Biosensors containing acetylcholinesterase and butyrylcholinesterase as recognition tools for detection of various compounds

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are enzymes expressed in the human body under physiological conditions. AChE is an important part of the cholinergic nerves where it hydrolyses neurotransmitter acetylcholine. Both cholinesterases are sensitive to inhibitors acting as neurotoxic compounds. In analytical applications, the enzymes can serve as a biorecognition element in biosensors as well as simple disposable sensors (dipsticks) and be used for assaying the neurotoxic compounds. In the present review, the mechanism of AChE and BChE inhibition by disparate compounds is explained and methods for assaying the enzymes activity are shown. Optical, electrochemical, and piezoelectric biosensors are described. Attention is also given to the application of sol-gel techniques and quantum dots in the biosensors’ construction. Examples of the biosensors are provided and the pros and cons are discussed.     

Determination of acetylcholinesterase and butyrylcholinesterase activity without dilution of biological samples

Two cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are known. The enzymes are important in the body and alteration of their activity has significant use in the diagnosis of poisoning, liver function, etc. Currently available methods for the determination of cholinesterases have some major drawbacks including various interferences and the inability to be used for decreasing the enzyme activity in the presence of reversible inhibitors due to sample dilution; hence, a method for dilution free assay of cholinesterases is desired. Here, microplates were modified with indoxylacetate (100 µL of 10 mmol L^?1 solution) and used for cholinesterases assay after drying at 37°C. The fact that indoxylacetate remains stable in dry state and serves simultaneously as a chromogen and substrate provide good prerequisites for the method. The limit of detection for BChE was 0.71 U while that for AChE was 2.8 U per a 100 µL sample (solution of enzyme or plasma sample). The limit of detection is low enough to allow standard examination of cholinesterasemia. The two cholinesterases can be distinguished from each other using selective inhibitors such as donepezil and iso-OMPA. The new method was also successfully validated for the standard Ellman’s assay using plasma samples with BChE activity adjusted by carbofuran. The new method based on indoxylacetate seems promising for routine tests.     

Isoquinoline Alkaloid Contents in Macleaya cordata Extracts and Their Acetylcholinesterase and Butyrylcholinesterase Inhibition

An important strategy for treating neurodegenerative disorders is to maintain the levels of acetylcholine in the synaptic cleft by blocking the cholinesterases. Searching for new effective compounds with inhibited acetylcholinesterase and butyrylcholinesterase activity is one of the most significant challenges of the modern scientific research. The aim of this study was the optimization of the condition for cholinesterase activity determination by high-performance liquid chromatography coupled with diode array detector (HPLC-DAD) in terms of concentrations of enzymatic reaction mixture components, temperature of incubation, and incubation time. In vitro investigation of acetylcholinesterase and butyrylcholinesterase activity inhibition by some isoquinoline alkaloids and extracts obtained from the aerial part and roots of Macleaya cordata collected in May, July, and September. Acetylcholinesterase and butyrylcholinesterase activity inhibition of the extracts obtained from the plant had not been tested previously. The application of the HPLC method allowed eliminating absorption of interfering components, for example, alkaloids such as sanguinarine and berberine. The HPLC method was successfully applied for the evaluation of the acetylcholinesterase inhibitory activity in samples such as plant extracts, especially those containing colored components adsorbing at the same wavelength as the adsorption wavelength of 5-thio-2-nitro-benzoic acid, which is the product of the reaction between thiocholine (product of the hydrolysis of acetyl/butyrylthiocholine reaction) with Ellman’s reagent. Moreover, liquid chromatography coupled with a triple quadrupole mass spectrometer (LC–QqQ–ESI–MS/MS) analysis allowed evaluating the identification of relevant bioactive compounds in the obtained plant extracts. The investigated alkaloids, especially sanguinarine and chelerythrine, and all the Macleaya cordata extracts, especially the extract obtained from the aerial part collected in May, exhibited very high cholinesterase activity inhibition. HPLC-DAD was also applied for the kinetics study of the most active alkaloids sanguinarine and chelerythrine. Our investigations demonstrated that these plant extracts can be recommended for further in vivo experiments to confirm their cholinesterase inhibition activity.     

CAGED CHOLINERGIC LIGANDS AND PHOTOREGULATION OF CHOLINESTERASE ACTIVITIES

Photolabile precursors of biologically interesting molecules, or "caged" compounds can provide control of temporal and spatial release of desired molecules by rapid photolysis, and are thus important tools in the study of fast biological processes. Acetylcholinesterase (AChE) is a particularly fast enzynie which hydrolyses the neurotransmitter acetylcholine with a turnover number approaching 20000 s^-1. Knowledge of the 3-D structure of AChE[1] has permitted a better understanding of structure-function relationships in the cholinesterases, but has also raised cogent new questions concerning the traffic of substrate and products to and from the active site. Time-resolved crystallography would present an ideal approach to investigate this issue at the atomic level and in real time,provided that suitable probes, which regulate the enzymatic activity by temporally and spatially controled release of enzyme substrates or product.     

1,2,4-Thiadiazoles as promising multifunctional agents for treatment of neurodegenerative diseases

Detailed studies of properties of new 3-substituted 5-anilino-1,2,4-thiadiazoles containing different substituents at position 3 of the thiadiazole ring were carried out, in particular, their esterase profile and antioxidant properties. It was found that the presence in the molecule of 2-aminopropyl fragment determines an efficient and selective inhibition of butyrylcholinesterase as compared to acetylcholinesterase and carboxylesterase, with radical-scavenging activity being weak. The compounds containing a 2-aminopropenyl fragment possess a high radicalscavenging activity, weakly inhibit cholinesterases, and exhibit anticarboxylesterase activity. A wide spectrum of activity of 3-substituted 5-anilino-1,2,4-thiadiazoles as inhibitors of cholinesterases and highly efficient scavengers of free radicals gives a basis for the optimization of structure and development in this series of original agents for therapy of neurodegenerative diseases.     

A direct method to visualise the aryl acylamidase activity on cholinesterases in polyacrylamide gels

Background  

In vertebrates, two types of cholinesterases exist, acetylcholinesterase and butyrylcholinesterase. The function of acetylcholinesterase is to hydrolyse acetylcholine, thereby terminating the neurotransmission at cholinergic synapse, while the precise physiological function of butyrylcholinesterase has not been identified. The presence of cholinesterases in tissues that are not cholinergically innervated indicate that cholinesterases may have functions unrelated to neurotransmission. Furthermore, cholinesterases display a genuine aryl acylamidase activity apart from their predominant acylcholine hydrolase activity. The physiological significance of this aryl acylamidase activity is also not known. The study on the aryl acylamidase has been, in part hampered by the lack of a specific method to visualise this activity. We have developed a method to visualise the aryl acylamidase activity on cholinesterase in polyacrylamide gels.     

Synthesis of a number of derivatives of alkaloids and of nitrogen-containing heterocycles and their anticholinesterase activities

N-Methyl- and N-phenylcarbamates based on a number of alkaloids and nitrogen-containing heterocycles have been synthesized, and they have proved to be weak irreversible inhibitors of acetylcholinesterase and butyrylcholinesterase. It has been shown that the choline fragments of the above-mentioned carbamates and their β-methylcholine analogs are reversible inhibitors of both cholinesterases and make a substantial contribution to the anticholinesterase activity. Selective inhibitors of ACE and BuCE have been found among the compounds synthesized.     

Spectroscopic Determination of Cholinesterase Activity and Inhibition in Sol-Gel Media

Biological activity of cholinesterases can be determined by optically monitoring the enzymatic reaction with indophenyl acetate, (N-4′-acetoxyphenyl)-4-quinone imine. At pH 8.0 cholinesterases hydrolyze this yellow dye to yield a blue reaction product. Cholinesterase inhibitors reduce the rate of this hydrolysis. Thus, by monitoring absorbance of the hydrolysis product at its maximum (630 nm) as a function of time, reaction rates of both cholinesterase activity and cholinesterase inhibition may be quantified spectroscopically. Using this technique, we measured the enzymatic activity of butyrylcholinesterase (BuChE) molecules encapsulated in tetramethyl orthosilicate (TMOS) silicate gel-glass prepared by hydrolysis and condensation. This activity is reduced, in a concentration-dependent manner, by the reversible cholinesterase inhibitors 1,5-bis(4-allyldimethyl-ammoniumphenyl) pentan 3-one dibromide (BADAPP) and 9-amino-1,2,3,4-tetrahydroacridine (THA; tacrine, Cognex). The gel-glasses are rigid, and compact, transparent and porous enough to allow reagents to diffuse in and out.     

Biphenylalkoxyamine Derivatives–Histamine H3 Receptor Ligands with Butyrylcholinesterase Inhibitory Activity

Neurodegenerative diseases, e.g., Alzheimer’s disease (AD), are a key health problem in the aging population. The lack of effective therapy and diagnostics does not help to improve this situation. It is thought that ligands influencing multiple but interconnected targets can contribute to a desired pharmacological effect in these complex illnesses. Histamine H₃ receptors (H₃Rs) play an important role in the brain, influencing the release of important neurotransmitters, such as acetylcholine. Compounds blocking their activity can increase the level of these neurotransmitters. Cholinesterases (acetyl- and butyrylcholinesterase) are responsible for the hydrolysis of acetylcholine and inactivation of the neurotransmitter. Increased activity of these enzymes, especially butyrylcholinesterase (BuChE), is observed in neurodegenerative diseases. Currently, cholinesterase inhibitors: donepezil, rivastigmine and galantamine are used in the symptomatic treatment of AD. Thus, compounds simultaneously blocking H₃R and inhibiting cholinesterases could be a promising treatment for AD. Herein, we describe the BuChE inhibitory activity of H₃R ligands. Most of these compounds show high affinity for human H₃R (K_i < 150 nM) and submicromolar inhibition of BuChE (IC₅₀ < 1 µM). Among all the tested compounds, 19 (E153, 1-(5-([1,1′-biphenyl]-4-yloxy)pentyl)azepane) exhibited the most promising in vitro affinity for human H₃R, with a K_i value of 33.9 nM, and for equine serum BuChE, with an IC₅₀ of 590 nM. Moreover, 19 (E153) showed inhibitory activity towards human MAO B with an IC₅₀ of 243 nM. Furthermore, in vivo studies using the Passive Avoidance Task showed that compound 19 (E153) effectively alleviated memory deficits caused by scopolamine. Taken together, these findings suggest that compound 19 can be a lead structure for developing new anti-AD agents.     

Conjugation of Aminoadamantane and γ-Carboline Pharmacophores Gives Rise to Unexpected Properties of Multifunctional Ligands

A new series of conjugates of aminoadamantane and γ-carboline, which are basic scaffolds of the known neuroactive agents, memantine and dimebon (Latrepirdine) was synthesized and characterized. Conjugates act simultaneously on several biological structures and processes involved in the pathogenesis of Alzheimer’s disease and some other neurodegenerative disorders. In particular, these compounds inhibit enzymes of the cholinesterase family, exhibiting higher inhibitory activity against butyrylcholinesterase (BChE), but having almost no effect on the activity of carboxylesterase (anti-target). The compounds serve as NMDA-subtype glutamate receptor ligands, show mitoprotective properties by preventing opening of the mitochondrial permeability transition (MPT) pore, and act as microtubule stabilizers, stimulating the polymerization of tubulin and microtubule-associated proteins. Structure–activity relationships were studied, with particular attention to the effect of the spacer on biological activity. The synthesized conjugates showed new properties compared to their prototypes (memantine and dimebon), including the ability to bind to the ifenprodil-binding site of the NMDA receptor and to occupy the peripheral anionic site of acetylcholinesterase (AChE), which indicates that these compounds can act as blockers of AChE-induced β-amyloid aggregation. These new attributes of the conjugates represent improvements to the pharmacological profiles of the separate components by conferring the potential to act as neuroprotectants and cognition enhancers with a multifunctional mode of action.     

Multifunctional approaches to provide potential pharmacophores for the pharmacy shelf: Heracleum sphondylium L. subsp. ternatum (Velen.) Brummitt.

Heracleum sphondylium L. subsp. ternatum (Velen.) Brummitt. commonly known as “hogweed” is traditionally used to manage several human ailments. This investigation assessed, for the first time, the enzyme inhibitory properties, antioxidant activity, phytochemical profile, antimutagenic, and antimicrobial potential of the ethyl acetate, methanol, and water extracts of H. sphondylium. We also established the possible interactions of identified phenolic compounds with cholinesterases, amylase, glucosidase, and tyrosinase using in silico docking studies. Chlorogenic acid was found in high amounts in the methanol extract of H. sphondylium. The methanol extract was an effective inhibitor of acetylcholinesterase (1.70 mg galantamine equivalent (GALAE)/g extract) while the ethyl acetate extract showed pronounced inhibitory action against butyrylcholinesterase (1.77 mg GALAE/g extract). The extracts exhibited low inhibition against amylase (0.12-0.84 mmol acarbose equivalent (ACAE)/g extract) and a more pronounced inhibition against glucosidase (2.29–3.65 mmol ACAE/g extract). In silico results showed that rutin and quercetin (-70.4 and -72.2 Kcal/mol, for rutin and quercetin respectively) docked to the enzymatic cavity of acetylcholinesterase but these phenolic compounds showed less affinity with butyrylcholinesterase (-15.0 and -5.2 Kcal/mol, for rutin and quercetin respectively). The extracts did not induce any mutations on the bacterial strains, while they have excellent antimutagenic capacity against well-known mutagens (inhibition values 98%, 97% and 96%). The methanol extract (0.78 mg/ml) showed moderate antifungal activity while the ethyl acetate extract (0.78–3.12 mg/ml) showed weak to moderate antimicrobial activity. This study provides valuable baseline data which might serve for the development of future pharmacophores for the management of human ailments.     

1,3-substituted imidazolidine-2,4,5-triones: synthesis and inhibition of cholinergic enzymes

A series of novel and highly active acetylcholinesterase and butyrylcholinesterase inhibitors derived from substituted benzothiazoles containing an imidazolidine-2,4,5-trione moiety were synthesized and characterized. The molecular structure of 1-(2,6-diisopropyl-phenyl)-3-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]-imidazolidine-2,4,5-trione (3g) was determined by single-crystal X-ray diffraction. Both optical isomers are present as two independent molecules in the triclinic crystal system. The lipophilicity of the compounds was determined as the partition coefficient log K(ow) using the traditional shake-flask method. The in vitro inhibitory activity on acetylcholinesterase from electric eel and butyrylcholinesterase isolated from equine serum was determined. The inhibitory activity on acetylcholinesterase was significantly higher than that of the standard drug rivastigmine. The discussed compounds are also promising inhibitors of butyrylcholinesterase, as some of the prepared compounds inhibit butyrylcholinesterase better than the internal standards rivastigmine and galanthamine. The highest inhibitory activity (IC?? = 1.66 μmol/L) corresponds to the compound 1-(4-isopropylphenyl)-3-[(R)-1-(6-fluorobenzo[d]thiazol-2-yl)ethyl]imidazolidine-2,4,5-trione (3d). For all the studied compounds, the relationships between the lipophilicity and the chemical structure as well as their structure-activity relationships are discussed.     

Synthesis and in vitro evaluation of N-(Bromobut-3-en-2-yl)-7-methoxy-1,2,3,4-tetrahydroacridin-9-amine as a cholinesterase inhibitor with regard to Alzheimer's disease treatment

A new tacrine based cholinesterase inhibitor, N-(bromobut-3-en-2-yl)-7-methoxy-1,2,3,4-tetrahydroacridin-9-amine (1), was designed and synthesized to interact with specific regions of human acetylcholinesterase and human butyrylcholinesterase. Its inhibitory ability towards cholinesterases was determined and compared to tacrine (THA) and 9-amino-7-methoxy-1,2,3,4-tetrahydroacridine (7-MEOTA). The assessment of IC50 values revealed 1 as a weak inhibitor of both tested enzymes.     

Modeling chemical transformations at the active sites of cholinesterases by quantum-based simulations

The significance of the quantum-mechanical–molecular-mechanical (QM/MM) method in modeling chemical transformations at the active sites of cholinesterases is discussed. Diverse versions of the QM/MM approach are applied to understand the molecular mechanisms of the reactivation reaction of butyrylcholinesterase phosphorylated by the catalytic serine residue.     

Synthesis of a number of derivatives of alkaloids and of nitrogen-containing heterocycles and their anticholinesterase activities

N-Methyl- and N-phenylcarbamates based on a number of alkaloids and nitrogen-containing heterocycles have been synthesized, and they have proved to be weak irreversible inhibitors of acetylcholinesterase and butyrylcholinesterase. It has been shown that the choline fragments of the above-mentioned carbamates and their -methylcholine analogs are reversible inhibitors of both cholinesterases and make a substantial contribution to the anticholinesterase activity. Selective inhibitors of ACE and BuCE have been found among the compounds synthesized.A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodynkh Soedinenii, No. 6, pp. 825–831, November–December, 1988.     

Covalently immobilized choline oxidase and cholinesterases on a methacrylate copolymer for disposable membrane biosensors

Bienzymatic sensors for the determination of esters of choline were prepared by covalent co-immobilization of cholinesterases and choline oxidase on polymer membranes, obtained by radiation-induced copolymerization of 2-hydroxyethyl methacrylate and glycidyl methacrylate at low temperature. Optimization of the covalent attachment of choline oxidase and acetyl-or butyrylcholinesterase to copolymer was explored. The enzyme-modified polymers were applied on platinum electrodes to form amperometric sensors, based on the electrochemical detection of enzymatically developed hydrogen peroxide. Acetyl-, acetylthiobutyryl-, and butyrylthiocholine contents in standard solutions were measured, and linear calibration curves were determined. Temperature and pH effects on the electrochemical response are described.     

Targeted synthesis and biological activity of polypharmacophoric agents for the treatment of neurodegenerative diseases

Synthetic approaches to the design of new multitarget agents for the treatment of neurodegenerative diseases are surveyed. These approaches are based on the conjugation of the pharmacophoric indole, phenothiazine, and aminoadamantane moieties via 1-oxopropylene and 2-hydroxypropylene spacers using reactions of acrylate- and epoxide-containing pharmacophores with tetrahydro-γ-carbolines, cycloalkaneindoles, carbazoles, and aminoadamantanes. The review considers data on biological activity of the compounds assessed within a complex screening system. This system includes neurotransmitter targets associated with cognitive compensation, such as cholinesterases and glutamate receptors, mitochondria, the influence on which can provide neuroprotective effects, and microtubules, the stabilization of which leads to the improvement of axonal transport. The ability of the compounds to act as free-radical scavengers was evaluated. Types of polypharmacophoric compounds promising for further development are proposed.     

Role of Natural Compounds and Target Enzymes in the Treatment of Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive neurological condition. The rising prevalence of AD necessitates the rapid development of efficient therapy options. Despite substantial study, only a few medications are capable of delaying the disease. Several substances with pharmacological activity, derived from plants, have been shown to have positive benefits for the treatment of AD by targeting various enzymes, such as acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), β-secretase, γ-secretase, and monoamine oxidases (MAOs), which are discussed as potential targets. Medicinal plants have already contributed a number of lead molecules to medicine development, with many of them currently undergoing clinical trials. A variety of medicinal plants have been shown to diminish the degenerative symptoms associated with AD, either in their raw form or as isolated compounds. The aim of this review was to provide a brief summary of AD and its current therapies, followed by a discussion of the natural compounds examined as therapeutic agents and the processes underlying the positive effects, particularly the management of AD.     

Anti-plasmodial and cholinesterase inhibiting activities of some constituents of Psorospermum glaberrimum

Glaberianthrone (1), a new bianthrone was isolated from the hexane extract of the stem bark of Psorospermum glaberrimum together with thirteen known compounds: 3-geranyloxyemodin anthrone (2), friedelan-3-one (3), 3-prenyloxyemodin anthrone (4), 3-geranyloxyemodin (5), 3-prenyloxyemodin (6), friedelan-3-ol (7), acetylvismione D (8), betulinic acid (9), 2-geranylemodin (10), bianthrone A2b (11), bianthrone 1a (12), emodin (13) and 2-prenylemodin (14). The structures of the isolated compounds were established by means of spectroscopic methods. The extracts and the isolated compounds were tested in vitro for their anti-plasmodial activity against Plasmodium falciparum (chloroquine resistant strain W2) and for their acetyl- and butyrylcholinesterase inhibitory properties. The n-hexane extract showed good anti-plasmodial activity against P. falciparum W2 strain, with IC(50) of 0.87 microg/ml. It also exhibited 65.5% and 98.2% of acetyl- and butyrylcholinesterase inhibition at 0.2 mg/ml, respectively. Compounds 2 and 8 showed the best potencies against P. falciparum W2 strain with IC(50) of 1.68 microM and 0.12 microM, (0.66 microg/ml and 0.054 microg/ml) respectively. All tested compounds showed good butyrylcholinesterase inhibition activities with compound 12 displaying the best potency (IC(50) 9.25+/-0.25 microM). All the tested compounds showed weak inhibitory activity against acetylcholinesterase.     

Determination of Anti-Alzheimer’s Disease Activity of Selected Plant Ingredients

Neurodegenerative diseases, among which one of the more common is Alzheimer’s disease, are the one of the biggest global public health challenges facing our generation because of the increasing elderly population in most countries. With the growing burden of these diseases, it is essential to discover and develop new treatment options capable of preventing and treating them. Neurodegenerative diseases, among which one of the most common is Alzheimer’s disease, are a multifactorial disease and therefore demand multiple therapeutic approaches. One of the most important therapeutic strategies is controlling the level of acetylcholine—a neurotransmitter in cholinergic synapses—by blocking the degradation of acetylcholine using acetylcholinesterase inhibitors such as tacrine, galantamine, donepezil and rivastigmine. However, these drugs can cause some adverse side effects, such as hepatotoxicity and gastrointestinal disorder. Thus, the search for new, more effective drugs is very important. In the last few years, different active constituents from plants have been tested as potential drugs in neurodegenerative disease therapy. The availability, lower price and less toxic effects of herbal medicines compared with synthetic agents make them a simple and excellent choice in the treatment of neurodegenerative diseases. The empirical approach to discovering new drugs from the systematic screening of plant extracts or plant-derived compounds is still an important strategy when it comes to finding new biologically active substances. The aim of this review is to identify new, safe and effective compounds that are potential candidates for further in vivo and clinical tests from which more effective drugs for the treatment of Alzheimer’s disease could be selected. We reviewed the methods used to determine anti-Alzheimer’s disease activity. Here, we have discussed the relevance of plant-derived compounds with in vitro activity. Various plants and phytochemical compounds have shown different activity that could be beneficial in the treatment of Alzheimer’s disorders. Most often, medicinal plants and their active components have been investigated as acetylcholinesterase and/or butyrylcholinesterase activity inhibitors, modifiers of β-amyloid processing and antioxidant agents. This study also aims to highlight species with assessed efficacy, usable plant parts and the most active plant components in order to identify species and compounds of interest for further study. Future research directions are suggested and recommendations made to expand the use of medicinal plants, their formulations and plant-derived active compounds to prevent, mitigate and treat Alzheimer’s disease.