Inhibitors of human 2,3-oxidosqualene cyclase (OSC) discovered by virtual screening

Five human 2,3-oxidosqualnene cyclase (OSC) inhibitors were discovered using the combination of a virtual screening based on a docking study and an isotope-free assay system. All of these inhibitors were revealed to have activities comparable or superior to that of LDAO, a known OSC inhibitor. The computational study of the newly identified inhibitors suggested that CH/π interactions with F444 and W581 contribute to the binding, and these interactions are candidates for additional structural filters in the next generation of virtual screening.     

Substrate activation in acetylcholinesterase induced by low pH or mutation in the π-cation subsite

Substrate inhibition is considered a defining property of acetylcholinesterase (AChE), whereas substrate activation is characteristic of butyrylcholinesterase (BuChE). To understand the mechanism of substrate inhibition, the pH dependence of acetylthiocholine hydrolysis by AChE was studied between pH 5 and 8. Wild-type human AChE and its mutants Y337G and Y337W, as well as wild-type Bungarus fasciatus AChE and its mutants Y333G, Y333A and Y333W were studied. The pH profile results were unexpected. Instead of substrate inhibition, wild-type AChE and all mutants showed substrate activation at low pH. At high pH, there was substrate inhibition for wild-type AChE and for the mutant with tryptophan in the π-cation subsite, but substrate activation for mutants containing small residues, glycine or alanine. This is particularly apparent in the B. fasciatus AChE. Thus a single amino acid substitution in the π-cation site, from the aromatic tyrosine of B. fasciatus AChE to the alanine of BuChE, caused AChE to behave like BuChE. Excess substrate binds to the peripheral anionic site (PAS) of AChE. The finding that AChE is activated by excess substrate supports the idea that binding of a second substrate molecule to the PAS induces a conformational change that reorganizes the active site.     

Discovery of acetylcholinesterase peripheral anionic site ligands through computational refinement of a directed library

The formation of beta-amyloid plaques in the brain is a key neurodegenerative event in Alzheimer's disease. Small molecules capable of binding to the peripheral anionic site of acetylcholinesterase (AChE) have been shown to inhibit the AChE-induced aggregation of the beta-amyloid peptide. Using the combination of a computational docking model and experimental screening, five compounds that completely blocked the amyloidogenic effect of AChE were rapidly identified from an approximately 200-member library of compounds designed to disrupt protein-protein interactions. Critical to this docking model was the inclusion of two explicit water molecules that are tightly bound to the enzyme. Interestingly, none of the tested compounds inhibited the related enzyme butyrylcholinesterase (BuChE) up to their aqueous solubility limits. These compounds are among the most potent inhibitors of amyloid beta-peptide aggregation and are equivalent only to propidium, a well-characterized AChE peripheral anionic site binder and aggregation inhibitor.     

Butyrylcholinesterase inhibitory activity of testosterone and some of its metabolites

Testosterone and ten of its metabolites were examined as inhibitors of butyrylcholinesterase. A significant enzyme inhibition activity (IC₅₀ = 1.55 μM) was observed for androst-4-en-3,7-dione. The kinetic parameters of butyrylcholinesterase inhibition were determined and molecular docking was carried out in order to develop a better understanding of the inhibitor-enzyme interactions. The results showed that the inhibition was non-competitive, stabilized mainly by hydrogen bonds and hydrophobic interactions between the inhibitor and butyrylcholinesterase.     

Horse serum butyrylcholinesterase kinetics: a molecular mechanism based on inhibition studies with dansylaminoethyltrimethylammonium

The kinetics of the hydrolysis of butyrylthiocholine by horse serum butyrylcholinesterase (acylcholine acylhydrolase; BuChE; EC 3.1.1.8) exhibit an activation phenomenon at high substrate concentrations. At least two mechanistic models can account for the enzyme kinetics: one assumes the binding of an additional substrate molecule on the acyl-enzyme intermediate, and the other hypothesizes the existence of a peripheral regulatory site for the substrate. (1-Dimethylaminonaphthalene-5-sulfonamidoethyl)-trimethylammonium perchlorate, a potent reversible inhibitor, appears to affect BuChE activity by binding to a peripheral site. The inhibition is of the mixed type at low substrate concentrations and of the competitive type at high substrate concentrations. This is consistent with a peripheral site for the binding of the substrate responsible for the activation phenomenon.     

Highly potent and selective aryl-1,2,3-triazolyl benzylpiperidine inhibitors toward butyrylcholinesterase in Alzheimer's disease

Acetylcholinesterase (AChE) is the key enzyme targeted in Alzheimer's disease (AD) therapy, nevertheless butyrylcholinesterase (BuChE) has been drawing attention due to its role in the disease progression. Thus, we aimed to synthesize novel cholinesterases inhibitors considering structural differences in their peripheral site, exploiting a moiety replacement approach based on the potent and selective hAChE drug donepezil. Hence, two small series of N-benzylpiperidine based compounds have successfully been synthesized as novel potent and selective hBuChE inhibitors. The most promising compounds (9 and 11) were not cytotoxic and their kinetic study accounted for dual binding site mode of interaction, which is in agreement with further docking and molecular dynamics studies. Therefore, this study demonstrates how our strategy enabled the discovery of novel promising and privileged structures. Remarkably, compound 11 proved to be one of the most potent (0.17?nM) and selective (>58,000-fold) hBuChE inhibitor ever reported.     

New cholinesterase inhibitors for Alzheimer’s disease: Structure Activity Studies (SARs) and molecular docking of isoquinolone and azepanone derivatives

A library of isoquinolinone and azepanone derivatives were screened for both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity. The strategy adopted included (a) in vitro biological assays, against eel AChE (EeAChE) and equine serum BuChE (EqBuChE) in order to determine the compounds IC₅₀ and their dose-response activity, consolidated by (b) molecular docking studies to evaluate the docking poses and interatomic interactions in the case of the hit compounds, validated by STD-NMR studies. Compound (1f) was identified as one of these hits with an IC₅₀ of 89.5 μM for EeAChE and 153.8 μM for EqBuChE, (2a) was identified as a second hit with an IC₅₀ of 108.4 μM (EeAChE) and 277.8 μM (EqBuChE). In order to gain insights into the binding mode and principle active site interactions of these molecules, (R)-(1f) along with 3 other analogues (also as the R-enantiomer) were docked into both RhAChE and hBuChE models. Galantamine was used as the benchmark. The docking study was validated by performing an STD-NMR study of (1f) with EeAChE using galantamine as the benchmark.     

Synthesis and characterization of CAPE derivatives as xanthine oxidase inhibitors with radical scavenging properties

Inhibitors of the enzyme xanthine oxidase (XO) with radical scavenging properties hold promise as novel agents against reperfusion injuries after ischemic events. By suppressing the formation of damaging reactive oxygen species (ROS) by XO or scavenging ROS from other sources, these compounds may prevent a buildup of ROS in the aftermath of a heart attack or stroke. To combine these two properties in a single molecule, we synthesized and characterized the non-purine XO inhibitor caffeic acid phenethylester (CAPE) and 19 derivatives using a convenient microwave-assisted Knoevenagel condensation protocol. Varying systematically the number and positions of the hydroxyl groups at the two phenyl rings, we derived structure-activity relationships based on experimentally determined XO inhibition data. Molecular docking suggested that critical enzyme/inhibitor interactions involved π-π interactions between the phenolic inhibitor ring and Tyr914, hydrogen bonds between inhibitor hydroxyl groups and Glu802, and hydrophobic interactions between the CAPE phenyl ring and non-polar residues located at the entrance of the binding site. To effectively scavenge the stable radical DPPH, two hydroxyl groups in 1,2- or 1,4-position at the phenyl ring were required. Among all compounds tested, E-phenyl 3-(3,4-dihydroxyphenyl)acrylate, a CAPE analog without the ethyl tether, showed the most promising properties.     

Generation of a monoclonal antibody that has reduced binding activity to VX-inactivated butyrylcholinesterase (BuChE) compared to BuChE by phage display

Organophosphate (OP) nerve agents are known as the most toxic chemical warfare agents that act by inhibiting the enzyme acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Because BuChE is present at a level of about 3,900 times higher than AChE in plasma, most OP agents first react with BuChE in plasma, suggesting that OP-inactivated BuChE (OP-iBuChE) may act as a biomarker of OP exposure. In this study, we generated an anti-BuChE monoclonal antibody (mAb) that has reduced binding activity to VX-inactivated BuChE compared to native BuChE by phage display. We performed subtractive biopanning of a synthetic human Fab library against native BuChE and soman-iBuChE or VX-iBuChE. As the results, we isolated four Fab clones that showed differential binding activities to VX-iBuChE and native BuChE in ELISAs. To confirm the antigen-binding specificity of the selected clones, the Fabs were converted to IgG1s, and the IgG antibodies were expressed in HEK293F cells and purified. One of them (A2) showed approximately 30% reduced binding activity to VX-iBuChE compared to BuChE in a dose-dependent manner, whereas the other three antibodies showed almost the same binding activities to VX-iBuChE and BuChE. In addition, the A2 antibody did not show reduced binding activity to sarin-iBuChE or soman-iBuChE compared to native BuChE. The results indicate that A2 antibody shows reduced binding activity only to VX-iBuChE. A2 antibody may be applied to specific diagnosis of VX exposure.     

Butyrylcholinesterase in the visual ganglia of the squid todarodes sagittatus I. (cephalopoda). isolation,molecular forms,interaction with substrates and inhibitors

1. Soluble butyrylcholinesterase (BuChE) was isolated from the visual ganglia of the squid Todarodes sagittatus L. Gel-chromatography on Sephadex G-200 columns resulted in its separation into three molecular forms.2. The major component with a molecular mass of 180kDa was used for kinetic study.3. The substrate analysis revealed squid enzyme to be BuChE of unusual type.4. Unlike typical BuChE (EC 3.1.1.8), squid enzyme splits acetyl-β-methylcholine (AMCh) with a relatively high rate, alongside with common BuChE substrates—butyrylcholine (BCh), propionylcholine (PCh), acetylcholine (ACh), butyrylthiocholine (BTCh) and acetylthiocholine (ATCh), the enzymic hydrolysis being suppressed by excess of all these substrates.5. Among them, the highest values of k_{cat and}k_cat/K_m were found for BCh and BTCh. Maximal activity of the enzyme was noticed at low BCh and BTCh concentrations (1–2 mM).6. Tetraalkylammonium ions exhibit a mixed type of inhibition and suppress the substrate inhibition of squid BuChE.7. Among organophosphorus inhibitors (OPI), the methylthiophosphonates are most potent for squid BuChE, and for some phosphates, selective OPI of typical BuChE, are potent as well.8. By the pattern of selectivity to OPI, squid enzyme differs from both typical BuChE of horse serum and acetylcholinesterase (EC 3.1.1.7) from bovine erythrocytes.9. Some details of the active center structure of squid BuChE compared to that of typical enzymes are discussed.     

Discovery of isoalloxazine derivatives as a new class of potential anti-Alzheimer agents and their synthesis

This article describes discovery of a novel and new class of cholinesterase inhibitors as potential therapeutics for Alzheimer’s disease. A series of novel isoalloxazine derivatives were synthesized and biologically evaluated for their potential inhibitory outcome for both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These compounds exhibited high activity against both the enzymes AChE as well as BuChE. Of the synthesized compounds, the most potent isoalloxazine derivatives (7m and 7q) showed IC₅₀ values of 4.72 μM and 5.22 μM respectively against AChE; and, 6.98 μM and 5.29 μM respectively against BuChE. These two compounds were further evaluated for their anti-aggregatory activity for β-amyloid (Aβ) in presence and absence of AChE by performing Thioflavin-T (ThT) assay and Congo red (CR) binding assay. In order to evaluate cytotoxic profile of these two potential compounds, cell viability assay of SH-SY5Y human neuroblastoma cells was performed. Further, to understand the binding behavior of these two compounds with AChE and BuChE enzymes, docking studies have been reported.     

Mechanism of stereoselective interaction between butyrylcholinesterase and ethopropazine enantiomers

Stereoselectivity of reversible inhibition of butyrylcholinesterase (BChE; EC 3.1.1.8) by optically pure ethopropazine [10-(2-diethylaminopropyl)phenothiazine hydrochloride] enantiomers and racemate was studied with acetylthiocholine (0.002–250 mM) as substrate. Molecular modelling resulted in the reaction between BChE and ethopropazine starting with the binding of ethopropazine to the enzyme peripheral anionic site. In the next step ethopropazine ‘slides down’ the enzyme gorge, resulting in interaction of the three rings of ethopropazine through π–π interactions with W82 in BChE. Inhibition mechanism was interpreted according to three kinetic models: A, B and C. The models differ in the type and number of enzyme–substrate, enzyme–inhibitor and enzyme–substrate–inhibitor complexes, i.e., presence of the Michaelis complex and/or acetylated BChE. Although, all three models reproduced well the BChE activity in absence of ethopropazine, model A was poor in describing inhibition with ethopropazine, while models B and C were better, especially for substrate concentrations above 0.2 mM. However model C was singled out because it approaches fulfilment of the one step-one event criteria, and confirms the inhibition mechanism derived from molecular modelling. Model C resulted in dissociation constants for the complex between BChE and ethopropazine: 61, 140 and 88 nM for R-enantiomer, S-enantiomer and racemate, respectively. The respective dissociation constants for the complexes between acetylated BChE and ethopropazine were 268, 730 and 365 nM. Butyrylcholinesterase had higher affinity for R-ethopropazine.     

Interaction of (benzylidene-hydrazono)-1,4-dihydropyridines with β-amyloid,acetylcholine, and butyrylcholine esterases

Approved drugs for the treatment of Alzheimer’s disease belong to the group of inhibitors of the acetylcholinesterase (AChE) and NMDA receptor inhibitors. However none of the drugs is able to combat or reverse the progression of the disease. Thus, the recently reported promising multitarget-directed molecule approach was applied here. Using the lead compound DUO3, which was found to be a potent inhibitor of the AChE and butyrylcholinesterase (BuChE) as well as an inhibitor of the formation of the amyloid (Aβ) plaque, new non-permanently positively charged derivatives were synthesized and biologically characterized. In contrast to DUO3 the new bisphenyl-substituted pyridinylidene hydrazones 5 are appropriate to cross the blood–brain barrier due to their pK_a values and lipophilicity, and to inhibit both the AChE and BuChE. More important some of the pyridinylidene hydrazones inhibit the Aβ fibril formation completely and destruct the already formed fibrils significantly.     

Novel isosorbide di-ester compounds as inhibitors of acetylcholinesterase

We report herein that a variety of isosorbide di-esters, previously reported to be novel substrates for butyrylcholinesterase (BuChE, EC 3.1.1.8), are in fact inhibitors of the homologous enzyme acetylcholinesterase (AChE), with IC(50) values in the micromolar range. In vitro studies show that they are mixed inhibitors of the enzyme, and thus the ternary enzyme-inhibitor-substrate complex can form in acetylcholinesterase. This is rationalised by molecular modelling which shows that the compounds bind in the mid-gorge area. In this position, simultaneous substrate binding might be possible, but the hydrolysis of this substrate is prevented. The di-esters dock within the butyrylcholinesterase gorge in a very different manner, with the ester sidechain at the 5-position occupying the acyl pocket at residues Leu286 and Val288, and the 2-ester binding to Trp82. The carbonyl group of the 2-ester is susceptible to nucleophilic attack by Ser198 of the catalytic triad. The larger residues of the acyl pocket in acetylcholinesterase prevent binding in this manner. The results complement each other and explain the differing behaviours of the esters in the cholinesterase enzymes. These findings may prove very significant for future work.     

Tailored Modeling of Rivastigmine Derivatives as Dual Acetylcholinesterase and Butyrylcholinesterase Inhibitors for Alzheimer's Disease Treatment

Rational modification of known drug candidates to design more potent ones using computational methods has found application in drug design, development, and discovery. Herein, we integrate computational and theoretical methodologies to unveil rivastigmine derivatives as dual inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) for Alzheimer's disease (AD) management. The investigation entails pharmacokinetics screening, density functional theory (DFT) mechanistic study, molecular docking, and molecular dynamics (MD) simulation. We designed over 20 rivastigmine substituents, subject them to some analyses, and identified RL2 with an appreciable blood-brain barrier score and no permeability glycoprotein binding. The compound shows higher acylation energy and a favored binding affinity to the cholinesterase enzymes. RL2 interacts with the AChE and BuChE active sites showing values of −41.1/−39.5 kcal mol⁻¹ while rivastigmine binds with −32.7/−30.7 kcal mol⁻¹ for these enzymes. The study revealed RL2 (4-fluorophenyl rivastigmine) as a potential dual inhibitor for AChE and BuChE towards Alzheimer's disorder management.     

Insights into the Role of Erythrina corallodendron L. in Alzheimer's Disease: in Vitro and in Silico Approach

Alzheimer's disease (AD) is a major health problem. Cholinergic transmission is greatly affected in AD. Phytochemical investigation of the alkaloid rich fraction (AF) of Erythrina corallodendron L leaves resulted in isolation of five known alkaloids: erysodine, erythrinine, 8-oxoerythrinine, erysovine N-oxide and erythrinine N-oxide. In this study, eysovine N-oxide was reported for the second time in nature. AF was assayed for cholinesterase inhibition at the concentration of 100 μg mL⁻¹. AF showed a higher percent inhibition for butyrylcholinesterase enzyme (BuChE) (83.28 %) compared to acetylcholinesterase enzyme (AChE) (64.64 %). The isolated alkaloids were also assayed for their anti-BuChE effect. In-silico docking study was done for the isolated compounds at the binding sites of AChE and BuChE to determine their binding pattern and interactions, also molecular dynamics were estimated for the compound displaying the best fit for AChE and BuChE. In addition, ADME parameters and toxicity were predicted for the isolated alkaloids compared to donepezil.     

In silico, NMR and pharmacological evaluation of an hydroxyoxindole cholinesterase inhibitor

From a screening study of various potential inhibitors for cholinesterases (ChEs), compound (rac)-1 (4-((3-hydroxy-2-oxo-3-phenylindolin-1-yl) methyl) piperidin-1-ium chloride) showed an IC₅₀ of 18?μM for butyrylcholinesterase (BuChE). Herein we present a toxicological and pharmacological evaluation of (rac)-1 to determine its potential for use as an alternative ChE inhibitor for the treatment of Alzheimer’s disease. The strategy adopted included in vivo and ex vivo studies with mouse models, Molecular Modelling and Saturation Transfer Difference (STD) NMR studies.Preliminary molecular docking studies were conducted with both (R) and (S)-1 with acetylcholinesterase (AChE) and BuChE, prior to advancing to the mouse model, and indeed favorable interactions were observed, with (R)-1 showing the best binding with AChE and (S)-1 with BuChE. STD-NMR studies were used to successfully validate these results. Toxicological studies were also conducted using the Artemia salina model, with donepezil as reference. It was found that in the in vivo mouse studies that (rac)-1 presented a slightly better inhibition of AChE (0.096?µmol.min^?1.mg^?1) than donepezil (0.112?µmol.min^?1.mg^?1) and the same level of inhibition for BuChE as donepezil (0.014?µmol.min^?1.mg^?1).     

Substrate activation in acetylcholinesterase induced by low pH or mutation in the pi-cation subsite

Substrate inhibition is considered a defining property of acetylcholinesterase (AChE), whereas substrate activation is characteristic of butyrylcholinesterase (BuChE). To understand the mechanism of substrate inhibition, the pH dependence of acetylthiocholine hydrolysis by AChE was studied between pH 5 and 8. Wild-type human AChE and its mutants Y337G and Y337W, as well as wild-type Bungarus fasciatus AChE and its mutants Y333G, Y333A and Y333W were studied. The pH profile results were unexpected. Instead of substrate inhibition, wild-type AChE and all mutants showed substrate activation at low pH. At high pH, there was substrate inhibition for wild-type AChE and for the mutant with tryptophan in the pi-cation subsite, but substrate activation for mutants containing small residues, glycine or alanine. This is particularly apparent in the B. fasciatus AChE. Thus a single amino acid substitution in the pi-cation site, from the aromatic tyrosine of B. fasciatus AChE to the alanine of BuChE, caused AChE to behave like BuChE. Excess substrate binds to the peripheral anionic site (PAS) of AChE. The finding that AChE is activated by excess substrate supports the idea that binding of a second substrate molecule to the PAS induces a conformational change that reorganizes the active site.     

Synthesis,biological activity and molecular modeling studies on 1H-benzimidazole derivatives as acetylcholinesterase inhibitors

A series of N-{2-[4-(1H-benzimidazole-2-yl)phenoxy]ethyl}substituted amine derivatives were designed to assess cholinesterase inhibitor activities. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitor activities were evaluated in vitro by using Ellman’s method. It was discovered that most of the compounds displayed AChE and/or BuChE inhibitor activity and few compounds were selective against AChE/BuChE. Compound 3c and 3e were the most active compounds in the series against eeAChE and hAChE, respectively. Molecular docking studies and molecular dynamics simulations were also carried out.     

Kinetic analysis of the inhibition of human butyrylcholinesterase with cymserine

Accompanying the gradual rise in the average age of the population of most industrialized countries is a regrettable progressive rise in the number of individuals afflicted with age-related neurodegenerative disorders, epitomized by Alzheimer's disease (AD) but, additionally, including Parkinson's disease (PD) and stroke. The primary therapeutic strategy, to date, involves the use of cholinesterases inhibitors (ChEIs) to amplify residual cholinergic activity. The enzyme, acetylcholinesterase (AChE), along with other elements of the cholinergic system is depleted in the AD brain. In contrast, however, its sister enzyme, butyrylcholinesterase (BuChE), that likewise cleaves acetylcholine (ACh), is elevated and both AChE and BuChE co-localize in high amounts with the classical pathological hallmarks of AD. The mismatch between increased brain BuChE and depleted levels of both ACh and AChE, particularly late in the disease, has supported the design and development of new ChEIs with a preference for BuChE; exemplified by the novel agent, cymserine, whose binding kinetics are characterized for the first time. Specifically, as assessed by the Ellman method, cymserine demonstrated potent concentration-dependent binding with human BuChE. The IC50 was determined as 63 to 100 nM at the substrate concentration range of 25 to 800 microM BuSCh. In addition, the following new binding constants were investigated for human BuChE inhibition by cymserine: T(FPnubeta), K(nubeta), K(Bs), K(MIBA), M(IC50), D(Sc), R(f), (O)K(m), OIC100, K(sl), theta(max) and R(i). These new kinetic constants may open new avenues for the kinetic study of the inhibition of a broad array of other enzymes by a wide variety of inhibitors. In synopsis, cymserine proved to be a potent inhibitor of human BuChE in comparison to its structural analogue, phenserine.