Molecular modelling and enzymatic studies of acetylcholinesterase and butyrylcholinesterase recognition with paraquat and related compounds

The potent herbicide paraquat and three other analogues MPP+, MPDP+ and MPTP have a known toxicological profile linked to the ability to damage dopaminergic neurons. Other biological effects were recently addressed to this class of compounds, including the ability to interact with enzymatic targets involved in the Central Nervous System, such as the acetylcholinesterase (AChE) and the butyrylcholinesterase (BuChE). A combined molecular modelling and enzymatic study focusing onto their interaction against the AChE and BuChE is reported. The former study was performed by docking techniques using target known co-crystallographic models. The latter study was carried out by the widely adopted Ellman's method. In both studies the anti-Alzheimer FDA approved drug tacrine was used as reference inhibitor. Our results indicate that paraquat, MPTP, MPDP+ and MPP+ recognize both enzymatic cleft in a similar fashion compared to the reference inhibitor. A structure-activity correlation was found with the net charge of the ligands, indicating a major role of the electrostatic term in the recognition and inhibition of these compounds. Our data completed their enzymatic profile, added new information on the molecular mechanisms underlying their neurotoxicity useful for the rational design of new cholinesterase inhibitors.     

Synthesis and molecular modeling study of Cu(Ⅱ) complexes derived from 2-(diphenylmethylene)hydrazinecarbothioamide derivatives with cholinesterase inhibitory activities

Thiosemicarbazones of 2-amino-5-chlorobenzophenone and 3-aminobenzophenone（L¹-L⁴） have been synthesized and their Cu（Ⅱ） complexes（1-4） were afforded via coordination with cupric chloride.All these compounds were characterized by UV-vis and IR spectroscopy together with CHN elemental analysis.NMR spectroscopy was also applied to characterize the ligands.In vitro chohnesterase inhibitory assays for the complexes（1-4） showed IC₅₀ values less than 10μmol/L,with complex 1 exhibiting the most activity,IC₅₀=2.15μmol/L and 2.16μmol/L for AChE and BuChE,respectively. Molecular modeling simulation revealed the binding interaction template for complex 1 with the AChE and BuChE receptors.In DPPH assay,the complexes also showed more in vitro antioxidant activities in comparison to their parent ligands.     

Tacrines as Therapeutic Agents for Alzheimer's Disease. V. Recent Developments

Herein we have reviewed our recent developments for the identification of new tacrine analogues for Alzheimer's disease (AD) therapy. Tacrine, the first cholinesterase inhibitor approved for AD treatment, did not stop the progression of AD, producing only some cognitive improvements, but exhibited secondary effects mainly due to its hepatotoxicity. Thus, the drug was withdrawn from the clinics administration. Since then, many publications have described non‐hepatotoxic tacrines, and in addition, important efforts have been made to design multitarget tacrines by combining their cholinesterase inhibition profile with the modulation of other biological targets involved in AD.     

Cholinesterase sensors based on screen-printed electrodes for detection of organophosphorus and carbamic pesticides

Cholinesterase sensors based on screen-printed electrodes modified with polyaniline, 7,7,8,8-tetracyanoquinodimethane (TCNQ), and Prussian blue have been developed and tested for detection of anticholinesterase pesticides in aqueous solution and in spiked grape juice. The influence of enzyme source and detection mode on biosensor performance was explored. It was shown that modification of the electrodes results in significant improvement of their analytical characteristics for pesticide determination. Thus, the slopes of the calibration curves obtained with modified electrodes were increased twofold and the detection limits of the pesticides were reduced by factors of 1.6 to 1.8 in comparison with the use of unmodified transducers. The biosensors developed make it possible to detect down to 2×10^–8 mol L^–1 chloropyrifos-methyl, 5×10^–8 mol L^–1 coumaphos, and 8×10^–9 mol L^–1 carbofuran in aqueous solution and grape juice. The optimal conditions for grape juice pretreatment were determined to diminish interference from the sample matrix.Abbreviations ChE Cholinesterase - TCNQ 7,7,8,8-Tetracyanoquinodimethane - ChO Choline oxidase - AChE Acetylcholinesterase - BChE Butyrylcholinesterase - BSA Bovine serum albumin - 2-PAM 2-Pyridine aldoxime methiodide     

Novel Enzymatic Potentiometric Approaches for Surfactant Analysis

The present study described a novel application of simple potentiometric enzymatic method for analysis of surfactants based on their inhibitory effect on acetylcholinesterase enzyme (AChE). The enzymatic activity was measured through monitoring hydrolysis of acetylcholine (ACh) with a disposable acetylcholine potentiometric sensor. Comprehensive investigations were carried out including the effect of incubation time, cholinesterase enzyme and the working calibration ranges. Based on inhibition of AChE, different cationic, anionic and nonionic surfactants were determined in the concentration range from 0 to 40 μg mL⁻¹ with detection limits reaching 0.07 μg mL⁻¹ depending on the nature of surfactants. The degree of AChE inhibition caused by different tested surfactants were as follows: cetylpyridinium chloride (CPC) > benzyldimethylhexadecyl ammonium chloride (BDHAC) > Hyamine (Hy)>cetyltrimethylammonium bromide (CTAB) > Triton X‐100 (TX‐100) > sodium dodecyl sulphate (SDS). The proposed method was applied for determination of surfactants in pharmaceutical formulation, detergents products and environmental samples with acceptable sensitivity and reproducibility.     

Cholinesterase-based dipstick assay for the detection of organophosphate and carbamate pesticides

A cholinesterase (ChE)-based dipstick-type assay for the class-specific detection of organophosphate (OP) and carbamate (CM) pesticides was developed. The principle of the assay is based on inhibition of the activity of a ChE by these two families of pesticides, which is dependent on the concentration of pesticides. The proposed assay system is composed of a test strip with an acetylcholinesterase (AChE)-coated membrane and an enzyme substrate solution. The assay protocol involves incubation of the enzyme-coated strip in the pesticide-containing sample solution followed by incubation of the sample-treated strip in a chromogenic enzyme substrate solution. The color intensity is estimated by the naked eye or a reflectometer. Of the membranes tested as the enzyme support, Hybond N⁺ was the most suitable. Among the compounds tested as the enzyme substrate, indophenyl acetate was the best. The detectable concentration range of the dipstick assay for the OP and CM pesticides was 10⁻⁶-10² and 10⁻⁶-10⁰ μg mL⁻¹, respectively. The sensitivity of the dipstick assay to the oxidized form of parathion (paraoxon) was higher than to parathion. The strip showed a large matrix effect with pesticide-spiked lettuce samples, whereas it showed a small matrix effect with pesticide-spiked rice samples.     

Planar Ni(II) 1,2-dithiolenes involving bidentate N -donor ligands

A series of planar Ni(II) dithiolenes derived from maleonitriledithiole (mnt), benzene-1,2-dithiole (bdt) and 1-toluene-3,4-dithiole (tdt) with bidentate N,N-ligands (bpy = 2,2′-bipyridine; phen = 1,10-phenanthroline, nphen = 5-nitro-1,10-phenanthroline) of the [Ni(N,N)(dithiol)] type have been synthesized. The compounds have been characterized by elemental analysis, IR and electronic spectroscopies, magnetochemical and conductivity measurements. Single crystal X-ray analysis of [Ni(phen)(bdt)] confirmed a planar geometry of NiN₂S₂. Possible practical applications such as use for vulcanization catalytic agents and for their anticholinesterase activity were evaluated.     

Conductimetric Measurement of Cholinesterase Inhibition by Some Pesticides

Abstract

In this present work, we have applied the conductimetric method to measure cholinesterase inhibition by pesticides.

When using acetylcholine as a substrate, we have demonstrated, that for a given incubation of 40 mn, the initial rate of the enzymatic hydrolysis can be related to pesticide concentration. In a given inhibitor concentration range, the activity/log (concentration) relationship is linear and may be used for pesticide quantitation.     

Dual effect of high electric field in capillary electrophoresis study of the conformational stability of Bungarus fasciatus acetylcholinesterase

The effect of high electric field in capillary zone electrophoresis (CZE) was evaluated for the study of the thermally induced unfolding of Bungarus fasciatus acetylcholinesterase. This monomer enzyme is characterised by two interdependent uncommon structural features, the asymmetrical distribution of charged residues and a relatively low thermal denaturation temperature. Both traits were presumed to interfere in the thermal unfolding of this enzyme as investigated by CZE. This paper analyses the effect of high electric field on the behaviour of the enzyme native state. It is shown that increasing the applied field causes denaturation-like transition of the enzyme at a current power which does not induce excessive Joule heating in the capillary. The susceptibility to electric field of proteins like cholinesterases, with charge distribution anisotropy, large permanent dipole moment and notable molecular flexibility associated with moderate thermal stability, was subsequently discussed.     

Cholinesterase inhibition based determination of pancuronium bromide in biological samples

Pancuronium bromide (PCBr) inhibition effect on enzyme cholinesterase from pooled human serum (Che, EC 3.1.1.8 acylcholine acylhydrolase) was used for development of a spectrophotometric kinetic method for PCBr determination in human serum and urine. Optimal conditions for the basic and inhibitor reactions were established: pH=7.7 and substrate concentration c(benzoylcholine chloride)=1.33 mmol/L. Kinetic parameters were also determined: Michaelis-Menten’s constant K_M=0.40 mmol/L, maximal reaction rate V_max=52.2 μmol/L min, inhibition constant K_i=0,56 μmol/L and IC₅₀=1.31 μmol/L. Linear dependence between the reaction rate and inhibitor concentration exists in PCBr concentration range 8.20–68.25 nmol/L, which corresponds to the real sample concentrations from 0.328 to 2.730 μmol/L. The method detection and quantification limits were 2.01 nmol/L and 6.67 nmol/L, respectively. Precision of the method was tested for three pancuronium concentrations (10.70, 29.35 and 51.25 nmol/L). Relative standard deviation (RSD) was in the range 0.15–7.45%. Accuracy was examined by standard addition method. Influence of the substances usually present in serum and urine on the reaction rate was tested. The developed method was applied for PCBr content determination in serum model samples, urine model samples and in urine taken during surgery. The method has good sensitivity, accuracy, precision and it is suitable for clinical practice.