Cholinesterase inhibitors Dotaz Zobrazit nápovědu
Currently, nerve agents are often used in terrorist attacks or assassinations. In such cases, it is necessary to detect them quickly, accurately and easily right in the field. Detection tubes, which are small devices containing pellets with immobilized cholinesterase and detection reagents, meet these conditions. Their detection mechanism is based on a highly sensitive enzymatic Ellman reaction, when in the absence of cholinesterase inhibitors the pellets develop a visible yellow color, whereas in their presence the carriers retain the original color. The rate of reaction, its sensitivity and the distinct color transition are the key points of the research. In this experiment, double-coated pellets were prepared. The first coating contained the butyrylcholinesterase immobilized in hypromellose, while the second coating consisted of ethylcellulose and triethyl citrate. Based on the properties of such carriers, samples containing lactose dispersed in the ethylcellulose coating were also prepared, which was expected to have an effect on increasing the permeability of the coating and hence the detection rate and color intensity. In addition to selected physicochemical properties, carriers were evaluated for enzyme activity, sensitivity and color transition intensity. Samples showing the best properties were subjected to a 24-months stability test at three different temperatures and humidity.
- Klíčová slova
- cholinesterase, detection tube, ethylcellulose, nerve agent, pellets,
- MeSH
- butyrylcholinesterasa chemie MeSH
- celulosa analogy a deriváty MeSH
- cholinesterasové inhibitory izolace a purifikace MeSH
- enzymy imobilizované chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- butyrylcholinesterasa MeSH
- celulosa MeSH
- cholinesterasové inhibitory MeSH
- enzymy imobilizované MeSH
- ethyl cellulose MeSH Prohlížeč
- Klíčová slova
- CHOLINESTERASE/chemistry *,
- MeSH
- cholinesterasové inhibitory * MeSH
- cholinesterasy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cholinesterasové inhibitory * MeSH
- cholinesterasy MeSH
Colorimetric biosensors of cholinesterase inhibitors are ideal for fast, reliable, and very simple detection of agents in air, in water, and on surfaces. This paper describes an innovation of the Czech Detehit biosensor, which is based on a biochemical enzymatic reaction visualized by using Ellman's reagent as a chromogenic indicator. The modification basically consists of a much more distinct color response of the biosensor, attained through optimization of the reaction system by using Guinea Green B as the indicator. The performance of the modified biosensor was verified on the chemical warfare agents (sarin, soman, cyclosarin, and VX) in water. The detection limits ascertained visually (with the naked eye) were about 0.001 µg/mL in water (exposure time 60 s, inhibition efficiency 25%).
- Klíčová slova
- Guinea Green B, biosensor of cholinesterase inhibitors, chemical warfare agents, enzymatic reaction, visual evaluation,
- MeSH
- biosenzitivní techniky metody MeSH
- chemické bojové látky analýza MeSH
- cholinesterasové inhibitory analýza MeSH
- kolorimetrie metody MeSH
- lissaminová zelená barviva chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chemické bojové látky MeSH
- cholinesterasové inhibitory MeSH
- guinea green B MeSH Prohlížeč
- lissaminová zelená barviva MeSH
Cholinesterase inhibitors are widely used as pesticides in agriculture, but also form a group of organophosphates known as nerve chemical warfare agents. This calls for close attention regarding their detection, including the use of various biosensors. One such biosensor made in the Czech Republic is the Detehit, which is based on a cholinesterase reaction that is assessed using a colour indicator-the Ellman's reagent-which is anchored on cellulose filter paper together with the substrate. With the use of this biosensor, detection is simple, quick, and sensitive. However, its disadvantage is that a less pronounced yellow discoloration occurs, especially under difficult light conditions. As a possible solution, a new indicator/substrate carrier has been designed. It is made of glass nanofibres, so the physical characteristics of the carrier positively influence reaction conditions, and as a result improve the colour response of the biosensor. The authors present and discuss some of the results of the study of this carrier under various experimental conditions. These findings have been used for the development of a modified Detehit biosensor.
- Klíčová slova
- Ellman’s reagent, biosensor, cellulose filter paper, cholinesterase reaction, glass fibre filter paper,
- MeSH
- biosenzitivní techniky metody MeSH
- cholinesterasové inhibitory chemie MeSH
- nanovlákna chemie MeSH
- sklo chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cholinesterasové inhibitory MeSH
Kinetics of hydrolysis of acetylcholine and acetylthiocholine by two types of acetylcholinesterase and butyrylcholinesterase inhibited by 13 new inhibitors (5 carbamates and 8 carbazates--hydrazinium derivatives) was measured in vitro in a batch reactor at 25 degrees C, pH 8, ionic strength 0.11 M and enzyme activity 3.5 U by four nondependent analytical methods. Sevin, rivastigmin (Exelon) and galantamin (Reminyl) served as comparative inhibiting standards. Kinetics of hydrolyses inhibited by all studied carbamates, sevin, carbazates (with exceptions) and rivastigmin (with exceptions) can be simulated by the competitive inhibition model with irreversible reaction between enzyme and inhibitor. Galantamin does not fulfil this model. In positive simulations, the value of inhibition (carbamoylation) rate constant k3 was calculated, describing the reaction velocity between the given enzyme and inhibitor. Physiologically important hydrolyses of acetylcholine catalyzed by acetylcholinesterase from electric eel or bovine erythrocytes and butyrylcholinesterase from horse plasma can be most quickly inhibited by carbamoylation of the mentioned enzymes by the 3-N,N-diethylaminophenyl-N'-(1-alkyl) carbamates 4 and 5. Probably this is due to a long enough hydrocarbon aliphatic substituent (hexyl and octyl) on the amidic nitrogen atom. The tested carbazates failed as inhibitors of cholinesterases. The regeneration ability of the inhibited enzymes was not measured.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- butyrylcholinesterasa metabolismus MeSH
- cholinesterasové inhibitory chemie farmakologie MeSH
- galantamin farmakologie MeSH
- karbaryl farmakologie MeSH
- kinetika MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- acetylcholinesterasa MeSH
- butyrylcholinesterasa MeSH
- cholinesterasové inhibitory MeSH
- galantamin MeSH
- karbaryl MeSH
Series of twenty-five benzyl (2S)-2-(arylcarbamoyl)pyrrolidine-1-carboxylates was prepared and completely characterized. All the compounds were tested for their in vitro ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and the selectivity of compounds to individual cholinesterases was determined. Screening of the cytotoxicity of all the compounds was performed using a human monocytic leukaemia THP-1 cell line, and the compounds demonstrated insignificant toxicity. All the compounds showed rather moderate inhibitory effect against AChE; benzyl (2S)-2-[(2-chlorophenyl)carbamoyl]pyrrolidine-1-carboxylate (IC50 = 46.35 μM) was the most potent agent. On the other hand, benzyl (2S)-2-[(4-bromophenyl)-] and benzyl (2S)-2-[(2-bromophenyl)carbamoyl]pyrrolidine-1-carboxylates expressed anti-BChE activity (IC50 = 28.21 and 27.38 μM, respectively) comparable with that of rivastigmine. The ortho-brominated compound as well as benzyl (2S)-2-[(2-hydroxyphenyl)carbamoyl]pyrrolidine-1-carboxylate demonstrated greater selectivity to BChE. The in silico characterization of the structure-inhibitory potency for the set of proline-based carbamates considering electronic, steric and lipophilic properties was provided using comparative molecular surface analysis (CoMSA) and principal component analysis (PCA). Moreover, the systematic space inspection with splitting data into the training/test subset was performed to monitor the statistical estimators performance in the effort to map the probability-guided pharmacophore pattern. The comprehensive screening of the AChE/BChE profile revealed potentially relevant structural and physicochemical features that might be essential for mapping of the carbamates inhibition efficiency indicating qualitative variations exerted on the reaction site by the substituent in the 3'-/4'-position of the phenyl ring. In addition, the investigation was completed by a molecular docking study of recombinant human AChE.
- Klíčová slova
- CoMSA, IVE-PLS, carbamates, in vitro cholinesterase inhibition, in vitro cytotoxicity assay, molecular docking study, proline,
- MeSH
- acetylcholinesterasa chemie MeSH
- butyrylcholinesterasa chemie MeSH
- cholinesterasové inhibitory chemická syntéza chemie farmakologie MeSH
- karbamáty chemická syntéza chemie farmakologie MeSH
- katalytická doména MeSH
- molekulární konformace MeSH
- prolin * chemie MeSH
- simulace molekulového dockingu MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetylcholinesterasa MeSH
- butyrylcholinesterasa MeSH
- cholinesterasové inhibitory MeSH
- karbamáty MeSH
- prolin * MeSH
- Klíčová slova
- CHOLINESTERASE/antagonists *, LIVER/chemistry *,
- MeSH
- cholinesterasové inhibitory * MeSH
- játra chemie MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cholinesterasové inhibitory * MeSH
A new and simple analytical method is described for the determination of the IC50 values of the inhibitors of the hydrolysis of acetylcholine (ACh) or acetylthiocholine (ATCh) by cholinesterases. The method is based on monitoring the time course of the pH value during the uninhibited and inhibited reaction. It requires only a pH meter with a suitable pH measuring cell and a small thermostated stirred batch reactor. The method has been validated for twelve different types of cholinesterase inhibitors. The determined IC50 values are comparable to those obtained by independent, more complicated, and expensive methods (Ellman's and pH-stat).
OBJECTIVES: The aim of the study was to use methods of pharmaceutical technology, and prepare carriers in the form of pellets suitable as a filling of detection tubes for enzymatic detection of cholinesterase inhibitors. The enzymatic detection was based on enzymatic hydrolysis of acetylthiocholine iodide and the subsequent colour reaction of its hydrolysis product with Ellman's reagent. The suitable carriers should be in the form of white, regular and sufficiently mechanically resistant particles of about 1 mm allowing it to capture the enzyme during the impregnation process and ensuring its high activity for enzymatic detection. METHODS: Carriers consisting of microcrystalline cellulose, lactose, povidone, and sodium carboxymethyl cellulose were prepared using extrusion-spheronization method under three different drying conditions in either a hot air oven or a microwave oven. Subsequently, the carriers were impregnated with acetylcholinesterase and their size, shape, mechanical resistance, bulk, tapped and pycnometric density, Hausner ratio, intraparticular and total tapped porosity, and activity were measured and recorded. RESULTS: In this procedure, carriers with different physical parameters and different acetylcholinesterase activity were evaluated. It was found that higher acetylcholinesterase activity was associated not only with a higher intraparticular porosity but also with more regular particles characterized by high sphericity and low total tapped porosity. CONCLUSION: This unique finding is important for the preparation of detection tubes based on enzymatic detection which is still irreplaceable especially in the field of detection and analysis of super-toxic cholinesterase inhibitors.
- MeSH
- acetylthiocholin analogy a deriváty metabolismus MeSH
- celulosa analýza MeSH
- cholinesterasové inhibitory metabolismus MeSH
- kyselina dithionitrobenzoová MeSH
- laktosa analýza MeSH
- poréznost MeSH
- povidon analýza MeSH
- sodná sůl karboxymethylcelulosy analýza MeSH
- sulfhydrylová reagencia MeSH
- testování materiálů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- acetylthiocholin MeSH
- acetylthiocholine iodide MeSH Prohlížeč
- celulosa MeSH
- cholinesterasové inhibitory MeSH
- kyselina dithionitrobenzoová MeSH
- laktosa MeSH
- microcrystalline cellulose MeSH Prohlížeč
- povidon MeSH
- sodná sůl karboxymethylcelulosy MeSH
- sulfhydrylová reagencia MeSH
Detection tubes are small devices for the colorimetric enzymatic detection of cholinesterase inhibitors such as sarin, soman, VX nerve agents and substances denoted as Novichok. These detectors contain carriers in the form of pellets with immobilized cholinesterase, substrate and detection reagent. Their advantages are portability, sensitivity and simplicity, enabling fast detection of such compounds from air and water in case of a terrorist attack or war. In general, maintaining the stability of an enzyme for a longer time is very problematic; therefore, its further enhancement is required for safety and financial reasons. In this study, the stability of our patented carriers in the form of pellets with immobilized butyrylcholinesterase containing an increasing amount of the unique sorbent Neusilin® US2 was evaluated. The samples containing Neusilin maintained the stability of the immobilized enzyme for a longer time even at higher temperature and humidity than the currently commercially used carrier without Neusilin, allowing improved detection of nerve agents.
- Klíčová slova
- Cholinesterase, Detection, Enzyme, Nerve agent, Neusilin, Stability,
- MeSH
- biosenzitivní techniky metody MeSH
- butyrylcholinesterasa chemie metabolismus MeSH
- cholinesterasové inhibitory analýza MeSH
- enzymy imobilizované chemie metabolismus MeSH
- kolorimetrie metody MeSH
- nosiče léků chemie MeSH
- silikáty metabolismus MeSH
- sloučeniny hliníku metabolismus MeSH
- sloučeniny hořčíku metabolismus MeSH
- stabilita enzymů účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aluminum magnesium silicate MeSH Prohlížeč
- butyrylcholinesterasa MeSH
- cholinesterasové inhibitory MeSH
- enzymy imobilizované MeSH
- nosiče léků MeSH
- silikáty MeSH
- sloučeniny hliníku MeSH
- sloučeniny hořčíku MeSH
