First principles calculations of thermodynamics and kinetic parameters and molecular dynamics simulations of acetylcholinesterase reactivators: can mouse data provide new insights into humans?

We have applied a theoretical methodology, previously developed to evaluate the association and kinetic reactivation constants of oximes, comparing theoretical data obtained for human acetylcholinesterase (HsAChE) with in vitro results from Mus musculus AChE (MmAChE) previously reported in the literature. Our results, further checked by additional molecular dynamics simulations steps, showed a good correlation between the theoretical and experimental data, supporting the methodology as appropriate for prediction of thermodynamic and kinetic parameters and corroborated MmAChE as a suitable model for studies with HsAChE.     

Potencies and selectivities of inhibitors of acetylcholinesterase and its molecular forms in normal and Alzheimer's disease brain

Eight inhibitors of acetylcholinesterase (AChE), tacrine, bis-tacrine, donepezil, rivastigmine, galantamine, heptyl-physostigmine, TAK-147 and metrifonate, were compared with regard to their effects on AChE and butyrylcholinesterase (BuChE) in normal human brain cortex. Additionally, the IC50 values of different molecular forms of AChE (monomeric, G1, and tetrameric, G4) were determined in the cerebral cortex in both normal and Alzheimer's human brains. The most selective AChE inhibitors, in decreasing sequence, were in order: TAK-147, donepezil and galantamine. For BuChE, the most specific was rivastigmine. However, none of these inhibitors was absolutely specific for AChE or BuChE. Among these inhibitors, tacrine, bis-tacrine, TAK-147, metrifonate and galantamine inhibited both the G1 and G4 AChE forms equally well. Interestingly, the AChE molecular forms in Alzheimer samples were more sensitive to some of the inhibitors as compared with the normal samples. Only one inhibitor, rivastigmine, displayed preferential inhibition for the G1 form of AChE. We conclude that a molecular form-specific inhibitor may have therapeutic applications in inhibiting the G1 form, which is relatively unchanged in Alzheimer's brain.     

Existence of Two Acetylcholinesterases in the Mosquito Culex pipiens (Diptera: Culicidae)

Abstract: Two acetylcholinesterases (AChEs), AChE1 and AChE2, differing in substrate specificity and in some aspects of inhibitor sensitivity, have been characterized in the mosquito Culex pipiens . The results of ultracentrifugation in sucrose gradients and nondenaturing gel electrophoresis of AChE activity peak fractions show that each AChE is present as two molecular forms: one amphiphilic dimer possessing a glycolipid anchor and one hydrophilic dimer that does not interact with nondenaturing detergents. Treatment by phosphatidylinositol-specific phospholipase C converts each type of amphiphilic dimer into the corresponding hydrophilic dimer. Molecular forms of AChE1 have a lower electrophoretic mobility than those of AChE2. However, amphiphilic dimers and hydrophilic dimers have similar sedimentation coefficients (5.5S and 6.5S, respectively). AChE1 and AChE2 dimers, amphiphilic or hydrophilic, resist dithiothreitol reduction under conditions that allow reduction of Drosophila AChE dimers. In the insecticide-susceptible strain S-LAB, AChE1 is inhibited by 5 × 10⁻⁴ M propoxur (a carbamate insecticide), whereas AChE2 is resistant. All animals are killed by this concentration of propoxur, indicating that only AChE1 fulfills the physiological function of neurotransmitter hydrolysis at synapses. In the insecticide-resistant strain, MSE, there is no mortality after exposure to 5 × 10⁻⁴ M propoxur: AChE2 sensitivity to propoxur is unchanged, whereas AChE1 is now resistant to 5 × 10⁻⁴ M propoxur. The possibility that AChE1 and AChE2 are products of tissue-specific posttranslational modifications of a single gene is discussed, but we suggest, based on recent results obtained at the molecular level in mosquitoes, that they are encoded by two different genes.     

Existence of two membrane-bound acetylcholinesterases in the honey bee head

Two acetylcholinesterase (EC 3.1.1.7) membrane forms AChE(m1) and AChE(m2), have been characterised in the honey bee head. They can be differentiated by their ionic properties: AChE(m1) is eluted at 220 mM NaCl whereas AChE(m2) is eluted at 350 mM NaCl in anion exchange chromatography. They also present different thermal stabilities. Previous processing such as sedimentation, phase separation, and extraction procedures do not affect the presence of the two forms. Unlike AChE(m1), AChE(m2) presents reversible chromatographic elution properties, with a shift between 350 to 220 mM NaCl, depending on detergent conditions. Purification by affinity chromatography does not abolish the shift of the AChE(m2) elution. The similar chromatographic behaviour of soluble AChE strongly suggests that the occurrence of the two membrane forms is not due to the membrane anchor. The two forms have similar sensitivities to eserine and BW284C51. They exhibit similar electrophoretic mobilities and present molecular masses of 66 kDa in SDS-PAGE and a sensitivity to phosphatidylinositol-specific phospholipase C in non-denaturing conditions, thus revealing the presence of a glycosyl-phosphatidylinositol anchor. We assume that bee AChE occurs in two distinct conformational states whose AChE(m2) apparent state is reversibly modulated by the Triton X-100 detergent into AChE(m1).     

Molecular modeling and in vitro reactivation study between the oxime BI-6 and acetylcholinesterase inhibited by different nerve agents

Nerve agents are organophosphates acting as potent inhibitors of acetylcholinesterase (AChE), the enzyme responsible for the hydrolysis of acetylcholine and, consequently, the termination of the transmission of nerve impulses. The inhibition of AChE by an organophosphate can be reversed by a nucleophilic agent able to dephosphorylate a serine residue in the active site of AChE. In this sense, the oximes are compounds capable of removing the nerve agent and reactivate the enzyme. Here, we have applied a methodology involving theoretical docking and Quantum Mechanics/Molecular Mechanics, using the softwares Molegro^® and Spartan^®, to evaluate the kinetic constants of reactivation and the interactions of the oxime BI-6 with AChE inhibited by different organophosphorus compounds in comparison to in vitro data. Results confirm that this method is suitable for the prediction of kinetic and thermodynamic parameters of oximes, which may be useful in the design and selection of new and more effective oximes.     

Cloning and sequence analysis of 2 different acetylcholinesterase genes in Rhopalosiphum padi and Sitobion avenae.

Two genes encoding different acetylcholinesterases (AChE) were successfully cloned from 2 species of aphid, Rhopalosiphum padi (L.) and Sitobion avenae (F.). They were named Rp.AChE1 (GenBank accession No. AY707318), Rp.AChE2 (AY667435), Sa.AChE1 (AY707319), and Sa.AChE2 (AY819704), and were 2133, 2363, 2131, and 2362 bp in length and encoded 664, 676, 664, and 676 amino acids, respectively. All of them shared the characteristics of the AChE family: catalytic tiads, 3 intra-chain disulfide bridges, an acyl pocket, and the conservative aromatic residues for the active site of the gorge. Sequence analysis revealed that Rp.AChE1 and Sa.AChE1 showed higher identity to the reported orthologous genes of Drosophila AChE, and Rp.AChE2 and Sa.AChE2 to paralogous genes. However, in each of the aphids, the 2 genes from the same species shared only 29% identity between one another. It was therefore concluded that each of the aphids has 2 different AChE genes, which are either orthologous or paralogous to Drosophila AChE. The high conservation of AChE1 and AChE2 indicated that 2 acetylcholinesterases exist popularly and that both might function in aphids.     

The effects of the acetylcholinesterase inhibitor ENA713 and the M1 agonist AF150(S) on apolipoprotein E deficient mice

Apolipoprotein E (apoE)-deficient and control mice were treated chronically with either the acetylcholinesterase (AChE) inhibitor ENA713, or the M1 muscarinic agonist AF150(S). Both treatments reversed the spatial working memory impairment of apoE-deficient mice but they differed in their effects on the levels of brain AChE activity. AF150(S) enhanced the brain AChE activity of apoE-deficient mice and rendered it similar to that of the untreated controls, whereas ENA713 reduced the brain AChE activity of control mice but had no effect on that of apoE-deficient mice. These findings suggest that AChE inhibition and M1 muscarinic activation have similar beneficial cognitive effects on apoE-deficient mice, but that the cellular and molecular mechanisms underlying these effects differ.     

Presenilin 1 interacts with acetylcholinesterase and alters its enzymatic activity and glycosylation

Presenilin 1 (PS1) plays a critical role in the gamma-secretase processing of the amyloid precursor protein to generate the beta-amyloid peptide, which accumulates in plaques in the pathogenesis of Alzheimer's disease (AD). Mutations in PS1 cause early onset AD, and proteins that interact with PS1 are of major functional importance. We report here the coimmunoprecipitation of PS1 and acetylcholinesterase (AChE), an enzyme associated with amyloid plaques. Binding occurs through PS1 N-terminal fragment independent of the peripheral binding site of AChE. Subcellular colocalization of PS1 and AChE in cultured cells and coexpression patterns of PS1 and AChE in brain sections from controls and subjects with sporadic or familial AD indicated that PS1 and AChE are located in the same intracellular compartments, including the perinuclear compartments. A PS1-A246E pathogenic mutation expressed in transgenic mice leads to decreased AChE activity and alteration of AChE glycosylation and the peripheral binding site, which may reflect a shift in protein conformation and disturbed AChE maturation. In both the transgenic mice and humans, mutant PS1 impairs coimmunoprecipitation with AChE. The results indicate that PS1 can interact with AChE and influence its expression, supporting the notion of cholinergic-amyloid interrelationships.     

Synthesis and screening for acetylcholinesterase inhibitor activity of some novel 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-ones: derivatives of irbesartan key intermediate

The association of bioactive nucleus with other pharmacological agents is hoped to improve the efficacy of the treatment by combining the effects of different pharmacological mechanisms of action. Keeping this in view, a series of 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-one derivatives have been synthesized by interaction of 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-one with different bioactive aralkyl halides in presence of powdered potassium carbonate by two different methods viz., conventional and microwave irradiation. The yields under conventional and microwave irradiation methods were in the range of 60-65% and 80-90%, respectively. The structure elucidation of the new compounds has been carried out with the help of elemental analysis and spectral data. All the synthesized compounds have been screened for their efficacy as acetylcholinesterase (AChE) inhibitor. AChE inhibitory activity study was carried out by using Ellman colorimetric assay with neostigmine as a reference standard against targets from different species, such as pure electric eel AChE, human serum AChE, and rat brain AChE. Among the compounds synthesized, compounds 5a, 5b, 5j showed good inhibition against AChE.     

A molecular dynamics study of components of the ginger (Zingiber officinale) extract inside human acetylcholinesterase: implications for Alzheimer disease

Components of ginger (Zingiber officinale) extracts have been described as potential new drug candidates against Alzheimer disease (AD), able to interact with several molecular targets related to the AD treatment. However, there are very few theoretical studies in the literature on the possible mechanisms of action by which these compounds can work as potential anti-AD drugs. For this reason, we performed here docking, molecular dynamic simulations and mmpbsa calculations on four components of ginger extracts former reported as active inhibitors of human acetylcholinesterase (HssAChE), and compared our results to the known HssAChE inhibitor and commercial drug in use against AD, donepezil (DNP). Our findings points to two among the compounds studied: (E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hept-4-en-3-on and 1-(3,4-dihydroxy-5-methoxyphenyl)-7-(4-hydroxy-3- ethoxyphenyl) heptane-3,5-diyl diacetate, as promising new HssAChE inhibitors that could be as effective as DNP. We also mapped the binding of the studied compounds in the different binding pockets inside HssAChE and established the preferred interactions to be favored in the design of new and more efficient inhibitors.     

Alpha7-nicotinic acetylcholine receptors mediate an Abeta(1-42)-induced increase in the level of acetylcholinesterase in primary cortical neurones

The beta-amyloid protein (Abeta) is the major protein component of amyloid plaques found in the Alzheimer brain. Although there is a loss of acetylcholinesterase (AChE) from both cholinergic and non-cholinergic neurones in the brain of Alzheimer patients, the level of AChE is increased around amyloid plaques. Previous studies using P19 cells in culture and transgenic mice which overexpress human Abeta have suggested that this increase may be due to a direct action of Abeta on AChE expression in cells adjacent to amyloid plaques. The aim of the present study was to examine the mechanism by which Abeta increases levels of AChE in primary cortical neurones. Abeta1-42 was more potent than Abeta1-40 in its ability to increase AChE in primary cortical neurones. The increase in AChE was unrelated to the toxic effects of the Abeta peptides. The effect of Abeta1-42 on AChE was blocked by inhibitors of alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) as well as by inhibitors of L- or N-type voltage-dependent calcium channels (VDCCs), whereas agonists of alpha7 nAChRs (choline, nicotine) increased the level of AChE. The results demonstrate that the effect of Abeta1-42 on AChE is due to an agonist effect of Abeta1-42 on the alpha7 nAChR.     

Tailored Modeling of Rivastigmine Derivatives as Dual Acetylcholinesterase and Butyrylcholinesterase Inhibitors for Alzheimer's Disease Treatment

Rational modification of known drug candidates to design more potent ones using computational methods has found application in drug design, development, and discovery. Herein, we integrate computational and theoretical methodologies to unveil rivastigmine derivatives as dual inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) for Alzheimer's disease (AD) management. The investigation entails pharmacokinetics screening, density functional theory (DFT) mechanistic study, molecular docking, and molecular dynamics (MD) simulation. We designed over 20 rivastigmine substituents, subject them to some analyses, and identified RL2 with an appreciable blood-brain barrier score and no permeability glycoprotein binding. The compound shows higher acylation energy and a favored binding affinity to the cholinesterase enzymes. RL2 interacts with the AChE and BuChE active sites showing values of −41.1/−39.5 kcal mol⁻¹ while rivastigmine binds with −32.7/−30.7 kcal mol⁻¹ for these enzymes. The study revealed RL2 (4-fluorophenyl rivastigmine) as a potential dual inhibitor for AChE and BuChE towards Alzheimer's disorder management.     

A theoretical model of the competition between hydrolase and carboxylesterase in protection against organophosphorus poisoning

A system of coupled non-linear differential equations describing interactions between organophosphorus compounds (OPs), OP hydrolase, acetylcholinesterase (AChE), and carboxylesterase (CaE) in a single compartment was derived incorporating irreversible combination of OP with AChE, hydrolytic breakdown of OP, and irreversible combination of OP with CaE. The equations were then uncoupled, providing non-linear differential equations on AChE, CaE and OP concentrations. One steady state solution of the AChE equation provided theoretical expressions for the amounts of OP hydrolyzed, bound with CaE, and bound with AChE. Assuming that the LD50 of an OP reflects the dose that depletes AChE to a 'minimal essential' level and that a single compartment model is applicable in vivo, the steady state solution becomes an equation predicting the LD50 from rate constants, initial enzyme levels, and the allowable AChE depletion. Normalization by initial AChE concentration produced a dimensionless relationship describing an 'OP toxicity surface' that clearly demonstrates regions where hydrolysis and CaE offer protection against OP poisoning. The surface can be used to theoretically predict an LD50 given only kinetic rate constants and effective whole-body AChE and CaE levels. Predictions of LD50s of seven OPs in rats were compared with published data. The relationship was found to adequately predict published LD50s spanning 5 orders of magnitude. The OP toxicity surface relationship provides a conceptual tool for use in OP toxicity research but should be particularly useful in predicting the relative protective effects of catalytic and stoichiometric scavenger mechanisms for an OP.     

Immunocytochemical localization of secretory acetylcholinesterase of the parasitic nematode Nippostrongylus brasiliensis

Various parasitic nematodes secrete acetylcholinesterase (AChE). In this study, the localization of AChE in the nematode Nippostrongylus brasiliensis and the secretory forms of AChE in culture fluid were examined. A thiocholine method revealed that AChE activity was localized in the subventral glands, which have a secretory and excretory function via a duct connected to the excretory pore. By electron microscopy, AChE activity was found mainly in the matrix of secretory granules, and sometimes in the Golgi apparatus in the subventral gland cells. These results show that nematode AChE is produced and stored in the subventral glands. Monoclonal antibodies against AChE of human erythrocytes or electric rays also bound to the nematode subventral gland, suggesting immuno-cross-reactivity of AChE among these species. When AChE activity in the nematode excretory-secretory product was examined by SDS polyacrylamide gel electrophoresis combined with the thiocholine method, intense activity was demonstrated as a single band at 74kDa. Immunoblot analysis showed specific recognition of this molecule by IgE and IgG1 antibodies, but not by IgG2a antibody, in nematode-infected rat sera. These results indicate that the nematode AChE molecule produced in and secreted from the subventral glands is antigenic for the production of IgE/IgG1 in host animals.     

Size and charge isomers of acetylcholinesterase in the cerebral cortex of young and aged rats

Previous studies in this laboratory showed an age-related decline of acetylcholinesterase (AChE) activity in the cerebral cortex of rats. In the present study the age-related differences in enzymatic activity were evaluated in terms of individual molecular forms. Extracts containing total, soluble and membrane-bound AChE were analyzed both by ultracentrifugation in sucrose gradient and by non-denaturing gradient polyacrylamide gel electrophoresis. By ultracentrifugation two molecular forms, namely 10S and 4S (corresponding to tetrameric-G4 and monomeric-G1 forms, respectively) were separated in extracts of total and soluble AChE, while only 10S forms were present in extracts of membrane-bound AChE. Electrophoresis of soluble AChE extracts revealed slowly- and fast-migrating bands, grouped in two clusters of at least three bands each; membrane-bound AChE contained only a single slowly-migrating band. Electrophoresis of the single forms isolated by ultracentrifugation showed that slowly- and fast-migrating bands corresponded to G4 and G1 forms, respectively. Therefore, in soluble AChE no one-to-one relationship between charge- and size-isomers was observed; on the contrary, such relationship has been shown for membrane-bound AChE. This implies that soluble G4 forms and membrane-bound-G4 form are electrophoretically different, being heterogeneous the former and homogeneous the latter. The age-related decline of total AChE, accompanied by a decrease of G4/G1 ratio, depended mainly on a decrease of membrane-bound AChE while soluble AChE and its G4/G1 ratio was unchanged. The qualitative pattern of charge isomers was not modified by aging.     

Molecular isoform distribution and glycosylation of acetylcholinesterase are altered in brain and cerebrospinal fluid of patients with Alzheimer's disease

The glycosylation of acetylcholinesterase (AChE) in CSF was analyzed by lectin binding. AChE from Alzheimer's disease (AD) patients was found to bind differently to two lectins, concanavalin A and wheat germ agglutinin, than AChE from controls. As multiple isoforms of AChE are present in both CSF and brain, we examined whether the abnormal glycosylation of AD AChE was due to changes in a specific molecular isoform. Globular amphiphilic dimeric (G2a) and monomeric (G1a) isoforms of AChE were found to be differentially glycosylated in AD CSF. Glycosylation of AChE was also altered in AD frontal cortex but not in cerebellum and was also associated with an increase in the proportion of light (G2 and G1) isoforms. This study demonstrates that the glycosylation of AChE is altered in the AD brain and that changes in AChE glycosylation in AD CSF may reflect changes in the distribution of brain isoforms. The study also suggests that glycosylation of AChE may be a useful diagnostic marker for AD.     

Altered Levels of Acetylcholinesterase in Alzheimer Plasma

Background

Many studies have been conducted in an extensive effort to identify alterations in blood cholinesterase levels as a consequence of disease, including the analysis of acetylcholinesterase (AChE) in plasma. Conventional assays using selective cholinesterase inhibitors have not been particularly successful as excess amounts of butyrylcholinesterase (BuChE) pose a major problem.

Principal Findings

Here we have estimated the levels of AChE activity in human plasma by first immunoprecipitating BuChE and measuring AChE activity in the immunodepleted plasma. Human plasma AChE activity levels were ∼20 nmol/min/mL, about 160 times lower than BuChE. The majority of AChE species are the light G₁+G₂ forms and not G₄ tetramers. The levels and pattern of the molecular forms are similar to that observed in individuals with silent BuChE. We have also compared plasma AChE with the enzyme pattern obtained from human liver, red blood cells, cerebrospinal fluid (CSF) and brain, by sedimentation analysis, Western blotting and lectin-binding analysis. Finally, a selective increase of AChE activity was detected in plasma from Alzheimer''s disease (AD) patients compared to age and gender-matched controls. This increase correlates with an increase in the G₁+G₂ forms, the subset of AChE species which are increased in Alzheimer''s brain. Western blot analysis demonstrated that a 78 kDa immunoreactive AChE protein band was also increased in Alzheimer''s plasma, attributed in part to AChE-T subunits common in brain and CSF.

Conclusion

Plasma AChE might have potential as an indicator of disease progress and prognosis in AD and warrants further investigation.     

Selective Complexing of Acetylcholinesterase in Brain by Intravenously Administered Monoclonal Antibody

Rats injected intravenously with monoclonal antibodies reactive with brain acetylcholinesterase (AChE) developed a prolonged depression of plasma AChE without changes in butyrylcholinesterase, lactic acid dehydrogenase, or hematocrit. One antibody, ZR1, accumulated in the brain and spinal cord. Within 3 days of injection, ZR1 bound to most of the AChE in cerebral cortex and certain other regions of the CNS. Examination of the molecular forms of cortical 10S AChE, whereas 4S AChE remained free. In vitro, however, ZR1 bound equally to solubilized 4S and 10S forms. These data provide direct evidence for the compartmentalization of different AChE forms in the CNS, 10S being mainly extracellular and 4S apparently intracellular. Development of a striking and persistent bilateral ptosis within hours of injection suggests that AChE in the autonomic nervous system is also accessible to antibodies and, furthermore, is the site of an immunopathological lesion. This novel model of cholinergic autoimmunity may have relevance for human neurological disorders of unknown etiology.     

Amyloid-beta increases acetylcholinesterase expression in neuroblastoma cells by reducing enzyme degradation

Amyloid-beta (Abeta) is the principal protein constituent of 'senile plaques' and is a suspected mediator in Alzheimer's disease (AD). Senile plaques also contain acetylcholinesterase (AChE; EC 3.1.1.7), which may have a role in promoting Alphabeta-toxicity. We have found that Alphabeta can affect AChE expression in a neuron-like line, the N1E.115 neuroblastoma cell. When 1 micro mAlphabeta 1-42 or 25-35 was added for 24 h to differentiating N1E.115 in culture, AChE activity increased 30-40% in adherent cells, and 100% or more in nonadherent cells. The changes in both tetrameric (G4) and monomeric (G1) AChE forms were comparable. Turnover studies indicated that the elevation of AChE activity reflected slowed AChE degradation rather than accelerated synthesis. With a similar time course, Alphabeta also increased the quantity of muscarinic receptors on the plasma membrane. Immunocytochemistry for a lysosomal membrane protein (LAMP-1) indicated no change in abundance or localization of lysosomes in treated cells. But decreased labeling by pH-sensitive fluorescent dye pointed to an impairment of lysosomal acidification. We consider that the alteration of AChE expression after Alphabeta-exposure could reflect lysosomal dysfunction, and might itself enhance Alphabeta-toxicity.