assays. Molecular docking experiments suggest that nantenine binds preferentially to the catalytic site of AChE but is also capable of interacting with the peripheral anionic site (PAS) of the enzyme, thus accounting for its mixed inhibition profile. The aporphine core of nantenine may thus be a useful template for the design of novel PAS or dual-site AChE inhibitors. Inhibiting the PAS is desirable for prevention of aggregation of the amyloid peptide Aβ, a major causative factor in the progression of Alzheimer’s disease (AD).     

Dyhidro-β-agarofurans natural and synthetic as acetylcholinesterase and COX inhibitors: interaction with the peripheral anionic site (AChE-PAS), and anti-inflammatory potentials

In order to find molecules of natural origin with potential biological activities, we isolate and synthesise compounds with agarofuran skeletons (epoxyeudesmanes). From the seeds of Maytenus disticha and Maytenus magellanica we obtained six dihydro-β-agarofurans, and by means of the Robinson annulation reaction we synthesised five compounds with the same skeleton. The structures were established on the basis of NMR, IR, and MS. The evaluated compounds showed inhibitory activity on the acetylcholinesterase enzyme and on the COX enzymes. Compound 4 emerged as the most potent in the acetylcholinesterase inhibition assay with IC₅₀ 17.0 ± 0.016 µM on acetylcholinesterase (AChE). The compounds evaluated were shown to be selective for AChE. The molecular docking, and the propidium displacement assay suggested that the compounds do not bind to the active site of the enzyme AChE, but rather bind to the peripheral anionic site (PAS) of the enzyme, on the other hand, the natural compound 8, showed the best inhibitory activity on the COX-2 enzyme with an IC₅₀ value of 0.04 ± 0.007 µM. The pharmacokinetic profile calculated in silico using the SWISSADME platform shows that these molecules could be considered as potential drugs for the treatment of neurodegenerative diseases such as AD.     

Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme

1. Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase. 2. The recombinant gene product exhibits biochemical traits similar to native "true" acetylcholinesterase as manifested by characteristic substrate inhibition, a Km of 117 microM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51. 3. The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization. 4. The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms. 5. In conclusion, the catalytic subunit expressed from the hydrophilic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.     

Study on dual-site inhibitors of acetylcholinesterase: Highly potent derivatives of bis- and bifunctional huperzine B

Natural (-)-huperzine B (HupB), isolated from Chinese medicinal herb, displayed moderate inhibitory activity of acetylcholinesterase (AChE). Based on the active dual-site of AChE, a series of novel derivatives of bis- and bifunctional HupB were designed and synthesized. The AChE inhibition potency of most derivatives of HupB was enhanced about 2-3 orders of magnitude as compared with the parental HupB. Among bis-HupB derivatives, 12h exhibited the most potent in the AChE inhibition and has been evaluated for its pharmacological actions in vivo on ChE inhibition, cognitive enhancement, and neuroprotection. The docking study on the bis-HupB derivatives 12 series with TcAChE has demonstrated that the ligands bound to the dual-site of the enzyme in different level.     

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.