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Confirmation of High-Throughput screening data and novel mechanistic insights into VDR-xenobiotic interactions by orthogonal assays
Mahapatra, D., Jill A. Franzosa, K. Roell, M. Kuenemann, Keith A. Houck, D. Reif, D. Fourches, AND S. Kullman. Confirmation of High-Throughput screening data and novel mechanistic insights into VDR-xenobiotic interactions by orthogonal assays. Scientific Reports. Nature Publishing Group, London, Uk, 8(8883):1-16, (2018). https://doi.org/10.1038/s41598-018-27055-3
In line with the continued surge in scientific interest in dissecting the roles played by NR’s specifically VDR in mechanisms associated with toxicity, we explored the utility of confirming high throughput analysis with orthogonal assays and the potential of VDR as a target of xenobiotics and endocrine disruptors.
Quantitative high-throughput (qHTS) testing formats have gained acceptance and widespread recognition in the field of toxicity testing of environmental compounds in recent years. As a result, significant progress has been made with respect to identification of compounds that modulate the function of nuclear receptors in vitro. Recent efforts through Tox21 and ToxCast programs have demonstrated that the Vitamin D receptor (VDR) is activated and/or antagonized by a wide range of structurally diverse chemicals including naturally occurring, synthetic, and environmental chemicals. Vitamin D has gained significant attention in recent years not only for its role in classical bone and mineral homeostatic functions but also for its roles in neurodevelopment, neuroprotection, cell proliferation and differentiation, immune function and inflammation. 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 generated by Tox21 qHTS library and followed up with additional orthogonal assays. Transient transactivation assay (TT) using a human VDR plasmid and Cyp241A1 luciferase reporter construct revealed 20/21 active VDR agonists and 18/19 active VDR antagonists. Compounds were then examined using a mammalian-2-hybrid assay (M2H) to evaluate VDR interactions with co-activators and co-regulators. With the exception of a select few compounds, VDR agonists exhibited minimal to moderate recruitment of co-regulators and co-activators whereas antagonists exhibited moderate to marked attenuation of co-activator recruitment by VDR both in the presence and absence of co-regulators. A unique set of compounds exhibiting synergistic activity in antagonist mode and no activity in agonist mode was identified. Additionally, cheminformatics modeling of VDR-ligand interactions were conducted. Overall, the data emphasizes the molecular complexity of VDR transcription machinery in terms of differential and preferential affinities of compounds for co-regulators/co-activators and respective sites of action.
Record Details:Record Type: DOCUMENT (JOURNAL/PEER REVIEWED JOURNAL)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL CENTER FOR COMPUTATIONAL TOXICOLOGY