Developmental toxicity assay using high content screening of zebrafish embryos

Typically, time‐consuming standard toxicological assays using the zebrafish (Danio rerio) embryo model evaluate mortality and teratogenicity after exposure during the first 2 days post‐fertilization. Here we describe an automated image‐based high content screening (HCS) assay to identify the teratogenic/embryotoxic potential of compounds in zebrafish embryos in vivo. Automated image acquisition was performed using a high content microscope system. Further automated analysis of embryo length, as a statistically quantifiable endpoint of toxicity, was performed on images post‐acquisition. The biological effects of ethanol, nicotine, ketamine, caffeine, dimethyl sulfoxide and temperature on zebrafish embryos were assessed. This automated developmental toxicity assay, based on a growth‐retardation endpoint should be suitable for evaluating the effects of potential teratogens and developmental toxicants in a high throughput manner. This approach can significantly expedite the screening of potential teratogens and developmental toxicants, thereby improving the current risk assessment process by decreasing analysis time and required resources. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

Screening of natural compounds and their derivatives as potential protein kinase C inhibitors

Protein kinase C (PKC) isozymes are important signal transducers in a number of cellular responses that makes PKC inhibition a topical target for drug discovery. In this study, a set of natural compounds and their derivatives (43 in total) were screened to establish their potential inhibitory effects on PKC, exploiting kinase activity and [³H]‐phorbol ester binding assays. Statistical evaluation of the assays yielded signal‐to‐background, signal‐to‐noise, and Z′ values of 16.1, 11.3, and 0.62, respectively, for the kinase activity assay and 47.4, 15.9, and 0.73 for the binding assay, demonstrating their suitability for screening. Of the compounds investigated, the most potent PKC inhibitor was (?)‐epigallocatechin gallate (EGCG), which had an IC₅₀ of 4.8 µM. In addition, (?)‐epicatechin gallate, dodecyl gallate, and the flavonoids myricetin, quercetin, rhamnetin, luteolin, isorhamnetin, and kaempferol were effective while naringin and scopoletin demonstrated negligible effects. None of the compounds was able to significantly inhibit the binding of phorbol ester to the regulatory domain of PKCα. The highest inhibition, 59%, was observed in the case of EGCG at a concentration of 150 µM. Taken together, nine PKC inhibitors were identified, none of which was able to compete with the binding of phorbol ester to PKCα, suggesting that the mechanism of PKC inhibition could be a result of binding to the catalytic domain of PKC. Drug Dev Res 63:76–87, 2004. © 2004 Wiley‐Liss, Inc.     

Structure‐based virtual screening toward the discovery of novel inhibitors of the DNA repair activity of the human apurinic/apyrimidinic endonuclease 1

The DNA repair activity of human apurinic/apyrimidinic endonuclease 1 (APE1) has been recognized as a promising target for the development of small‐molecule inhibitors to be used in combination with anticancer agents. In an attempt to identify novel inhibitors of APE1, we present a structure‐based virtual screening (SBVS) study based on molecular docking analysis of the compounds of NCI database using the GOLD 5.1.0 (Genetic Optimization for Ligand Docking) suite of programs. Compounds selected in this screening were tested with a fluorescence‐based APE1 endonuclease activity assay. Two compounds ( 37 and 41 ) were able to inhibit the multifunctional enzyme APE1 in the micromolar range, while compound 22 showed inhibitory effects at nanomolar concentrations. These results were confirmed by a plasmid DNA nicking assay. In addition, the potential APE1 inhibitors did not affect the cell viability of non‐tumor MCF10A cells. Overall, compounds 22 , 37, and 41 appear to be important scaffolds for the design of novel APE1 inhibitors and this study highlights the relevance of in silico‐based approaches as valuable tools in drug discovery.     

Confocal fluorescence microscopy for high-throughput screening of G-protein coupled receptors

In the pharmaceutical industry, G-protein coupled receptors (GPCRs) are the most successful group of therapeutic targets. Finding compounds that interfere with the ligand-GPCR interaction in a specific and selective way is a major focus of pharmaceutical research today. As compound libraries of large pharmaceutical companies have increased to hundreds of thousands of test compounds, there is a growing need for miniaturization of drug discovery assays to save bioreagents and to reduce the consumption of test compounds. Due to its high sensitivity combined with a femtoliter-sized measurement volume, confocal fluorescence microscopy enables designs for GPCR binding assays with tiny sample volumes. The GPCRs are prepared in the form of plasma membrane fragments from GPCR-overexpressing cells or may be integrated into virus-like particles (VLiPs). One technique to extract binding data from confocal fluorescence experiments is the so-called fluorescence intensity distribution analysis (FIDA). In this review article, we describe the applicability of FIDA to GPCR-focussed high-throughput screening (HTS) and compare FIDA to two other GPCR-adaptable drug discovery techniques for ligand binding studies, the scintillation proximity assay (SPA) and macroscopic fluorescence polarization (FP) measurements. FIDA measures the absolute concentrations of both GPCR-bound and unbound ligand, thereby providing an internal control to the drug screening data. FIDA is amenable to work with relatively low amounts of GPCRs so that the assay may be carried out with biomembranes of a low GPCR density. Moreover, the fluorescence intensity readout of the FIDA technique may be combined with other confocal fluorescence readouts such as fluorescence anisotropy or lifetime. The combination of a low sample volume with an information-rich measurement means that confocal fluorescence spectroscopy can bring substantial benefits as a bioassay platform to pharmaceutical GPCR-directed research.     

药物筛选技术的最新进展--高内涵筛选

化合物活性筛选是创新药物研究过程的起点和具有决定意义的关键步骤。基于细胞的高内涵药物筛选技术实现了对化合物多靶点多参数的同时检测，代表着创新药物研究技术发展的必然趋势，将在未来的新药研发过程中发挥重要作用。笔者介绍了高内涵筛选技术的概念、系统组成，分析了其优势特点，并简要讨论了其在新药研究尤其是抗肿瘤药物研究中的实际应用。     

Set‐up of an infrared fast behavioral assay using zebrafish (Danio rerio) larvae,and its application in compound biotoxicity screening

Zebrafish (Danio rerio) is increasingly employed for evaluating toxicity and drug discovery assays. Commonly experimental approaches for biotoxicity assessment are based on visual inspection or video recording. However, these techniques are limited for large‐scale assays, as they demand either a time‐consuming detailed inspection of the animals or intensive computing resources in order to analyze a considerable amount of screenshots. Recently, we have developed a simple methodology for tracking the locomotor activity of small animals cultured in microtiter plates. In this work, we implemented this automatic methodology, based on infrared (IR) microbeam scattering, for measuring behavioral activity in zebrafish larvae. We determined the appropriate culture conditions, number of animals and stage of development to get robust results. Furthermore, we validated this methodology as a rapid test for evaluating toxicity. By measuring the effects of reference compounds on larvae activity, we were able to estimate the concentration that could cause a 50% decrease in activity events values (AEC₅₀), showing a strong linear correlation (R² = 0.91) with the LC₅₀ values obtained with the standard DarT test. The toxicity order of the measured compounds was CuSO₄ > 2,4‐dinitrophenol > 3,4‐dichloroaniline > SDS > sodium benzoate > EDTA > K₂CrO₄; regarding solvents, EtOH ≈ DMSO. In this study, we demonstrate that global swimming behavior could be a simple readout for toxicity, easy to scale‐up in automated experiments. This approach is potentially applicable for fast ecotoxicity assays and whole‐organism high‐throughput compound screening, reducing the time and money required to evaluate unknown samples and to identify leading pharmaceutical compounds. Copyright © 2013 John Wiley & Sons, Ltd.     

Biocompatibility assessment of nanomaterials for environmental safety screening

In view of the extensive use of nanoparticles in countless applications, a fast and effective method for assessing their potential adverse effects on the environment and human health is extremely important. At present, in vitro cell‐based assays are the standard approach for screening chemicals for cytotoxicity because of their relative simplicity, sensitivity, and cost‐effectiveness compared with animal studies. Regrettably, such cell‐based viability assays encounter limitations when applied to determining the biological toxicity of nanomaterials, which often interact with assay components and produce unreliable outcomes. We have established a cell‐impedance‐based, label‐free, real‐time cell‐monitoring platform suitable for use in a variety of mammalian cell lines that displays results as cell index values. In addition to this real‐time screening platform, other traditional cytotoxicity assays were employed to validate cytotoxicity assessments. We suggest that the cell impedance measurement approach is effective and better suited to determining the cytotoxicity of nanomaterials for environmental safety screening. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1170–1182, 2017.     

Identification of Dipeptidyl Peptidase IV Inhibitors: Virtual Screening,Synthesis and Biological Evaluation

Inhibition of dipeptidyl peptidase IV is an important approach for the treatment of type‐2 diabetes. In this study, we reported a multistage virtual screening workflow that integrated 3D pharmacophore models, structural consensus docking, and molecular mechanics/generalized Born surface area binding energy calculation to identify novel dipeptidyl peptidase IV inhibitors. After screening our in‐house database, two hit compounds, HWL‐405 and HWL‐892, having persistent high performance in all stages of virtual screening were identified. These two hit compounds together with several analogs were synthesized and evaluated for in vitro inhibition of dipeptidyl peptidase IV. The experimental data indicated that most designed compounds exhibited significant dipeptidyl peptidase IV inhibitory activity. Among them, compounds 35f displayed the greatest potency against dipeptidyl peptidase IV in vitro with the IC₅₀ value of 78 nm . In an oral glucose tolerance test in normal male Kunming mice, compound 35f reduced blood glucose excursion in a dose‐dependent manner.     

High-Throughput screeening assays for CYP2B6 metabolism and inhibition usuing fluorogenic vivid substrates

CYP2B6 is a highly polymorphic P450 isozyme involved in the metabolism of endo-and xenobiotics with known implications for the activation of many procarcinogens resulting in carcinogenesis. However, lack of validated high-throughput screening (HTS) CYP2B6 assays has limited the current understanding and full characterization of this isozyme’s involvement in human drug metabolism. Here, we have developed and characterized a fluorescence-based HTS assay employing recombinant human CYP2B6 and 2 novel fluorogenic substrates (the Vivid CYP2B6 Blue and Cyan Substrates). Assay validation included testing the inhibitory potency of a panel of drugs and compounds known to be metabolized by this isozyme, including CYP2B6 substrates, inhibitors, and known inducers. Compound rankings based on inhibitory potency in the Vivid CYP2B6 Blue and Cyan Assays matched compound rankings based on relative affinity measurements from previously published data (K_i, K_d, or K_m values) for the CYP2B6 isozyme. In conclusion, these assays are proven to be robust and sensitive, with broad dynamic ranges and kinetic parameters allowing screening in HTS mode of a large panel of compounds for CYP2B6 metabolism and inhibition, and are a valuable new tool for CYP2B6 studies.     

SVMDLF: A novel R‐based Web application for prediction of dipeptidyl peptidase 4 inhibitors

Dipeptidyl peptidase 4 (DPP4) is a well‐known target for the antidiabetic drugs. However, currently available DPP4 inhibitor screening assays are costly and labor‐intensive. It is important to create a robust in silico method to predict the activity of DPP4 inhibitor for the new lead finding. Here, we introduce an R‐based Web application SVMDLF (SVM‐based DPP4 Lead Finder) to predict the inhibitor of DPP4, based on support vector machine (SVM) model, predictions of which are confirmed by in vitro biological evaluation. The best model generated by MACCS structure fingerprint gave the Matthews correlation coefficient of 0.87 for the test set and 0.883 for the external test set. We screened Maybridge database consisting approximately 53,000 compounds. For further bioactivity assay, six compounds were shortlisted, and of six hits, three compounds showed significant DPP4 inhibitory activities with IC₅₀ values ranging from 8.01 to 10.73 μm . This application is an OpenCPU server app which is a novel single‐page R‐based Web application for the DPP4 inhibitor prediction. The SVMDLF is freely available and open to all users at http://svmdlf.net/ocpu/library/dlfsvm/www/ and http://www.cdri.res.in/svmdlf/ .     

Sense and sensibility: the use of cell death biomarker assays in high-throughput anticancer drug screening and monitoring treatment responses

Cell-based screening allows identification of biologically active compounds, for example, potential anticancer drugs. In this review, various screening assays are discussed in terms of what they measure and how this affects interpretation and relevance. High-throughput (HT) assays of viability based on the reduction of exogenous substrates do not always reflect viability or cell number levels. Membrane integrity assays can be used for HT quantification of cell death, but are non-specific as to the death mode. Several HT assays monitor end point apoptosis. Screening libraries at a single concentration (micromolar) can prevent detection of potent apoptosis inducers, as high concentrations may induce mainly necrosis. Using monolayer cultures limits the significance of cell-based screening as the properties of monolayer cells differ from tumours in vivo. Spheroid cultures are more physiological, but are impractical for screening by conventional methods. The authors have developed an assay quantifying accumulation of a caspase-cleaved protein specific for epithelial cells. It provides an integrated measure of apoptosis in two- and three-dimensional cultures and can be used as a blood biomarker assay for tumour apoptosis in vivo.     

Identification of novel nematode succinate dehydrogenase inhibitors: Virtual screening based on ligand-pocket interactions

To discover new nematicidal succinate dehydrogenase (SDH) inhibitors with novel structures, we conducted a virtual screening of the ChemBridge library with 1.7 million compounds based on ligand-pocket interactions. The homology model of Caenorhabditis elegans SDH was established, along with a pharmacophore model based on ligand-pocket interactions. After the pharmacophore-based and docking-based screening, 19 compounds were selected for the subsequent enzymatic assays. The results showed that compound 1 (ID: 7607321) exhibited inhibitory activity against SDH with a determined IC₅₀ value of 19.6 μM. Structural modifications and nematicidal activity studies were then carried out, which provided further evidence that compound 1 exhibited excellent nematicidal activity. Molecular dynamics simulations were then conducted to investigate the underlying molecular basis for the potency of these inhibitors against SDH. This work provides a reliable strategy and useful information for the future design of nematode SDH inhibitors.     

Development of a quantitative morphological assessment of toxicant‐treated zebrafish larvae using brightfield imaging and high‐content analysis

One of the rate‐limiting procedures in a developmental zebrafish screen is the morphological assessment of each larva. Most researchers opt for a time‐consuming, structured visual assessment by trained human observer(s). The present studies were designed to develop a more objective, accurate and rapid method for screening zebrafish for dysmorphology. Instead of the very detailed human assessment, we have developed the computational malformation index, which combines the use of high‐content imaging with a very brief human visual assessment. Each larva was quickly assessed by a human observer (basic visual assessment), killed, fixed and assessed for dysmorphology with the Zebratox V4 BioApplication using the Cellomics® ArrayScan® V^TI high‐content image analysis platform. The basic visual assessment adds in‐life parameters, and the high‐content analysis assesses each individual larva for various features (total area, width, spine length, head–tail length, length–width ratio, perimeter–area ratio). In developing the computational malformation index, a training set of hundreds of embryos treated with hundreds of chemicals were visually assessed using the basic or detailed method. In the second phase, we assessed both the stability of these high‐content measurements and its performance using a test set of zebrafish treated with a dose range of two reference chemicals (trans‐retinoic acid or cadmium). We found the measures were stable for at least 1 week and comparison of these automated measures to detailed visual inspection of the larvae showed excellent congruence. Our computational malformation index provides an objective manner for rapid phenotypic brightfield assessment of individual larva in a developmental zebrafish assay. Copyright © 2016 John Wiley & Sons, Ltd.     

In vitro screening assays to identify natural or synthetic acetylcholinesterase inhibitors: thin layer chromatography versus microplate methods.

Acetylcholinesterase inhibitors (AChEI) are currently still the best available pharmacotherapy for Alzheimer patients. Successful screening for new AChEI relies on effective and fast assays. Two colorimetric screening assays frequently used to search for new AChEI, namely a thin layer chromatography (TLC) assay with Fast Blue B salt as reagent and a 96-well plate assay based on Ellman's method, were compared. For the majority (83%) of the 138 test compounds of natural and synthetic origin, the results obtained with the two assays converged and both screening assays were considered suitable for the generation of new hits. Fifteen percent of investigated compounds were classified as active with the microplate assay but were shown to be inactive by TLC and about 2% were measured active by TLC but showed to be inactive with the microplate assay. These divergences were not due to the main differences between the experimental protocols of the two screening assays, namely the different colorimetric methods and pre-incubation of test compounds with acetylcholinesterase (AChE). They might be explained by the interaction of either AChE or test compounds with the silica of the TLC plates, resulting in an altered affinity of the enzyme for the compounds.     

Consideration of dosimetry in evaluation of ToxCast™ data

The US Environmental Protection Agency (US EPA) Toxcast? program has the stated goal of predicting hazard, characterizing toxicity pathways and prioritizing the toxicity testing of environmental chemicals through the use of in vitro high‐throughput screening (HTS) assays. This analysis integrates data from biomonitoring and from in vivo toxicity and pharmacokinetic studies to examine the physiological relevance of the tested and responding in vitro concentrations for five case study chemicals: triclosan, 2,4‐dichlorophenoxyacetic acid, perfluorooctanoic acid, monobutyl phthalate and mono‐2(ethylhexyl)phthalate. This analysis also examines the ToxCast? phase 1 data set for approximately 50 chemicals belonging to four ‘common mechanism groups’ which have been the subject of cumulative risk assessments by the US EPA for both the pattern of key responses and the relative potencies of included chemicals compared with the in vivo relative potencies. Responding concentrations in vitro were generally in the range of serum or plasma concentrations associated with no‐observed to lowest‐observed effect levels for the case study chemicals, while available biomonitoring data demonstrating actual exposures were generally lower. ToxCast? assay endpoints related to acetylcholinesterase (AChE) inhibition had low sensitivity for detecting organophosphate pesticides but good sensitivity for detecting N‐methyl carbamates. However, in vitro relative potencies did not correlate with in vivo potency. Both qualitative and quantitative predictive power is probably affected by the lack of comprehensive metabolic activity in most current in vitro systems explored in the ToxCast? program, and this remains a fundamental challenge for high‐throughput toxicity screening efforts. Copyright © 2011 John Wiley & Sons, Ltd.     

Retrospective analysis for valproate screening targets with liquid chromatography–high resolution mass spectrometry with positive electrospray ionization: An omics‐based approach

Liquid chromatography coupled with high‐resolution mass spectrometry (LC–HRMS) is an important analytical tool in the systematic toxicological analysis performed in forensic toxicology. However, some important compounds, such as the antiepileptic drug valproate (valproic acid; VPA), cannot be directly detected with positive electrospray ionization (ESI⁺) due to poor ionization. Here we demonstrate an omics‐based retrospective analysis for the identification of indirect screening targets for VPA in whole blood with LC–ESI⁺–HRMS. Analysis was performed utilizing data acquired across four years from LC–ESI⁺–HRMS, with VPA results from a quantitative LC–MS/MS method. The combined data with VPA results were split into an exploration set (n = 68; 28% positive) and a test set (n = 37; 32% positive). Eight indirect targets for VPA were identified in the exploration set. The evaluation of these targets was confirmed with retrospective target analysis of the test set. Using a combination of two out of the eight indirect targets, we attained a sensitivity of 92% (n = 12; VPA concentration range: 4.4–29.7 mg/kg) and 100% specificity (n = 25) for VPA with LC–ESI⁺–HRMS. VPA screening targets were identified with retrospective data analysis and could be appended to the existing screening procedure. A sensitive and specific screening with LC–ESI⁺–HRMS was achieved with targets corresponding to the sodium adducts of C₇H₁₄O₃ and C₈H₁₄O₃. Three chromatographic resolved isomer peaks were observed for the latter, and the consistently most intense peak was tentatively identified as 3‐hydroxy‐4‐en‐VPA.     

Yeast: bridging the gap between phenotypic and biochemical assays for high-throughput screening

ABSTRACT

Introduction: Both in vitro biochemical and phenotypic assay platforms have clear limitations in high throughput screening (HTS) for drug discovery. The use of genetically tractable model yeast as a vehicle for target-based HTS overcomes many of these by allowing the identification of on-target compounds that function within a eukaryotic cellular context.

Areas covered: In this special report, the use of yeast-based assays in HTS is discussed with reference to the various platforms that have been utilized over the past 20 years. The specific issues considered are the necessity to employ counter and secondary screening approaches to ensure the on-target activity of hits, and the recent developments in detection systems that have facilitated miniaturization and ultra-HTS.

Expert opinion: It is difficult at present to predict the future. That being said, the demonstrable possibilities of optimizing yeast-based HTS, coupled with the demonstration of utility in an industrial setting, shows that these platforms have the potential to bridge the gap between phenotypic and biochemical assays for HTS.