Confirmation of high-throughput screening data and novel mechanistic insights into VDR-xenobiotic interactions by orthogonal assays

Debabrata Mahapatra,Denis Fourches,Kyle Roell,Melaine Agnes Kuenemann,David M Reif,Jill A Franzosa,Keith A Houck,Seth W Kullman

doi:10.1038/s41598-018-27055-3

Debabrata Mahapatra, Denis Fourches + Show 6 more

Open Access

https://doi.org/10.1038/s41598-018-27055-3

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Abstract

High throughput screening (HTS) programs have demonstrated that the Vitamin D receptor (VDR) is activated and/or antagonized by a wide range of structurally diverse chemicals. In this study, we examined the Tox21 qHTS data set generated against VDR for reproducibility and concordance and elucidated functional insights into VDR-xenobiotic interactions. Twenty-one potential VDR agonists and 19 VDR antagonists were identified from a subset of >400 compounds with putative VDR activity and examined for VDR functionality utilizing select orthogonal assays. Transient transactivation assay (TT) using a human VDR plasmid and Cyp24 luciferase reporter construct revealed 20/21 active VDR agonists and 18/19 active VDR antagonists. Mammalian-2-hybrid assay (M2H) was then used to evaluate VDR interactions with co-activators and co-regulators. With the exception of a select few compounds, VDR agonists exhibited significant recruitment of co-regulators and co-activators whereas antagonists exhibited considerable attenuation of recruitment by VDR. A unique set of compounds exhibiting synergistic activity in antagonist mode and no activity in agonist mode was identified. Cheminformatics modeling of VDR-ligand interactions were conducted and revealed selective ligand VDR interaction. Overall, data emphasizes the molecular complexity of ligand-mediated interactions with VDR and suggest that VDR transactivation may be a target site of action for diverse xenobiotics.

Highlights

Following National Research Council’s recommendations[1] for a shift from traditional low throughput in vivo rodent assays to less expensive in vitro high throughput methods, core regulatory bodies such as the U.S Environmental Protection Agency (EPA), National Toxicology Program (NTP), National Institute of Health (NIH), NIH National Center for Advancing Translational Sciences (NCATS), US Food and Drug Administration (FDA) responded to the urgency with the initiation of ToxCastTM and Tox[21] programs[2,3]
In the wake of the above-mentioned targeted nuclear receptor (NR) studies, and the emergence of newly identified environmental compounds with potential endocrine disrupting properties, we focused our attention to the library of screened compounds that altered the transactivational activity of vitamin D receptor (VDR)
The list of active agonists and antagonists was reduced to compounds with ratio values greater than 5 and assessed in relation to curve fit parameters and flag criteria, which are available via the ToxCast Dashboard

Summary

Introduction

Following National Research Council’s recommendations[1] for a shift from traditional low throughput in vivo rodent assays to less expensive in vitro high throughput methods, core regulatory bodies such as the U.S Environmental Protection Agency (EPA), National Toxicology Program (NTP), National Institute of Health (NIH), NIH National Center for Advancing Translational Sciences (NCATS), US Food and Drug Administration (FDA) responded to the urgency with the initiation of ToxCastTM and Tox[21] programs[2,3] These programs were aimed at prioritizing toxicity evaluations through promoting the increasing use of in vitro high throughput screening assays for large numbers of chemicals already in commercial use for which little or no toxicity data was available[4,5]. Molecular modeling was employed to forecast and study the molecular interactions of the most potent compounds once docked in the VDR binding site

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