Targeting acetylcholinesterase to treat neurodegeneration

Neurodegenerative disorders, such as Alzheimer’s disease, are often characterised by the degeneration of the cholinergic system. Thus, the aim of many treatment regimens is to support this system either by means of muscarinic agonists or by inhibitors of acetylcholinesterase (AChE), the latter being able to increase the concentration of acetylcholine. However, both pharmacological groups of drugs can only help in the beginning of the progressive disease. The finding that the occupation of the peripheral anionic site of AChE is able to stop the formation of the amyloid plaque led to the development of bivalent ligands that occupy both the active and the peripheral site. This dual action might be more beneficial for treatment of Alzheimer´s disease than simple inhibition of the acetylcholine hydrolysis. Thus, the new bivalent ligands are the focus of this review.     

Bivalent ligands derived from Huperzine A as acetylcholinesterase inhibitors

The naturally occurring alkaloid Huperzine A (HupA) is an acetylcholinesterase (AChE) inhibitor that has been used for centuries as a Chinese folk medicine in the context of its source plant Huperzia Serrata. The potency and relative safety of HupA rendered it a promising drug for the ameliorative treatment of Alzheimer's disease (AD) vis-à-vis the "cholinergic hypothesis" that attributes the cognitive decrements associated with AD to acetylcholine deficiency in the brain. However, recent evidence supports a neuroprotective role for HupA, suggesting that it could act as more than a mere palliative. Biochemical and crystallographic studies of AChE revealed two potential binding sites in the active-site gorge of AChE, one of which, the "peripheral anionic site" at the mouth of the gorge, was implicated in promoting aggregation of the beta amyloid (Abeta) peptide responsible for the neurodegenerative process in AD. This feature of AChE facilitated the development of dual-site binding HupA-based bivalent ligands, in hopes of concomitantly increasing AChE inhibition potency by utilizing the "chelate effect", and protecting neurons from Abeta toxicity. Crystal structures of AChE allowed detailed modeling and docking studies that were instrumental in enhancing the understanding of underlying principles of bivalent inhibitor-enzyme dynamics. This monograph reviews two categories of HupA-based bivalent ligands, in which HupA and HupA fragments serve as building blocks, with a focus on the recently solved crystallographic structures of Torpedo californica AChE in complex with such bifunctional agents. The advantages and drawbacks of such structured-based drug design, as well as species differences, are highlighted and discussed.     

Heterocyclic inhibitors of AChE acylation and peripheral sites

Notwithstanding the criticism to the so called " cholinergic hypothesis", the therapeutic strategies for the treatment of Alzheimer's disease (AD) have been mainly centered on the restoration of cholinergic functionality and, until the last year, the only drugs licensed for the management of AD were the acetycholinesterase (AChE) inhibitors. Target enzyme AChE consists of a narrow gorge with two separate ligand binding sites: an acylation site at the bottom of the gorge containing the catalytic triad and a peripheral site located at the gorge rim, which encompasses binding sites for allosteric ligands. The aim of this short review is to update the knowledge on heterocyclic AChE inhibitors able to interact with the two sites of enzymes, structurally related to the well known inhibitors physostigmine, rivastigmine and propidium. The therapeutic potential of the dual site inhibithors in inhibiting amyloid-beta aggregatrion and deposition is also briefly summarised.     

治疗阿尔茨海默病的乙酰胆碱酯酶抑制剂的分子设计: 从多位点抑制剂到一药多靶

阿尔茨海默病 (Alzheimer’s disease, AD) 是一种病因尚不明确且致病机制极为复杂的神经退行性疾病, 严重威胁老年人的健康并对整个社会发展带来沉重的负担。设计开发治疗阿尔茨海默病的药物一直是药物研发领域的热点和难点, 其中尤以乙酰胆碱酯酶 (acetylcholinesterase, AChE) 抑制剂的研究最为活跃并已在临床中成功应用。然而, 现有商品化AChE抑制剂的临床治疗效果有限, 并且都伴随不同程度的毒副作用。因此,  寻找高效、低毒的多重结合位点的AChE抑制剂和针对多重作用靶标的多功能抑制剂 (即一药多靶) 成为AChE抑制剂分子设计的主要发展方向。本文结合近年来的研究进展, 从代表性AChE抑制剂的化学结构和结合模式出发, 对AChE抑制剂分子设计的发展历程及最新成果进行了综述。     

Huprine X is a novel high-affinity inhibitor of acetylcholinesterase that is of interest for treatment of Alzheimer's disease

Inhibitors of the enzyme acetylcholinesterase (AChE) slow and sometimes reverse the cognitive decline experienced by individuals with Alzheimer's disease. Huperzine A, a natural product used in traditional Chinese herbal medicine, and tacrine (Cognex) are among the potent AChE inhibitors used in this treatment, but the search for more selective inhibitors continues. We report herein the synthesis and characterization of (-)-12-amino-3-chloro-9-ethyl-6,7, 10,11-tetrahydro-7,11-methanocycloocta[b]quinoline hydrochloride (huprine X), a hybrid that combines the carbobicyclic substructure of huperzine A with the 4-aminoquinoline substructure of tacrine. Huprine X inhibited human AChE with an inhibition constant K(I) of 26 pM, indicating that it binds to this enzyme with one of the highest affinities yet reported. Under equivalent assay conditions, this affinity was 180 times that of huperzine A, 1200 times that of tacrine, and 40 times that of E2020 (donepezil, Aricept), the most selective AChE inhibitor currently approved for therapeutic use. The association and dissociation rate constants for huprine X with AChE were determined, and the location of its binding site on the enzyme was probed in competition studies with the peripheral site inhibitor propidium and the acylation site inhibitor edrophonium. Huprine X showed no detectable affinity for the edrophonium-AChE complex. In contrast, huprine X did form a ternary complex with propidium and AChE, although its affinity for the free enzyme was found to be 17 times its affinity for the propidium-AChE complex. These data indicated that huprine X binds to the enzyme acylation site in the active site gorge but interferes slightly with the binding of peripheral site ligands.     

Recent advances in acetylcholinesterase Inhibitors and Reactivators: an update on the patent literature (2012-2015)

Introduction: Acetylcholinesterase (AChE) is the major enzyme that hydrolyzes acetylcholine, a key neurotransmitter for synaptic transmission, into acetic acid and choline. Mild inhibition of AChE has been shown to have therapeutic relevance in Alzheimer’s disease (AD), myasthenia gravis, and glaucoma among others. In contrast, strong inhibition of AChE can lead to cholinergic poisoning. To combat this, AChE reactivators have to be developed to remove the offending AChE inhibitor, restoring acetylcholine levels to normal.

Areas covered: This article covers recent advances in the development of acetylcholinesterase modulators, including both inhibitors of acetylcholinesterase for the efforts in development of new chemical entities for treatment of AD, as well as re-activators for resurrection of organophosphate bound acetylcholinesterase.

Expert opinion: Over the past three years, research efforts have continued to identify novel small molecules as AChE inhibitors for both CNS and peripheral diseases. The more recent patent activity has focused on three AChE ligand design areas: derivatives of known AChE ligands, natural product based scaffolds and multifunctional ligands, all of which have produced some unique chemical matter with AChE inhibition activities in the mid picomolar to low micromolar ranges. New AChE inhibitors with polypharmacology or dual inhibitory activity have also emerged as highlighted by new AChE inhibitors with dual activity at L-type calcium channels, GSK-3, BACE1 and H3, although most only show low micromolar activity, thus further research is warranted. New small molecule reactivators of organophosphate-inhibited AChE have also been disclosed, which focused on the design of neutral ligands with improved pharmaceutical properties and blood-brain barrier (BBB) penetration. Gratifyingly, some research in this area is moving away from the traditional quaternary pyridinium oximes AChE reactivators, while still employing the necessary reactivation group (oximes). However, selectivity over inhibition of native AChE enzyme, effectiveness of reactivation, broad-spectrum reactivation against multiple organophosphates and reactivation of aged-enzyme continue to be hurdles for this area of research.     

Peripheral site ligands accelerate inhibition of acetylcholinesterase by neutral organophosphates

The rates of inhibition of mouse acetylcholinesterase (AChE; EC 3.1.1.7) by paraoxon, haloxon, DDVP and enantiomers of neutral alkyl methylphosphonyl thioates and cationic alkyl methylphosphonyl thiocholines were measured in the presence and absence of AChE peripheral site inhibitors: gallamine, d-tubocurarine, propidium, atropine and derivatives of coumarin. All ligands, except the coumarins, at submillimolar concentrations enhanced the rates of inhibition by neutral organophosphates, whereas inhibition rates by cationic organophosphates were decreased. When peripheral site ligand concentrations extended to millimolar concentrations the extent of the enhancement decreased, creating a well-shaped activation profile. Analysis of inhibition by DDVP revealed that peripheral site inhibitors increase the second-order reaction rates by increasing maximal rates of phosphorylation. These observations suggest that peripheral site ligands are capable of allosterically affecting the conformation of residues in the choline binding site of AChE, thus optimizing the position of the leaving group of uncharged organophosphates during the inhibition reaction.     

Effect of tissue-specific acetylcholinesterase inhibitor C-547 on α3β4 and αβεδ acetylcholine receptors in COS cells

The C-547 is the most effective muscle and tissue-specific anticholinesterase among alkylammonium derivatives of 6-methyluracil (ADEMS) acting in nanomolar concentrations on locomotor muscles but not on respiratory muscles, smooth muscles and heart and brain acetylcholine esterases (AChE). When applied systematically it could influence peripheral acetylcholine receptors. The aim of the present study was to investigate the effect of C-547 on rat α3β4 (ganglionic type) and αβεδ (muscle type) nicotinic receptors expressed in COS cells. Currents evoked by rapid application of acetylcholine or nicotine were recorded in whole-cell mode by electrophysiological patch-clamp technique 2-4 days after cell transfection by plasmids coding the α3β4 or αβεδ combination of receptor subunits. In cells sensitive to acetylcholine, the application of C-547 evoked no responses. When acetylcholine was applied during an already running application of C-547, acetylcholine responses were only inhibited at concentrations higher than 10(-7)M. This inhibition is not voltage-dependent, but is accompanied by an increased rate of desensitization. Thus in both types of receptors, effective doses are approximately 100 times higher than those inhibiting AChE in leg muscles and similar to those inhibiting respiratory diaphragm muscles and external intercostal muscles. These observations show that C-547 can be considered for symptomatic treatment of myasthenia gravis and other congenital myasthenic syndromes as an inhibitor of AChE in leg muscles at concentrations much lower than those inhibiting muscle and ganglion types of acetylcholine receptors.     

An inhibitory monoclonal antibody to rabbit brain acetylcholinesterase. Studies on interaction with the enzyme

A recently isolated monoclonal antibody was found to be a potent and powerful inhibitor of the catalytic activity of rabbit brain acetylcholinesterase (AChE; acetylcholine acetylhydrolase, EC 3.1.1.7), with an IC50 of about 1 nM and a maximal inhibition of at least 90%. The antibody increased the optimal concentration of acetylthiocholine as much as 50-fold, but analysis of the substrate kinetics did not indicate a simple competitive interaction. The antibody markedly reduced the labeling of purified rabbit brain AChE by tritiated diisopropyl fluorophosphate (DFP) and also impeded the binding of propidium iodide, a fluorescent probe thought to be directed toward the peripheral anionic site. The antibody's affinity for enzyme with active sites that were phosphorylated with DFP or occupied by reversible ligands was measurably less than for native enzyme. It is possible that the mechanism of inhibition involves antibody-induced conformational changes that are unfavorable for catalysis.     

Hybrid-based multi-target ligands for the treatment of Alzheimer's disease

Progresses in medicinal chemistry over the last few years have focused on the design and synthesis of hybrid compounds, molecules encompassing in a single scaffold two pharmacophores from known entities endowed with well established biological activities. The interest in this topic is related to the increasing emphasis on the identification of the different factors involved in a number of disorders, such as the complex multifactorial Alzheimer's disease (AD), and hybrid- based strategy has become a focal point in this medicinal chemistry field since it could lead to derivatives with an improved biological profile. Using this strategy, acetylcholinesterase inhibitors (AChEIs) have been extensively coupled with properly selected bioactive molecules to obtain homo- and heterodimers endowed with increased potency together with supplementary actions. In the past decade the inhibition of the AChE induced aggregation of the -amyloid peptide into the senile plaques, which is a key event in the neurotoxic cascade of AD, has been considered a relevant approach leading to several dual binding site inhibitors, able to contact both the peripheral anionic site of AChE and the active site. In recent years, pioneering efforts have been performed to obtain novel AChEIs that, beyond the capability to inhibit AChE, were able to hit a number of specific AD targets. In particular, these compounds proved to possess antioxidant, anti-inflammatory, or neuroprotective activities, useful to block or revert the progression of the disease. This review summarizes the progresses that have been made in the design of hybrid molecules for the treatment of AD.     

An overview of the current and novel drugs for Alzheimer's disease with particular reference to anti-cholinesterase compounds

Several cellular processes could be targeted if the complex nature of Alzheimer's disease (AD) was already understood. Most of AD treatments have been focused on the inhibition of acetylcholinesterase (AChE) in order to raise the levels of its substrate, i.e. the neurotransmitter acetylcholine (ACh), to augment cognitive functions of affected patients. Effectiveness in AChE inhibition and side-effect issues of clinical (tacrine, donepezil, galanthamine and rivastigmine) as well as of novel inhibitors is reviewed here. Novel design methods for the inhibition of AChE include the use of in silico tools to predict the interactions between AChE and the desired compound, both at the active site of the enzyme, responsible of hydrolysing ACh and with the peripheral anionic site (PAS), which has been described as a promoting agent of the amyloid beta-peptide (A beta) aggregation present in the senile plaques of the brain of AD individuals.     

A step further towards multitarget drugs for Alzheimer and neuronal vascular diseases: targeting the cholinergic system, amyloid-β aggregation and Ca(2+) dyshomeostasis

Alzheimer's disease (AD) is the most common neurodegenerative disease, affecting mainly elderly people. The reasons why AD occurs are complex and multifactorial and several biochemical targets are thought to play a key role in its progress and development. This fact has led to the development of a multitarget-directed ligand strategy as a logical approach for designing a suitable therapy. Currently, most prescribed drugs for treating AD are acetylcholinesterase inhibitors (AChEI), although these inhibitors represent solely palliative treatment. This account will summarize our current therapeutic approach for the design of multitarget drugs primarily aimed at inhibiting AChE using the key features of tacrine, which was the first approved drug for AD treatment. Secondly, as calcium homeostasis is directly related to the cell death-survival equilibrium, suitable therapy might include an action that regulates calcium homeostasis by means of targeting voltage dependent calcium channels. It is, therefore, hoped that targeting calcium homeostasis will lead directly to the development of potential neuroprotective agents. Thus, 1,4-dihydropyridines, well-known voltage-dependent calcium channel (VDCC) ligands, will be incorporated into the new molecules as a second structural feature in order to bring about this action. As a result of this development, herein, we describe the synthetic and pharmacological profile of new [1,8]-naphthyridine analogues, which are hybrids of tacrine and 1,4-dihydropyridines. Some of our molecules have shown improved inhibitory action against cholinesterases, whilst maintaining their VDCC modulating activity, and have good characteristics as neuroprotective agents. Based on kinetic analysis of the AChE inhibition experiments, it has been shown that many of the compounds bind at the peripheral anionic site (PAS). Since the AChE PAS is linked to β-amyloid aggregation, this would give a third biological target for further preclinical development, making these molecules highly interesting targets in the search to obtain better treatments for AD.     

Nature: A Substantial Source of Auspicious Substances with Acetylcholinesterase Inhibitory Action

Acetylcholinesterase (AChE) (EC 3.1.1.7) is an important enzyme that breaks down of acetylcholine in synaptic cleft in neuronal junctions. Inhibition of AChE is associated with treatment of several diseases such as Alzheimer’s disease (AD), myasthenia gravis, and glaucoma as well as the mechanisms of insecticide and anthelmintic drugs. Several AChE inhibitors are available in clinical use currently for the treatment of AD; however, none of them has ability, yet, to seize progress of the disease. Consequently, an extensive research has been going on finding new AChE inhibitors. In this sense, natural inhibitors have gained great attention due to their encouraging effects toward AChE. In this review, promising candidate molecules with marked AChE inhibition from both plant and animal sources will be underlined.     

Allosteric modulation of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are receptors for the neurotransmitter acetylcholine and are members of the ‘Cys-loop’ family of pentameric ligand-gated ion channels (LGICs). Acetylcholine binds in the receptor extracellular domain at the interface between two subunits and research has identified a large number of nAChR-selective ligands, including agonists and competitive antagonists, that bind at the same site as acetylcholine (commonly referred to as the orthosteric binding site). In addition, more recent research has identified ligands that are able to modulate nAChR function by binding to sites that are distinct from the binding site for acetylcholine, including sites located in the transmembrane domain. These include positive allosteric modulators (PAMs), negative allosteric modulators (NAMs), silent allosteric modulators (SAMs) and compounds that are able to activate nAChRs via an allosteric binding site (allosteric agonists). Our aim in this article is to review important aspects of the pharmacological diversity of nAChR allosteric modulators and to describe recent evidence aimed at identifying binding sites for allosteric modulators on nAChRs.     

Cholinesterase reactivators: the fate and effects in the organism poisoned with organophosphates/nerve agents

Understanding the mechanism of action of organophosphates (OP)/nerve agents -- irreversible acetylcholinesterase (AChE, EC 3.1.1.7) inhibition at the cholinergic synapses followed by metabolic dysbalance of the organism -- two therapeutic principles for antidotal treatment are derived. The main drugs are anticholinergics that antagonize the effects of accumulated acetylcholine at the cholinergic synapses and cholinesterase reactivators (oximes) reactivating inhibited AChE. Anticonvulsants such as diazepam are also used to treat convulsions. Though there are experimental data on a good therapeutic effects of reactivators, some attempts to underestimate the role of reactivators as effective antidotes against OP poisoning have been made. Some arguments on the necessity of their administration following OP poisoning are discussed. Their distribution patterns and some metabolic and pharmacological effects are described with the aim to resolve the question on their effective use, possible repeated administration in the treatment of OP poisoning, their peripheral and central effects including questions on their penetration through the blood brain barrier as well as a possibility to achieve their effective concentration for AChE reactivation in the brain. Reactivation of cholinesterases in the peripheral and central nervous system is described and it is underlined its importance for the survival or death of the organism poisoned with OP. Metabolization and some other effects of oximes (not connected with AChE reactivation) are discussed (e.g. forming of the phosphonylated oxime, parasympatholytic action, hepatotoxicity, behavioral changes etc.). An universality of oximes able to reactivate AChE inhibited by all OP is questioned and therefore, needs of development of new oximes is underlined.     

Two new dilactonized glycerol glycosides of the dual anticholinesterase active extract from Ocotea daphnifolia using bioguided fractionation and molecular docking studies

The dual inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) is considered as an important strategy for the treatment of Alzheimer's disease. In this study, we applied the bioguided fractionations of Ocotea daphinifolia ethyl acetate active extract to furnish a fraction with high inhibitory activity for AChE and BuChE (82% and 92%, respectively). High-performance liquid chromatography semipreparative purification of this fraction provided two new natural products: 1-β-D-galactopyranosyl-glycerol-2,3-heptanedionate, ( 1 ) whose complete chemical structural elucidation was made with spectrometric analysis (MS, 1D, and 2D NMR) and its minor derivative 1-β-D-gulopyranosyl-glycerol-2,3-heptanedionate; ( 2 ) which could be characterized by 2D ¹H-¹³C heteronuclear single-quantum correlation spectra analysis. Investigation of the intermolecular interactions with cholinesterases was carried out by molecular docking studies, and results suggested that both compounds are capable to interact with the catalytic site of both enzymes. Compounds 1 and 2 interact with residues of catalytic domains and the peripheral anionic binding site of AChE and BuChE. The results are comparable to those achieved with rivastigmine and galantamine. Thus, this study provides evidence for consideration of the glycosylglycerol from O. daphnifolia as new valuable dual cholinesterases inhibitor.     

Can cholinesterase inhibitors affect neural development?

Accumulating evidence supports the view that acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can influence the proliferation and differentiation of nerve cells. AChE in particular has been found to promote neurite outgrowth in a variety of model systems, possibly by serving as an adhesion molecule. Thus one might suspect that cholinesterase inhibitors would disturb neuronal development, with long-term implications for structure and function in the central and peripheral nervous systems. The actual picture is more complex because AChE's effects on neurite outgrowth may reflect protein-protein interactions that are not directly related to catalytic function but are nonetheless influenced by ligands with special structural features. The putative structural interactions have not yet been rigorously defined, but they are likely to involve enzyme regions at or near the peripheral anionic site. In addition to such effects, some organophosphorus anticholinesterases have been reported to act by still other mechanisms to depress macromolecule synthesis and cell survival in the developing brain. Taken together, this emerging information highlights the potential importance of anticholinesterase agents in developmental neurotoxicology.     

Structures of Human Acetylcholinesterase Bound to Dihydrotanshinone I and Territrem B Show Peripheral Site Flexibility

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Huprine-tacrine heterodimers as anti-amyloidogenic compounds of potential interest against Alzheimer's and prion diseases

A family of huprine-tacrine heterodimers has been developed to simultaneously block the active and peripheral sites of acetylcholinesterase (AChE). Their dual site binding for AChE, supported by kinetic and molecular modeling studies, results in a highly potent inhibition of the catalytic activity of human AChE and, more importantly, in the in vitro neutralization of the pathological chaperoning effect of AChE toward the aggregation of both the β-amyloid peptide (Aβ) and a prion peptide with a key role in the aggregation of the prion protein. Huprine-tacrine heterodimers take on added value in that they display a potent in vitro inhibitory activity toward human butyrylcholinesterase, self-induced Aβ aggregation, and β-secretase. Finally, they are able to cross the blood-brain barrier, as predicted in an artificial membrane model assay and demonstrated in ex vivo experiments with OF1 mice, reaching their multiple biological targets in the central nervous system. Overall, these compounds are promising lead compounds for the treatment of Alzheimer's and prion diseases.     

Structural requirements of acetylcholinesterase reactivators

Nerve agents (sarin, soman, cyclosarin, tabun and VX agent) and pesticides (paraoxon, chlorpyrifos, TEPP) represent extremely toxic group of organophosphorus compounds (OPCs). These compounds inhibit enzyme acetylcholinesterase (AChE, EC 3.1.1.7) via its phosphorylation or phosphonylation at the serine hydroxy group in its active site. Afterwards, AChE is not able to serve its physiological function and intoxicated organism is died due to overstimulation of cholinergic nervous system. The current standard treatment of poisoning with highly toxic OPCs usually consists of the combined administration of anticholinergic drugs (preferably atropine) and AChE reactivators (called "oximes"). Anticholinergic drugs block effects of accumulated neurotransmitter acetylcholine at nicotinic and muscarinic receptor sites, while oximes reactivate AChE inhibited by OPCs. Unfortunately, none from the currently used oximes is sufficiently effective against all known nerve agents and pesticides. Therefore, to find new oximes able to sufficiently reactivate inhibited AChE (regardless of the type of OPCs) is still very important task for medicinal chemistry with the aim to improve the efficacy of antidotal treatment of the acute poisonings mentioned. In this paper, the relationship between chemical structure of AChE reactivators and their ability to reactivate AChE inhibited by several nerve agents and pesticides is summarized. It is shown that there are several structural fragments possibly involving in the structure of proposed AChE reactivators. Finally, an attempt of a future course of new AChE reactivators development is discussed.