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Profiling of the Tox21 10K compound library for agonists and antagonists of the estrogen receptor alpha signaling pathway.

ABSTRACT: The U.S. Tox21 program has screened a library of approximately 10,000 (10K) environmental chemicals and drugs in three independent runs for estrogen receptor alpha (ER?) agonist and antagonist activity using two types of ER reporter gene cell lines, one with an endogenous full length ER? (ER-luc; BG1 cell line) and the other with a transfected partial receptor consisting of the ligand binding domain (ER-bla; ER? ?-lactamase cell line), in a quantitative high-throughput screening (qHTS) format. The ability of the two assays to correctly identify ER? agonists and antagonists was evaluated using a set of 39 reference compounds with known ER? activity. Although both assays demonstrated adequate (i.e. >80%) predictivity, the ER-luc assay was more sensitive and the ER-bla assay more specific. The qHTS assay results were compared with results from previously published ER? binding assay data and showed >80% consistency. Actives identified from both the ER-bla and ER-luc assays were analyzed for structure-activity relationships (SARs) revealing known and potentially novel ER? active structure classes. The results demonstrate the feasibility of qHTS to identify environmental chemicals with the potential to interact with the ER? signaling pathway and the two different assay formats improve the confidence in correctly identifying these chemicals.

SUBMITTER: Huang R

PROVIDER: S-EPMC4092345 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Profiling of the Tox21 10K compound library for agonists and antagonists of the estrogen receptor alpha signaling pathway.

Huang Ruili R Sakamuru Srilatha S Martin Matt T MT Reif David M DM Judson Richard S RS Houck Keith A KA Casey Warren W Hsieh Jui-Hua JH Shockley Keith R KR Ceger Patricia P Fostel Jennifer J Witt Kristine L KL Tong Weida W Rotroff Daniel M DM Zhao Tongan T Shinn Paul P Simeonov Anton A Dix David J DJ Austin Christopher P CP Kavlock Robert J RJ Tice Raymond R RR Xia Menghang M

Scientific reports 20140711

The U.S. Tox21 program has screened a library of approximately 10,000 (10K) environmental chemicals and drugs in three independent runs for estrogen receptor alpha (ERα) agonist and antagonist activity using two types of ER reporter gene cell lines, one with an endogenous full length ERα (ER-luc; BG1 cell line) and the other with a transfected partial receptor consisting of the ligand binding domain (ER-bla; ERα β-lactamase cell line), in a quantitative high-throughput screening (qHTS) format. T ...[more]

PMID: 25012808

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Project description:Cytotoxicity is a commonly used in vitro endpoint for evaluating chemical toxicity. In support of the U.S. Tox21 screening program, the cytotoxicity of ~10K chemicals was interrogated at 0, 8, 16, 24, 32, & 40 hours of exposure in a concentration dependent fashion in two cell lines (HEK293, HepG2) using two multiplexed, real-time assay technologies. One technology measures the metabolic activity of cells (i.e., cell viability, glo) while the other evaluates cell membrane integrity (i.e., cell death, flor). Using glo technology, more actives and greater temporal variations were seen in HEK293 cells, while results for the flor technology were more similar across the two cell types. Chemicals were grouped into classes based on their cytotoxicity kinetics profiles and these classes were evaluated for their associations with activity in the Tox21 nuclear receptor and stress response pathway assays. Some pathways, such as the activation of H2AX, were associated with the fast-responding cytotoxicity classes, while others, such as activation of TP53, were associated with the slow-responding cytotoxicity classes. By clustering pathways based on their degree of association to the different cytotoxicity kinetics labels, we identified clusters of pathways where active chemicals presented similar kinetics of cytotoxicity. Such linkages could be due to shared underlying biological processes between pathways, for example, activation of H2AX and heat shock factor. Others involving nuclear receptor activity are likely due to shared chemical structures rather than pathway level interactions. Based on the linkage between androgen receptor antagonism and Nrf2 activity, we surmise that a subclass of androgen receptor antagonists cause cytotoxicity via oxidative stress that is associated with Nrf2 activation. In summary, the real-time cytotoxicity screen provides informative chemical cytotoxicity kinetics data related to their cytotoxicity mechanisms, and with our analysis, it is possible to formulate mechanism-based hypotheses on the cytotoxic properties of the tested chemicals.

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Project description:Endocrine disrupting chemicals (EDCs) are exogenous compounds that interfere with the endocrine system of vertebrates, often through direct or indirect interactions with nuclear receptor proteins. Estrogen receptors (ERs) are particularly important protein targets and many EDCs are ER binders, capable of altering normal homeostatic transcription and signaling pathways. An estrogenic xenobiotic can bind ER as either an agonist or antagonist to increase or inhibit transcription, respectively. The receptor conformations in the complexes of ER bound with agonists and antagonists are different and dependent on interactions with co-regulator proteins that vary across tissue type. Assessment of chemical endocrine disruption potential depends not only on binding affinity to ERs, but also on changes that may alter the receptor conformation and its ability to subsequently bind DNA response elements and initiate transcription. Using both agonist and antagonist conformations of the ER?, we developed an in silico approach that can be used to differentiate agonist versus antagonist status of potential binders.The approach combined separate molecular docking models for ER agonist and antagonist conformations. The ability of this approach to differentiate agonists and antagonists was first evaluated using true agonists and antagonists extracted from the crystal structures available in the protein data bank (PDB), and then further validated using a larger set of ligands from the literature. The usefulness of the approach was demonstrated with enrichment analysis in data sets with a large number of decoy ligands.The performance of individual agonist and antagonist docking models was found comparable to similar models in the literature. When combined in a competitive docking approach, they provided the ability to discriminate agonists from antagonists with good accuracy, as well as the ability to efficiently select true agonists and antagonists from decoys during enrichment analysis.This approach enables evaluation of potential ER biological function changes caused by chemicals bound to the receptor which, in turn, allows the assessment of a chemical's endocrine disrupting potential. The approach can be used not only by regulatory authorities to perform risk assessments on potential EDCs but also by the industry in drug discovery projects to screen for potential agonists and antagonists.

Dataset Information

Profiling of the Tox21 10K compound library for agonists and antagonists of the estrogen receptor alpha signaling pathway.

Publications

Profiling of the Tox21 10K compound library for agonists and antagonists of the estrogen receptor alpha signaling pathway.

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