NIH Chemical Genomics Center [NCGC]
U.S. Environmental Protection Agency [EPA]
National Institutes of Environmental Health Sciences [NIEHS]
National Toxicology Program [NTP]

NCGC Assay Overview

The Retinoid X receptor (RXR), a nuclear hormone receptor is expressed in liver, spleen, placenta, epidermis, central nervous system. These receptors are able to form homo and heterodimers with other nuclear receptors and due to this heterodimerizing ability, RXR functions as master regulators producing diverse physiological effects. Three subtypes of RXR exist; alpha, beta and gamma. Retinoid X receptor alpha (RXR-alpha) has potential roles in metabolic signaling pathways, skin alopecia, dermal cysts, cardiac development, insulin sensitization etc. Therefore screening environmental chemicals on RXR-alpha signaling can be used for the studies focused on such therapeutic applications.

NCGC Assay Protocol Summary

Using a beta-lactamase reporter-gene under control of Retinoid X receptor--alpha (RXR-alpha) response element, a cell-based assay [GeneBLAzer (RXR-alpha -UAS-bla assay developed by Invitrogen] was used to measure RXR-alpha ligands signaling. GeneBLAzer RXR-alpha -UAS-bla HEK293T cells were used with assay medium containing 2% charcoal-stripped FBS. The assay was performed in clear bottom black Greiner 1536-well plates. 9-cis-retinoic acid was used in the screen as the positive control. Library compounds were measured for their ability to stimulate the reporter gene activity. Compounds were screened in a titration series in 1536-well format. The fluorescence intensity (405 nm excitation, 460/530 nm emission) was measured using an EnVision plate reader. Data was normalized relative to 9-cis-retinoic acid control (500 nM, 100%), and DMSO only wells (basal, 0%). Concentration-response titration points for each compound were fitted to the Hill equation yielding concentrations of half-maximal stimulation (EC50) and maximal response (efficacy) values.

qHTS protocol for RXR-alpha beta-lactamase assay in agonist mode

[Step] [Parameter] [Value] [Description]
1. Reagent; 6 uL; 2500 cells/well
2. Incubation; 37 C for 4 hrs
3. Compounds; 23 nL; 0.59 nM to 92 uM
4. Controls (9-cis-retinoic acid); 23 nL,
- One column with dose response, start 38 uM, 1:3 dilutions in duplicates
- Final concentrations of 500 nM and 50 nM in two columns
5. Incubation; 37 C for 18 hrs
6. Detection reagent; 1 uL of CCF4-dye
7. Incubation; RT for 2 hrs
8. Detection; Fluorescence; Envision 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, active compounds are expected to have the activator phenotype and apparent activators are ranked higher than compounds that showed apparent inhibition.
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.