High‐Throughput Screen of Natural Product Extracts in A Yeast Model of Polyglutamine Proteotoxicity

Proteins with expanded polyglutamine (polyQ) segments cause a number of fatal neurodegenerative disorders, including Huntington's disease (HD). Previous high‐throughput screens in cellular and biochemical models of HD have revealed compounds that mitigate polyQ aggregation and proteotoxicity, providing insight into the mechanisms of disease and leads for potential therapeutics. However, the structural diversity of natural products has not yet been fully mobilized toward these goals. Here, we have screened a collection of ~11 000 natural product extracts for the ability to recover the slow growth of ΔProQ103‐expressing yeast cells in 384‐well plates (Z' ~ 0.7, CV ~ 8%). This screen identified actinomycin D as a strong inhibitor of polyQ aggregation and proteotoxicity at nanomolar concentrations (~50–500 ng/mL). We found that a low dose of actinomycin D increased the levels of the heat‐shock proteins Hsp104, Hsp70 and Hsp26 and enhanced binding of Hsp70 to the polyQ in yeast. Actinomycin also suppressed aggregation of polyQ in mammalian cells, suggesting a conserved mechanism. These results establish natural products as a rich source of compounds with interesting mechanisms of action against polyQ disorders.     

Virtual Screening of CB2 Receptor Agonists from Bayesian Network and High‐Throughput Docking: Structural Insights into Agonist‐Modulated GPCR Features

The relevance of CB₂‐mediated therapeutics is well established in the treatment of pain, neurodegenerative and gastrointestinal tract disorders. Recent works such as the crystallization of class‐A G‐protein‐coupled receptors in a range of active states and the identification of specific anchoring sites for CB₂ agonists challenged us to design a reliable agonist‐bound homology model of CB₂ receptor. Docking‐scoring enrichment tests of a high‐throughput virtual screening of 140 compounds led to 13 hits within the micromolar affinity range. Most of these hits behaved as CB₂ agonists, among which two novel full agonists emerged. Although the main challenge was a high‐throughput docking run targeting an agonist‐bound state of a CB₂ model, a prior 2D ligand‐based Bayesian network was computed to enrich the input commercial library for 3D screening. The exclusive discovery of agonists illustrates the reliability of this agonist‐bound state model for the identification of polar and aromatic amino acids as new agonist‐modulated CB₂ features to be integrated in the wide activation pathway of G‐protein‐coupled receptors.     

High‐throughput Screening for Identification of Inhibitors of EpCAM‐Dependent Growth of Hepatocellular Carcinoma Cells

The cancer stem cell marker, EpCAM, is an important indicator of Wnt/β‐catenin signaling activation and a functional component of hepatocellular tumor‐initiating cells. A high‐throughput screening assay was developed to identify inhibitors of EpCAM‐dependent growth of hepatocellular carcinoma (HCC) cells. EpCAM(+) and EpCAM(?) HCC cell lines were assessed for differential sensitivity to a Wnt/β‐catenin pathway inhibitor. Libraries comprising 22 668 pure compounds and 107 741 crude or partially purified natural product extracts were tested, and 12 pure compounds and 67 natural product extracts were identified for further study. Three active compounds and the positive control were further characterized in terms of effects on EpCAM expression. Treatment of EpCAM(+) Hep3B cells resulted in loss of EpCAM expression as assessed by flow cytometry. This reduction was incomplete (most cells continued to express EpCAM), but resulted in generation of cell populations expressing lower levels of EpCAM. Sublethal concentrations (~IC₅₀) reduced median EpCAM expression to 28% of control after 1 day and 19% of control after 2 days. Reduction in EpCAM expression preceded growth inhibition suggesting that a threshold of EpCAM expression may be required for growth of EpCAM‐dependent cells. The identification of compounds with a variety of possible molecular targets suggests a likelihood of multiple mechanisms for modulation of EpCAM‐dependent cell growth.     

LC—MS／MS高通量快速筛查中成药和保健品中未知非法添加药物

目的建立LC—MS／MS的筛查方法,高通量快速筛查中成药和保健品中的非法添加药物。方法色谱条件采用ZORBAXXDB—C18柱,乙腈：1mM甲酸铵（含0．1％甲酸）梯度洗脱;质谱条件采用电喷雾离子化源正离子化筛查方式（ESI＋）,通过相关信息扫描模式（IDA）,最终在已建立的谱库中自动搜索出中成药和保健品中未知的非法添加物。结果对于怀疑有未知的非法添加物样本,经18min的快速筛查,可以同时筛查到包括降糖、镇静催眠、解热镇痛、非甾体抗炎、抗生素、抗癫痫等多种类的非法添加药物。结论本文建立的LC—MS／MS方法简单、快速、准确、高通量,可用于筛查中成药和保健品中未知的非法添加物。     

Screening protocol for the identification of modulators by immunofluorescent cell‐based assay

High‐throughput assays are a common strategy for the identification of compounds able to modulate a certain cellular activity. Here, we show an automatized analysis platform for the quantification of nuclear foci as inhibitory effect of compounds on a target protein labeled by fluorescent antibodies. Our experience led us to a fast analysis platform that combines cell‐based assays, high‐content screening, and confocal microscopy, with an automatic and user‐friendly statistical analysis of plate‐based assays including positive and negative controls, able to identify inhibitory effect of compounds tested together with the Z‐prime and Window of individual plate‐based assays to assess the reliability of the results. The platform integrates a web‐based tool implemented in Pipeline Pilot and R, and allows computing the inhibition values of different parameters obtained from the high‐content screening and confocal microscopy analysis. This facilitates the exploration of the results using the different parameters, providing information at different levels as the number of foci observed, the sum of intensity of foci, area of foci, etc, the detection and filtering of outliers over the assay plate, and finally providing a set of statistics of the parameters studied together with a set of plots that we believe significantly helps to the interpretation of the assay results.     

Ultra‐fast LC–MS/MS in therapeutic drug monitoring: Quantification of clozapine and norclozapine in human plasma

A novel approach to high‐throughput, targeted liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis has been developed. A single chromatographic system can be used for the analysis of a range of 20 drugs and metabolites with a total analysis time of 36 s (one 96‐well plate of prepared samples per hour). To demonstrate the applicability of this approach to quantitative analysis, a method has been validated for the therapeutic drug monitoring of clozapine and norclozapine following automated extraction from human plasma. Chromatographic retention times were 11.4 and 12.4 s for norclozapine and clozapine, respectively (for both analytes the chromatographic peak width was less than 1 s). Comparison with a conventional LC–MS/MS method (5 min analysis time) showed excellent agreement. This new approach offers analysis times more akin to flow‐injection analysis, but is likely to be more widely applicable because of chromatographic resolution from residual matrix components and isobaric interferences.     

Expression levels of human P‐glycoprotein in In Vitro cell lines: correlation between mRNA and protein levels for P‐glycoprotein expressed in cells

The purpose of this study was to investigate the correlation between mRNA and protein levels for P‐glycoprotein (P‐gp) expressed in various cell lines to validate the estimation of P‐gp activity from its mRNA levels. P‐gp expression levels in various cell monolayers, normal, P‐gp‐induced, P‐gp‐highly induced, (multidrug resistance, MDR) MDR1‐knockdown (A2‐2) and MDR1‐knockdown (B2‐2) Caco‐2 cells and MDCKII/MDR1 cells, were quantified by real‐time quantitative polymerase chain reaction (PCR) and western blot analysis. Both mRNA and protein levels of P‐gp were lowest in the MDR1‐knockdown (B2‐2) Caco‐2 cells, followed by the MDR1‐knockdown (A2‐2) Caco‐2, normal Caco‐2, P‐gp‐induced Caco‐2 and P‐gp‐highly induced Caco‐2 cells, and highest in the MDCKII/MDR1 cells. Except for the MDCKII/MDR1 cells, the protein levels of P‐gp in all Caco‐2 cell lines showed a linear correlation with its mRNA levels; however, although the MDR1 mRNA level in MDCKII/MDR1 cells was much higher than in the P‐gp‐highly induced Caco‐2 cells, the protein levels were almost the same in both cells. From these findings, it was suggested that P‐gp activity in MDCKII/MDR1 cells could not be estimated from its mRNA levels. Copyright © 2009 John Wiley & Sons, Ltd.     

Screening method for stimulants in urine by UHPLC‐MS/MS: identification of isomeric compounds

A fast screening method for the detection of more than 60 stimulants in urine was developed. The method consisted of a dilution of the urine (1:5 v/v) and analysis by ultra high performance liquid chromatography coupled to tandem mass spectrometry, using a C18 column (1.7 µm particle size), a mobile phase containing deionized water and acetonitrile with formic acid, and gradient elution. The chromatographic run time was 5 min. The detection was performed in positive mode electrospray ionization, monitoring one or two specific ion transitions for each analyte. Appropriate repeatability was obtained, with relative standard deviation (RSD) values below 25% for most of the analytes. Regarding intermediate precision, estimated during routine work, higher RSDs were obtained, probably due to between‐day differences in the status of the mass spectrometer and in the chromatographic system. Matrix effect ranged from 60 to 255% with RSD lower than 35% for the majority of compounds. Despite the matrix effect observed, the signal/noise ratio of the analytes spiked at 50 ng/mL was greater than three in all tested samples, allowing a correct detection of all substances at the minimum required performance levels required by the World Anti‐Doping Agency and demonstrating the suitability of the method. The method was tested in administration study samples and satisfactorily in operation for more than one year with routine doping samples. The presence of isomeric stimulants with closely similar chromatographic and/or mass spectrometric properties did not allow the unequivocal identification of these compounds after the first analysis. Different possibilities for separation and identification of isomeric compounds are presented. Copyright © 2015 John Wiley & Sons, Ltd.     

Virtual and In Vitro Bioassay Screening of Phytochemical Inhibitors from Flavonoids and Isoflavones Against Xanthine Oxidase and Cyclooxygenase‐2 for Gout Treatment

Synthetic drugs such as allopurinol and benzbromarone are commonly used to treat the complex pathogenesis of gout, a metabolic disease that results from an inflammation of the joints caused by precipitation of uric acid. We seek to discover novel phytochemicals that could treat gout, by targeting the xanthine oxidase and cyclooxygenase‐2 enzymes. In this study, we report the screening of nine compounds of flavonoids from the ZINC and PubChem databases (containing 2092 flavonoids) using the igemdock software tool against the xanthine oxidase and cyclooxygenase‐2 3D protein structures. Each compound was also evaluated by an in vitro bioassay testing the inhibition of xanthine oxidase and cyclooxygenase‐2. Myricetin and luteolin were found to be the potential dual inhibitors of xanthine oxidase and cyclooxygenase‐2 as demonstrated by IC₅₀: 62.7 and 3.29 μg/mL (xanthine oxidase)/70.8 and 16.38 μg/mL (cyclooxygenase‐2), respectively. In addition, structure–activity relationships and other important factors of the flavonoids binding to the active site of xanthine oxidase and cyclooxygenase‐2 were discussed, which is expected for further rational drug design.     

Screening of non‐steroidal anti‐inflammatory drugs for inhibitory effects on the activities of six UDP‐glucuronosyltransferases (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) using LC‐MS/MS

Non‐steroidal anti‐inflammatory drugs (NSAIDs) are used widely to relieve pain and to decrease inflammation. Several clinical studies have reported that NSAIDs inhibit uridine 5′‐diphospho‐glucuronosyltransferase (UGT) enzymes. Therefore, the study evaluated the inhibitory potential of 15 NSAIDs on the activities of six UGT isoforms (i.e. UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) in human liver microsomes (HLMs). Among the 15 NSAIDs tested here, mefenamic acid and diclofenac inhibited all UGTs tested in this study. Piroxicam and niflumic acid inhibited UGT1A9 activity (IC₅₀ = 73.8 μm and 0.38 μm , respectively) and naproxen selectively inhibited UGT2B7 activity (IC₅₀ = 53.1 μm ), whereas it did not inhibit the other UGTs tested (IC₅₀ > 200 μm ). Diflunisal inhibited the UGT1A1 (IC₅₀ = 33.0 μm ) and UGT1A9 (IC₅₀ = 19.4 μm ). Acetaminophen, fenoprofen, ibuprofen, ketoprofen, meloxicam, phenylbutazone, salicylic acid and sulindac showed negligible inhibitory effects on the six UGTs (IC₅₀ > 100 μm ). These results suggest that some NSAIDs have the potential to inhibit UGTs in vitro. Copyright © 2014 John Wiley & Sons, Ltd.     

Application of electrospray ionization product ion spectra for identification with atmospheric pressure matrix‐assisted laser desorption/ionization mass spectrometry – a case study with seized drugs

Product ion spectra obtained with liquid chromatography‐electrospray ionization tandem mass spectrometry (LC‐ESI/MS/MS) were applied to the identification of seized drug samples from atmospheric pressure matrix‐assisted laser desorption/ionization product ion spectra (AP‐MALDI‐MS/MS spectra). Data acquisition was performed in the information‐dependent acquisition (IDA) mode, and the substance identification was based on a spectral library previously created with LC‐ESI/MS/MS using protonated molecules as precursor ions. A total of 39 seized drug samples were analyzed with both AP‐MALDI and LC‐ESI techniques using the same triple‐quadrupole instrument (AB Sciex 4000QTRAP). The study shows that ESI‐MS/MS spectra can be directly utilized in AP‐MALDI‐MS/MS measurements as the average fit and purity score percentages with AP‐MALDI were 90% and 85%, respectively, being similar to or even better than those obtained with the reference LC/ESI‐MS/MS method. This fact enables the possibility to use large ESI spectral libraries, not only to ESI analyses but also to analyses with other ionization techniques which produce protonated molecules as the base peak. The data obtained shows that spectral library search works also for analytical techniques which produce multi‐component mass spectra, such as AP‐MALDI, unless isobaric compounds are encountered. The spectral library search was successfully applied to rapid identification of confiscated drugs by AP‐MALDI‐IDA‐MS/MS. Copyright © 2012 John Wiley & Sons, Ltd.