Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder worldwide. Currently, the only strategy for palliative treatment of AD is to inhibit acetylcholinesterase (AChE) in order to ...increase the concentration of acetylcholine in the synaptic cleft. Evidence indicates that AChE also interacts with the β-amyloid (Aβ) protein, acting as a chaperone and increasing the number and neurotoxicity of Aβ fibrils. It is known that AChE has two binding sites: the peripheral site, responsible for the interactions with Aβ, and the catalytic site, related with acetylcholine hydrolysis. In this work, we reported the synthesis and biological evaluation of a library of new tacrine-donepezil hybrids, as a potential dual binding site AChE inhibitor, containing a triazole-quinoline system. The synthesis of hybrids was performed in four steps using the click chemistry strategy. These compounds were evaluated as hAChE and hBChE inhibitors, and some derivatives showed IC50 values in the micro-molar range and were remarkably selective towards hAChE. Kinetic assays and molecular modeling studies confirm that these compounds block both catalytic and peripheral AChE sites. These results are quite interesting since the triazole-quinoline system is a new structural scaffold for AChE inhibitors. Furthermore, the synthetic approach is very efficient for the preparation of target compounds, allowing a further fruitful new chemical library optimization.
Snakebites cause upwards of 1.8 million envenomings, 138,000 deaths and 500,000 cases of long term morbidity each year. Viper snake venoms (family Viperidae) generally contain a high proportion of ...proteases which can cause devastating effects such as hemorrhage, coagulopathy, edema, necrosis, and severe pain, in envenomed victims. In this study, analytical techniques were combined with enzymatic assays to develop a novel method for the detection of snake venom protease activity by using rhodamine-110-peptide substrate. In the so called at-line nanofractionation set up, crude venoms were first separated with reversed phase liquid chromatography, after which fractions were collected onto 384-well plates. Protease activity assays were then performed in the 384-well plates and bioassay chromatograms were constructed revealing protease activity. Parallel obtained UV absorbance, MS and proteomics data from a previous study facilitated toxin identification. The application of the rhodamine-110-peptide substrate assay showed significantly greater sensitivity compared to prior assays using casein-FITC as the substrate. Moreover, cross referencing UV and MS data and resulted in the detection of a number of tentative proteases suspected to exhibit protease activity, including snake venom serine proteases from Calloselasma rhodostoma and Daboia russelli venom and a snake venom metalloproteinase from the venom of Echis ocellatus. Our data demonstrate that his methodology can be a useful tool for selectively identifying snake venom proteases, and can be applied to provide a better understanding of protease-induced pathologies and the development of novel therapeutics for treating snakebite.
•A post-column assay was used for assessing protease activity of snake venom toxins in crude and fractionated venoms.•We developed a new and sensitive bioassay for measuring protease activity in venoms.•Analytics for identifying and studying proteases in venoms were applied.•A rhodamine-110 based substrate is preferred for measuring proteolytic activities in venoms.
In this work, twelve analogues of piperidine alkaloids (-)-cassine and (-)-spectaline were synthesized, as well as the racemic forms of these natural products. The compounds were evaluated for their ...inhibition of electric eel acetylcholinesterase (AChEee) and human butyrylcholinesterase (BChEhu) by on-flow mass-spectrometry-based dual-enzyme assay, and the inhibition mechanisms for the most potent analogues were also determined. Our results showed a preference for BChEhu inhibition with compounds 10c (Ki = 5.24 μM), 12b (Ki = 17.4 μM), 13a (Ki = 13.2 μM) and 3 (Ki = 11.3 μM) displaying the best inhibitory activities.
A chemical study of the EtOAc extract of Nemania bipapillata (AT-05), an endophytic fungus isolated from the marine red alga Asparagopsis taxiformis - Falkenbergia stage, led to the isolation of five ...new botryane sesquiterpenes, including the diastereomeric pair (+)-(2R,4S,5R,8S)-(1) and (+)-(2R,4R,5R,8S)-4-deacetyl-5-hydroxy-botryenalol (2), (+)-(2R,4S,5R,8R)-4-deacetyl-botryenalol (3), one pair of diastereomeric botryane norsesquiterpenes bearing an unprecedented degraded carbon skeleton, (+)-(2R,4R,8R)-(4) and (+)-(2R,4S,8S)-(5), which were named nemenonediol A and nemenonediol B, respectively, in addition to the known 4β-acetoxy-9β,10β,15α-trihydroxyprobotrydial (6). Their structures were elucidated using 1D and 2D NMR, HRESIMS and comparison with literature data of similar known compounds. The absolute configurations of 2, 3 and 4 were deduced by comparison of experimental and calculated electronic circular dichroism (ECD) spectra, while those of 1 and 5 were assigned from vibrational circular dichroism (VCD) data. Compound 4 weakly inhibited acetylcholinesterase, whereas compound 1 inhibited both acetylcholinesterase and butyrylcholinesterase. Compounds 1, 3, 5 and 6 were tested against two carcinoma cell lines (MCF-7 and HCT-116), but showed no significant citotoxicity at tested concentrations (IC
> 50 µM).
The parasite
Schistosoma mansoni
(Sm) depends exclusively on the salvage pathway for its purine requirements. The enzyme purine nucleoside phosphorylase (PNP) is, therefore, a promising target for ...development of antischistosomal agents and an assay for screening of inhibitors. To enable this, immobilized SmPNP reactors were produced. By quantification of hypoxanthine by liquid chromatography, kinetic constants (
K
M
) for the substrate inosine were determined for the free and immobilized enzyme as 110 ± 6.90 μmol L
−1
and 164 ± 13.4 μmol L
−1
, respectively, indicating that immobilization did not affect enzyme activity. Furthermore, the enzyme retained 25 % of its activity after four months. Non-Michaelis kinetics for the phosphate substrate, and capacity for P
i
-independent hydrolysis were also demonstrated, despite the low rate of enzymatic catalysis. Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated with a small library of 9-deazaguanine analogues. The method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives.
Figure
A cartoon illustrating the online SmPNP-IMER activity assay
The use of immobilized capillary enzyme reactors (ICERs) for online ligand screening has been adopted as a new technique for high-throughput screening (HTS). In this work, the selected target was the ...enzyme acetylcholinesterase (AChE), and the AChE-ICERs produced were used in a liquid chromatograph–tandem ion-trap mass spectrometer. The activity and kinetic parameters were evaluated by monitoring the choline’s precursor ion (M + H)+ m/z 104.0 and its ion fragment (C2H3OH) – (M + H)+ m/z 60.0. The assay method was validated using the reference AChE inhibitors tacrine and galanthamine. Two new ligands, out of a library of 17 coumarin derivatives, were identified, and the half-maximal inhibitory concentration (IC50), inhibition constant (K i), and the inhibition mechanism were determined. A coumarin derivative with IC50 similar to tacrine was highlighted.
New trends in LC protein ligand screening de Moraes, Marcela C.; Vanzolini, Kenia L.; Cardoso, Carmen L. ...
Journal of pharmaceutical and biomedical analysis,
01/2014, Volume:
87
Journal Article
Peer reviewed
•A critical view of bioaffinity-based strategies for protein ligand screening.•Zonal, frontal and nonlinear chromatography with immobilized proteins is presented.•Ligand fishing with magnetic beads ...is an innovative tool also discussed.
The diversity of small molecules available to produce truly innovative drugs associated with the wealth of known biological targets calls for key strategies in protein ligand screening. This review encompasses the recently developed bioaffinity-based strategies. A critical view of the use of zonal, frontal, and nonlinear chromatography with immobilized proteins is given. The association of these elution modes with the ligand fishing method, which uses nanomagnetics particles, is also addressed. A series of applications and how these new screening strategies can be used to determine the function, affinity, and activity parameters of proteins is discussed.
The aim of this study was to evaluate the transitory stress levels and the anxiety state in children submitted to conventional and computerized dental anesthesia. Twenty children (7 to 12 years) were ...randomly assigned to receive conventional and computerized dental anesthesia. To investigate the hypothesis that transitory stress could be lower after using computerized anesthesia compared to conventional anesthesia, cortisol levels in saliva were measured before and after each technique. Anxiety was also evaluated individually by answering the State-Trait Anxiety Inventory for Children (STAIC). Numerical data were analyzed statistically by the Mann-Whitney non-parametric test (5% significance level). Salivary cortisol levels increased in 8 (40%) patients after conventional anesthesia and in 9 (45%) patients after computerized anesthesia, with no statistically significant difference between the two types (p=0.34). In the same way, no statistically significant difference was found between the techniques (p=0.39) related to the psychological analysis based on the STAIC scores. Local anesthesia using either conventional anesthesia or a computerized delivery system produced similar level of stress/anxiety in pediatric patients, using both quantitative and qualitative analyses.
O objetivo deste estudo foi avaliar os níveis de estresse transitório e o estado de ansiedade em crianças submetidas à anestesia dental convencional e computadorizada. Vinte crianças (7 a 12 anos de idade) foram randomicamente designadas para receber anestesia dental convencional e computadorizada. Para investigar a hipótese que o estresse transitório poderia ser menor após a anestesia computadorizada, comparada à anestesia convencional, os níveis de cortisol na saliva foram medidos antes e depois de cada técnica. A ansiedade também foi avaliada individualmente por meio do State-Trait Anxiety Inventory for Children (STAIC). Os dados numéricos foram analisados estatisticamente pelo teste não paramétrico de Mann-Whitney (nível de significância de 5%). Os níveis salivares de cortisol aumentaram em 8 (40%) pacientes após anestesia convencional e em 9 (45%) pacientes após anestesia computadorizada, sem diferença estatisticamente significante entre os dois tipos (p=0,34). Da mesma maneira, não foi encontrada diferença estatisticamente significante entre as técnicas (p=0,39) com relação à análise psicológica baseada nos escores STAIC. Anestesia local usando tanto a anestesia convencional quanto o sistema de aplicação computadorizado produziu nível similar de estresse/ansiedade em pacientes infantis, utilizando análises quantitativa e qualitativa.
This review deals with two-dimensional liquid chromatography (2D-LC) separations encompassing target heart-cut (LC-LC), multiple heart-cut (mLC-LC), non-targeted comprehensive (LC × LC), and ...selective comprehensive (sLC × LC) analysis. It presents an overview of basic concepts and emphasizes the versatility of the applications gained by going from one-(1D) to two-dimensional (2D) separations. This review also discusses target analysis of achiral and chiral drugs for different applications and the use of 2D-LC in zonal bioaffinity chromatography. Advances in instrumental and column technologies have widened the application of LC × LC and sLC × LC separations, and we will discuss some of them.
Acetylcholinesterase and butyrylcholinesterase are the two enzymes involved in the cholinergic system and its modulation. These enzymes have been recognized as therapeutic targets for certain disease ...states. This article presents a critical overview of both functional and non-functional assays employed in the identification of cholinesterase ligands. Various approaches, including colorimetric assays, zonal and frontal bioaffinity liquid chromatography, affinity selection mass spectrometry, and nuclear magnetic resonance, are discussed. Furthermore, the most innovative and recent applications in drug discovery involving synthetic and natural libraries are highlighted.
•Cholinesterases are target proteins to develop new drugs for a variety of disease states.•Functional colorimetric methods are widely applied but can furnish false results.•MS-based assays allow the direct monitoring of cholinesterase activity.•Non-functional assays are effective for isolating ligands from complex libraries.•NMR reveals molecular-level insights into the protein-ligand interaction.
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