National Center for Advancing Translational Sciences [NCATS]
NIH Chemical Genomics Center [NCGC]
National Institutes of Environmental Health Sciences [NIEHS]
National Toxicology Program [NTP]

Assay Overview:

Nuclear steroid hormone receptors are involved in many biological processes including inflammatory responses, cell proliferation, and apoptosis. Environmental factors or compounds disrupting these nuclear receptor pathways and gene expression may promote susceptibility to immunotoxicants and autoimmune diseases. One important group of these expression factors are the retinoid-related orphan receptors. To assess compounds that may interfere with the retinoid-related orphan receptor gamma (ROR-gamma) transcription factor, a cell-based luciferase assay was used.
The CHO cell-based assay utilizes a TET-inducible ROR expression factor and ROR response element-LUC reporter gene. Reporter activity was induced using doxycycline and measured after 16hr incubation. Luminescence was read using Viewlux (PerkinElmer), where a decrease in reporter activity would show a decrease in luminescence.

Assay Protocol Summary:

1,000 cells in 4 uL/well were dispensed into white wall/solid bottom 1536-well plates using a Multidrop Combi (Thermo Scientific) dispenser. Following transfer of 23 nL compound or DMSO vehicle by a pin tool, the plates were treated with 1 ul doxycycline to induce reporter gene activity. After 16hr incubation at 37C and 5% CO2, 5uL one-glo (promega) was added to each plate and following 30 min incubation at room temperature, luminescence was quantified on a ViewLux (PerkinElmer) plate reader.

Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.