Quantitative High-Throughput Screening and Orthogonal Assays to Identify Modulators of the Vitamin D Receptor (SETAC)

The Vitamin D nuclear receptor (VDR) is a selective, ligand-inducible transcription factor involved in numerous biological processes such as cell proliferation, differentiation, detoxification, calcium homeostasis, neurodevelopment, immune system regulation, cardiovascular function and cancer. Potential environmental chemical disruptors of VDR signaling were investigated by quantitative high-throughput screening as part of the U.S. Tox21 collaboration. An 8.5K chemical library was tested for agonist and antagonist activity using a quantitative 1536-well plate high-throughput screening (qHTS) platform with a VDR beta-lactamase reporter gene assay in HEK 293T cells (15 concentrations run in triplicate). Results were analyzed using the U.S. EPA ToxCast Data Analysis Pipeline (invitroDB_v1) and identified 78 compounds as potential VDR agonists. Only 11 of the these compounds were active at < 10 µM. Analysis also revealed that 342 of the compounds screened functioned as VDR antagonists; of which 30% were potent at less than 10 µM. All cadmium compounds (n = 4) were potent at sub-micromolar concentrations in the antagonist assay. To evaluate the specificity of the active compounds, comparisons were made with available Tox21 and ToxCast data from assays of permissive (PPARs, LXR, PXR, FXR) and non-permissive (TR, RAR) RXR heterodimerization partners. Ratios of the activity in the VDR assays and complementary cytotoxicity activity data from qHTS in agonist and antagonist mode were used to rank the chemicals based on activity. These data were used to select an initial set of chemicals (15 = agonist; 15 = antagonist) to be tested in follow-up in vitro assays. The chemicals were run in concentration-response (0.012 nM – 120 µM) in a transient transactivation assay with a luciferase reporter derived from the human CYP24 promoter. In addition, the effect of agonists/antagonists on interactions of VDR with RXR and co-activator (SRC-1) was also evaluated using a mammalian 2 hybrid system. Overall, these results illustrate the application of robotic qHTS assays for testing environmental chemicals and identifying VDR activity. This approach facilitates the assessment and characterization of chemical-induced toxicity pathways to support risk assessments. This abstract does not necessarily reflect US EPA policy.