DHR3 selectivity assay for inhibitors of ROR gamma transcriptional activity
The retinoic acid-related orphan receptor (ROR) gamma is a transcription factor that has a central role in the differentiation of Th17 cells, a subset of T helper cells that secrete the inflammatory cytokines IL-17A, IL-17F, and IL-22. Th17 cells have been implicated in graft versus host disease, autoimmune disease and asthma. RORgamma is induced in naive T helper cells in the presence of more ..
BioActive Compounds: 21
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: R03 DA026211-01
Assay Provider: Dan Littman, New York University
The retinoic acid-related orphan receptor (ROR) gamma is a transcription factor that has a central role in the differentiation of Th17 cells, a subset of T helper cells that secrete the inflammatory cytokines IL-17A, IL-17F, and IL-22. Th17 cells have been implicated in graft versus host disease, autoimmune disease and asthma. RORgamma is induced in naive T helper cells in the presence of TGF-beta combined with IL-6, IL-21, or IL-23, and thereafter directs the expression of the Th17 lineage cytokines. To identify nonselective inhibitors, a Drosophila Schneider cell line was stably transfected with two vectors: a gene expressing a fusion of the Gal4 DNA binding domain and the transactivation domain of the Drosophila Hormone Receptor 3 (DHR3), under the control of the metallothionine promoter and a Photinus luciferase reporter regulated by the Gal4 binding site enhancer, UAS. The addition of copper to the medium induced expression of the Gal4-DHR3 fusion that subsequently induced the UAS-luciferase reporter. Small molecule inhibitors that inhibit components common to the RORgamma and DHR3 assays, such as proteins that interact with general nuclear hormone receptors, will be active in both assays and thus scored as nonselective.
Assay Protocol Summary:
For screening, 600 cells in 4 uL/well were dispensed into white solid 1536-well plates (Grenier) using a solenoid-based dispenser. Following transfer of 23 nL compound or DMSO vehicle by a pin tool, the plates were incubated 1 hr at ambient temperature, and 1 uL/well copper sulfate (700 uM final concentration) was added. The plates were centrifuged 15 s at 1000 RPM and incubated 20 hr at ambient temperature. After addition of 1.5 uL Photinus luciferase detection reagent, the plates were incubated 10 min at ambient temperature and then read by an ViewLux (Perkin Elmer) to detect luminescence.
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)