4-Phenethyl-1-Propargylpiperidine-Derived Dual Inhibitors of Butyrylcholinesterase and Monoamine Oxidase B

The multi-target-directed ligands (MTDLs) strategy is encouraged for the development of novel modulators targeting multiple pathways in the neurodegenerative cascade typical for Alzheimer’s disease (AD). Based on the structure of an in-house irreversible monoamine oxidase B (MAO-B) inhibitor, we aimed to introduce a carbamate moiety on the aromatic ring to impart cholinesterase (ChE) inhibition, and to furnish multifunctional ligands targeting two enzymes that are intricately involved in AD pathobiology. In this study, we synthesized three dual hMAO-B/hBChE inhibitors 13–15, with compound 15 exhibiting balanced, low micromolar inhibition of hMAO-B (IC₅₀ of 4.3 µM) and hBChE (IC₅₀ of 8.5 µM). The docking studies and time-dependent inhibition of hBChE confirmed the initial expectation that the introduced carbamate moiety is responsible for covalent inhibition. Therefore, dual-acting compound 15 represents an excellent starting point for further optimization of balanced MTDLs     

Aging of cholinesterases phosphylated by tabun proceeds through O-dealkylation

Human butyrylcholinesterase (hBChE) hydrolyzes or scavenges a wide range of toxic esters, including heroin, cocaine, carbamate pesticides, organophosphorus pesticides, and nerve agents. Organophosphates (OPs) exert their acute toxicity through inhibition of acetylcholinesterase (AChE) by phosphorylation of the catalytic serine. Phosphylated cholinesterase (ChE) can undergo a spontaneous, time-dependent process called "aging", during which the OP-ChE conjugate is dealkylated. This leads to irreversible inhibition of the enzyme. The inhibition of ChEs by tabun and the subsequent aging reaction are of particular interest, because tabun-ChE conjugates display an extraordinary resistance toward most current oxime reactivators. We investigated the structural basis of oxime resistance for phosphoramidated ChE conjugates by determining the crystal structures of the non-aged and aged forms of hBChE inhibited by tabun, and by updating the refinement of non-aged and aged tabun-inhibited mouse AChE (mAChE). Structures for non-aged and aged tabun-hBChE were refined to 2.3 and 2.1 A, respectively. The refined structures of aged ChE conjugates clearly show that the aging reaction proceeds through O-dealkylation of the P(R) enantiomer of tabun. After dealkylation, the negatively charged oxygen forms a strong salt bridge with protonated His438N epsilon2 that prevents reactivation. Mass spectrometric analysis of the aged tabun-inhibited hBChE showed that both the dimethylamine and ethoxy side chains were missing from the phosphorus. Loss of the ethoxy is consistent with the crystallography results. Loss of the dimethylamine is consistent with acid-catalyzed deamidation during the preparation of the aged adduct for mass spectrometry. The reported 3D data will help in the design of new oximes capable of reactivating tabun-ChE conjugates.     

Outlining In Vitro and In Silico Cholinesterase Inhibitory Activity of Twenty-Four Natural Products of Various Chemical Classes: Smilagenin,Kokusaginine, and Methyl Rosmarinate as Emboldening Inhibitors

Cholinesterase (ChE) inhibition is an important treatment strategy for Alzheimer’s disease (AD) as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are involved in the pathology of AD. In the current work, ChE inhibitory potential of twenty-four natural products from different chemical classes (i.e., diosgenin, hecogenin, rockogenin, smilagenin, tigogenin, astrasieversianins II and X, astragalosides I, IV, and VI, cyclocanthosides E and G, macrophyllosaponins A-D, kokusaginin, lamiide, forsythoside B, verbascoside, alyssonoside, ipolamide, methyl rosmarinate, and luteolin-7-O-glucuronide) was examined using ELISA microtiter assay. Among them, only smilagenin and kokusaginine displayed inhibitory action against AChE (IC₅₀ = 43.29 ± 1.38 and 70.24 ± 2.87 µg/mL, respectively). BChE was inhibited by only methyl rosmarinate and kokusaginine (IC₅₀ = 41.46 ± 2.83 and 61.40 ± 3.67 µg/mL, respectively). IC₅₀ values for galantamine as the reference drug were 1.33 ± 0.11 µg/mL for AChE and 52.31 ± 3.04 µg/mL for BChE. Molecular docking experiments showed that the orientation of smilagenin and kokusaginine was mainly driven by the interactions with the peripheral anionic site (PAS) comprising residues of hAChE, while kokusaginine and methyl rosmarinate were able to access deeper into the active gorge in hBChE. Our data indicate that similagenin, kokusaginine, and methyl rosmarinate could be hit compounds for designing novel anti-Alzheimer agents.     

Assessing the Role of a Malonamide Linker in the Design of Potent Dual Inhibitors of Factor Xa and Cholinesterases

The rational discovery of new peptidomimetic inhibitors of the coagulation factor Xa (fXa) could help set more effective therapeutic options (to prevent atrial fibrillation). In this respect, we explored the conformational impact on the enzyme inhibition potency of the malonamide bridge, compared to the glycinamide one, as a linker connecting the P1 benzamidine anchoring moiety to the P4 aryl group of novel selective fXa inhibitors. We carried out structure–activity relationship (SAR) studies aimed at investigating para- or meta-benzamidine as the P1 basic group as well as diversely decorated aryl moieties as P4 fragments. To this end, twenty-three malonamide derivatives were synthesized and tested as inhibitors of fXa and thrombin (thr); the molecular determinants behind potency and selectivity were also studied by employing molecular docking. The malonamide linker, compared to the glycinamide one, does significantly increase anti-fXa potency and selectivity. The meta-benzamidine (P1) derivatives bearing 2′,4′-difluoro-biphenyl as the P4 moiety proved to be highly potent reversible fXa-selective inhibitors, achieving inhibition constants (K_i) in the low nanomolar range. The most active compounds were also tested against cholinesterase (ChE) isoforms (acetyl- or butyrylcholinesterase, AChE, and BChE), and some of them returned single-digit micromolar inhibition potency against AChE and/or BChE, both being drug targets for symptomatic treatment of mild-to-moderate Alzheimer’s disease. Compounds 19h and 22b were selected as selective fXa inhibitors with potential as multimodal neuroprotective agents.     

Hydrazones of 4-(Trifluoromethyl)benzohydrazide as New Inhibitors of Acetyl- and Butyrylcholinesterase

Based on the broad spectrum of biological activity of hydrazide–hydrazones, trifluoromethyl compounds, and clinical usage of cholinesterase inhibitors, we investigated hydrazones obtained from 4-(trifluoromethyl)benzohydrazide and various benzaldehydes or aliphatic ketones as potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). They were evaluated using Ellman’s spectrophotometric method. The hydrazide–hydrazones produced a dual inhibition of both cholinesterase enzymes with IC₅₀ values of 46.8–137.7 µM and 19.1–881.1 µM for AChE and BuChE, respectively. The majority of the compounds were stronger inhibitors of AChE; four of them (2-bromobenzaldehyde, 3-(trifluoromethyl)benzaldehyde, cyclohexanone, and camphor-based 2o, 2p, 3c, and 3d, respectively) produced a balanced inhibition of the enzymes and only 2-chloro/trifluoromethyl benzylidene derivatives 2d and 2q were found to be more potent inhibitors of BuChE. 4-(Trifluoromethyl)-N’-[4-(trifluoromethyl)benzylidene]benzohydrazide 2l produced the strongest inhibition of AChE via mixed-type inhibition determined experimentally. Structure–activity relationships were identified. The compounds fit physicochemical space for targeting central nervous systems with no apparent cytotoxicity for eukaryotic cell line together. The study provides new insights into this CF₃-hydrazide–hydrazone scaffold.     

Real-time assessment of cigarette smoke particle deposition in vitro

ABSTRACT: BACKGROUND: Recently there has been a rapid increase in approaches to assess the effects of cigarette smoke in vitro. Despite a range of gravimetric and chemical methods, there is a requirement to identify simpler and more reliable methods to quantify in vitro whole smoke dose, to support extrapolation and comparisons to human/in vivo dose. We have previously characterised an in vitro exposure system using a Borgwaldt RM20S smoking machine and a chamber exposing cellular cultures to whole smoke at the air-liquid interface. In this study we demonstrate the utility of a quartz crystal microbalance (QCM), using this exposure system, to assess real-time cigarette smoke particulate deposition during a 30 minute smoke exposure. Smoke was generated at various dilutions (1:5--1:400, smoke:air) using two cigarette products, 3R4F Kentucky reference and 1 mg commercially available cigarettes. The QCM, integrated into the chamber, assessed particulate deposition and data generated was compared to traditional chemical spectrofluorometric analysis. RESULTS: The QCM chamber was able to detect mass differences between the different products within the nanogram range. 3R4F reference cigarette smoke deposition ranged from 25.75 [PLUS-MINUS SIGN]2.30 mug/cm2 (1:5) to 0.22 [PLUS-MINUS SIGN]0.03 mug/cm2 (1:400). 1 mg cigarette smoke deposition was less and ranged from 1.42 [PLUS-MINUS SIGN]0.26 mug/cm2 (1:5), to 0.13 [PLUS-MINUS SIGN]0.02 mug/cm2 (1:100). Spectrofluorometric analysis demonstrated statistically significant correlation of particulate deposition with the QCM (p < 0.05), and regression R2 value were 97.4 %. The fitted equation for the linear model which describes the relationship is: QCM = [MINUS SIGN]0.6796 + 0.9744 chemical spectrofluorescence CONCLUSIONS: We suggest the QCM is a reliable, effective and simple tool that can be used to quantify smoke particulate deposition in real-time, in vitro and can be used to quantify other aerosols delivered to our chamber for assessment.     

First-in-Class Isonipecotamide-Based Thrombin and Cholinesterase Dual Inhibitors with Potential for Alzheimer Disease

Recently, the direct thrombin (thr) inhibitor dabigatran has proven to be beneficial in animal models of Alzheimer’s disease (AD). Aiming at discovering novel multimodal agents addressing thr and AD-related targets, a selection of previously and newly synthesized potent thr and factor Xa (fXa) inhibitors were virtually screened by the Multi-fingerprint Similarity Searching aLgorithm (MuSSeL) web server. The N-phenyl-1-(pyridin-4-yl)piperidine-4-carboxamide derivative 1, which has already been experimentally shown to inhibit thr with a K_i value of 6 nM, has been flagged by a new, upcoming release of MuSSeL as a binder of cholinesterase (ChE) isoforms (acetyl- and butyrylcholinesterase, AChE and BChE), as well as thr, fXa, and other enzymes and receptors. Interestingly, the inhibition potency of 1 was predicted by the MuSSeL platform to fall within the low-to-submicromolar range and this was confirmed by experimental K_i values, which were found equal to 0.058 and 6.95 μM for eeAChE and eqBChE, respectively. Thirty analogs of 1 were then assayed as inhibitors of thr, fXa, AChE, and BChE to increase our knowledge of their structure-activity relationships, while the molecular determinants responsible for the multiple activities towards the target enzymes were rationally investigated by molecular cross-docking screening.     

A New Bienzymatic Biosensor Based on Butyrylcholine Esterase‐Sulfhydryl Oxidase Enzymes

Abstract

Over the last decades, cholinesterase (ChE) biosensors have emerged as a sensitive and rapid technique for toxicity analysis in environmental monitoring, food, and quality control. These systems have the potential to complement or replace the classical analytical methods by simplifying or eliminating sample preparation protocols and making field testing easier and faster with significant decrease in costs per analysis. In this study, a new bienzymatic biosensor based on butyrylcholinesterase (BuChE) and sulfhydryl oxidase (SOX) enzymes was developed. This system makes use of the biocatalyzed hydrolysis of butyrylthiocholine to butyric acid and thiocholine that acts as a SOX substrate. Measurements were performed by following of the consumed oxygen level related to butyrylthiocholine concentration in the enzymatic reactions. Bienzymatic system was characterized and applied for detection of donepezil which is a reversible inhibitor of cholinesterase and belongs to a new class of cholinesterase inhibitors having an N‐benzylpiperidine and an indanone moiety, by following of the decrease in biosensor response as a result of competitive inhibition.     

Design and synthesis of 2,4-dioxochroman-pyridinium-phenylacetamide derivatives as new anti-Alzheimer agents: in vitro and in silico studies

2,4-Dioxochroman-pyridinium-phenylacetamide derivatives 7a–n were synthesized and evaluated for their in vitro cholinesterase (ChE) inhibitory activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Obtained results demonstrated that, among the synthesized compounds, two compounds, 7j and 7k , were more potent than the standard drug donepezil against BuChE and did not show cytotoxicity and carcinogenicity. Furthermore, through molecular modeling and molecular dynamic studies. we showed that these compounds can be located deep in the gorge cavity of BuChE and that they interacted with catalytic residues, acyl, and cholin-binding pockets of this enzyme. Support information     

Synthesis, characterization, X-ray crystallography, acetyl cholinesterase inhibition and antioxidant activities of some novel ketone derivatives of gallic hydrazide-derived Schiff bases

Alzheimer's disease (AD) is the most common form of dementia among older people and the pathogenesis of this disease is associated with oxidative stress. Acetylcholinesterase inhibitors with antioxidant activities are considered potential treatments for AD. Some novel ketone derivatives of gallic hydrazide-derived Schiff bases were synthesized and examined for their antioxidant activities and in vitro and in silico acetyl cholinesterase inhibition. The compounds were characterized using spectroscopy and X-ray crystallography. The ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays revealed that all the compounds have strong antioxidant activities. N-(1-(5-bromo-2-hydroxyphenyl)-ethylidene)-3,4,5-trihydroxybenzohydrazide (2) was the most potent inhibitor of human acetyl cholinesterase, giving an inhibition rate of 77% at 100 μM. Molecular docking simulation of the ligand-enzyme complex suggested that the ligand may be positioned in the enzyme's active-site gorge, interacting with residues in the peripheral anionic subsite (PAS) and acyl binding pocket (ABP). The current work warrants further preclinical studies to assess the potential for these novel compounds for the treatment of AD.     

Immobilized butyrylcholinesterase in the characterization of new inhibitors that could ease Alzheimer's disease

Focus of this work was the development and characterization of a new immobilized enzyme reactor (IMER) containing human recombinant butyrylcholinesterase (rBChE) for the on-line kinetic characterization of specific, pseudo-irreversible and brain-targeted BChE inhibitors as potential drug candidates for Alzheimer's disease (AD). Specifically, a rBChE-IMER containing 0.99 U of covalently bound target enzyme was purposely developed and inserted into a HPLC system connected to a UV-vis detector. Selected reversible cholinesterase inhibitors, (-)-phenserine and (-)-cymserine analogues, were then kinetically characterized by rBChE-IMER, and by classical in solution assays and their carbamoylation and decarbamoylation constants were determined. The results support the elucidation of the potency, inhibition duration, mode of action and specific structure/activity relations of these agents and allow cross-validation of the two assay techniques.     

Bio-Guided Fractionation of Stem Bark Extracts from Phyllanthus muellarianus: Identification of Phytocomponents with Anti-Cholinesterase Activity

A combination of flash chromatography, solid phase extraction, high-performance liquid chromatography, and in vitro bioassays was used to isolate phytocomponents endowed with anticholinesterase activity in extract from Phyllanthus muellarianus. Phytocomponents responsible for the anti-cholinesterase activity of subfractions PMF1 and PMF4 were identified and re-assayed to confirm their activity. Magnoflorine was identified as an active phytocomponent from PMF1 while nitidine was isolated from PMF4. Magnoflorine was shown to be a selective inhibitor of human butyrylcholinesterase—hBChE (IC₅₀ = 131 ± 9 μM and IC₅₀ = 1120 ± 83 μM, for hBuChE and human acetylcholinesterase—hAChE, respectively), while nitidine showed comparable inhibitory potencies against both enzymes (IC₅₀ = 6.68 ± 0.13 μM and IC₅₀ = 5.31 ± 0.50 μM, for hBChE and hAChE, respectively). When compared with the commercial anti-Alzheimer drug galanthamine, nitidine was as potent as galanthamine against hAChE and one order of magnitude more potent against hBuChE. Furthermore, nitidine also showed significant, although weak, antiaggregating activity towards amyloid-β self-aggregation.     

GC/MS analysis of three Amaryllidaceae species and their cholinesterase activity

Alzheimer's disease (AD), characterized by the death of nerve cells in the cerebral cortex, is the most common subtype of dementia. Despite the exponential growth in the number of AD patients, acetylcholinesterase (AChE) inhibitors are currently used to treat AD. Plants of the Amaryllidaceae family are known to synthesize a particular type of bioactive compounds, named Amaryllidaceae alkaloids, which have shown AChE inhibitory activity. Alkaloid extracts of three species of Amaryllidaceae were studied with respect to their acetylcholinesterase and butyrylcholinesterase inhibitory activity and alkaloid patterns. Eleven alkaloids were identified by GC/MS. Significant cholinesterase inhibitory activity was demonstrated by the alkaloid extract of N. undulata (IC50,(HuAChE) = 14.3 +/- 1.2 microg/mL; IC50,(HuBuChE) = 33.9 +/- 1.9 microg/mL).     

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.     

Amperometric biosensors based on nafion coated screen-printed electrodes for the determination of cholinesterase inhibitors

Screen-printed electrodes coated with the nafion layer have been investigated for cholinesterase biosensor design. The butyrylcholinesterase (ChE) from horse serum was immobilised onto the nafion layer by cross-linking with glutaraldehyde vapours. The biosensors obtained showed better long-term stability and lower working potential in comparison to those obtained with no nafion coating. The sensitivity of a biosensor toward organophosphate pesticides is not affected by the nafion coating. The detection limits were found to be 3.5x10(-7) M for trichlorfon and 1.5x10(-7) M for coumaphos.     

4-N-苯胺基喹啉衍生物的合成及胆碱酯酶抑制活性

设计合成了一系列4-N-苯胺基喹啉类衍生物,采用Ellman法测定了目标化合物对乙酰胆碱酯酶(AChE)和丁酰胆碱酯酶(BChE)的抑制活性.结果表明,当喹啉环上连有伸长的吡啶季铵盐片段时,可显著提高目标化合物的胆碱酯酶抑制作用.化合物16对AChE和BChE具有明显的双重抑制作用,其IC_(50)值分别为0.92和14.20μmol/L,抑制效果强于阳性对照药加兰他敏.     

Design of active-site-directed fluorescent probes and their reactions with biopolymers

Fluorescent probes which are active-site-directed, reversible, competitive inhibitors of serum cholinesterase (ChE) enzymes have been designed and synthesized. Reversible inhibitors of enzyme active sites have a unique importance when they act as fluorescent probes, allowing fluorescence spectroscopic detection of conformation changes and activesite dynamics. 5-Dimethylamino-naphthalene-1-sulfonamido-N,N-dimethyl-n-propyl-amine and its aliphatic quaternary derivative are fluorescent probes for serum cholinesterase. The quaternary probe forms complexes with acetylcholinesterase (AChE). The dissociation constants K_d for the two probes with serum ChE are 6.0 × 10^?7 and 6.5 × 10^?7M. The inhibition constants K_i are 3.1 × 10^?6 and 6.3 × 10^?6M from the slopes of Lineweaver-Burk plots. The Michelis constant K_m for the enzyme was 8.8 × 10^?4M.     

Synthesis, characterization, acetylcholinesterase inhibition, molecular modeling and antioxidant activities of some novel Schiff bases derived from 1-(2-ketoiminoethyl)piperazines

Some novel Schiff bases derived from 1-(2-ketoiminoethyl)piperazines were synthesized and characterized by mass spectroscopy, FTIR, UV-Visible, 1H and 13C-NMR. The compounds were tested for inhibitory activities on human acetylcholinesterase (hAChE), antioxidant activities, acute oral toxicity and further studied by molecular modeling techniques. The study identified the compound (DHP) to have the highest activity among the series in hAChE inhibition and DPPH assay while the compound LP revealed the highest activity in the FRAP assay. The hAChE inhibitory activity of DHP is comparable with that of propidium, a known AChE inhibitor. This high activity of DHP was checked by molecular modeling which showed that DHP could not be considered as a bivalent ligand due to its incapability to occupy the esteratic site (ES) region of the 3D crystal structure of hAChE. The antioxidant study unveiled varying results in 1,1-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. This indicates mechanistic variations of the compounds in the two assays. The potential therapeutic applications and safety of these compounds were suggested for use as human acetylcholinesterase inhibitors and antioxidants.     

Discovery of potential inhibitor against human acetylcholinesterase: a molecular docking and molecular dynamics investigation

Alzheimer’s disease (AD) is a progressive neurodegenerative disease of central nervous system among elderly people. Human acetylcholinesterase (hAChE), an important enzyme in neuronal signaling, is responsible for the degradation of acetylcholine which in turn prevents the post synaptic signal transmissions. hAChE has been an attractive target of drug discovery for the search of therapeutics against AD. In the recent past hAChE has become hot target for the investigation of new potential therapeutics. We performed virtual screening of entire database against hAChE. Further, the extra precision molecular docking was carried out to refine the docking results and the best complex was passed for molecular dynamics simulations in order of understanding the hAChE dynamics and its behavior in complex with the ligand which corroborate the outcomes of virtual screening. This also provides binding free energy data that establishes the ligands efficiency for inhibiting hAChE. The computational findings discussed in this paper provide initial information of inhibitory effects of ligand, (drugbank entry DB00983), over hAChE.     

Non-Alkaloid Cholinesterase Inhibitory Compounds from Natural Sources

Alzheimer’s disease (AD) is a severe neurodegenerative disorder of different brain regions accompanied by distresses and affecting more than 25 million people in the world. This progressive brain deterioration affects the central nervous system and has negative impacts on a patient’s daily activities such as memory impairment. The most important challenge concerning AD is the development of new drugs for long-term treatment or prevention, with lesser side effects and greater efficiency as cholinesterases inhibitors and the ability to remove amyloid-beta(Aβ) deposits and other related AD neuropathologies. Natural sources provide promising alternatives to synthetic cholinesterase inhibitors and many have been reported for alkaloids while neglecting other classes with potential cholinesterase inhibition. This review summarizes information about the therapeutic potential of small natural molecules from medicinal herbs, belonging to terpenoids, coumarins, and phenolic compounds, and others, which have gained special attention due to their specific modes of action and their advantages of low toxicity and high efficiency in the treatment of AD. Some show superior drug-like features in comparison to synthetic cholinesterase inhibitors. We expect that the listed phytoconstituents in this review will serve as promising tools and chemical scaffolds for the discovery of new potent therapeutic leads for the amelioration and treatment of Alzheimer’s disease.