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BioAssay: AID 492954

Mouse Th17 T cell differentiation assay for inhibitors of ROR gamma transcriptional activity

Keywords: NIH Roadmap, MLPCN, MLI, MLSMR, qHTS, NCGC, qHTS, nuclear hormone receptor, retinoic acid-related orphan receptor, ROR gamma ..more
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 Tested Compounds
 Tested Compounds
All(36)
 
 
Active(17)
 
 
Inactive(8)
 
 
Inconclusive(11)
 
 
 Tested Substances
 Tested Substances
All(37)
 
 
Active(18)
 
 
Inactive(8)
 
 
Inconclusive(11)
 
 
AID: 492954
Data Source: NCGC (RORG545)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-12-02
Hold-until Date: 2011-12-01
Modify Date: 2011-12-02

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 17
Depositor Specified Assays
AIDNameTypeComment
2551qHTS for inhibitors of ROR gamma transcriptional activityconfirmatory
2604Quantitative high throughput screen for inhibitors of ROR gamma transcriptional activity: Summarysummary
Description:
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]

MLPCN Grant: R03 DA026211-01
Assay Provider: Dan Littman, New York University

Keywords: NIH Roadmap, MLPCN, MLI, MLSMR, qHTS, NCGC, qHTS, nuclear hormone receptor, retinoic acid-related orphan receptor, ROR gamma

Assay Overview:

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. ROR gamma 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 confirm inhibition of ROR gamma, compounds were tested in a mouse Th17 T cell differentiation assay. Th17 differentiation was tracked by expression of a GFP reporter under the control of the endogenous IL-17a promoter using flow cytometry. Compounds that block ROR gamma activity prevent expression of the IL17a-GFP reporter.
Protocol
Assay Protocol Summary:

Cells from lymph nodes and spleens derived from six to eight week old IL17a-GFP mice (Biocytogen LLC) were used for T cell purification. B220- cells were isolated on an autoMACS Pro with bead depletion of B220+ cells (Miltenyi). Naive CD4+ T cell were further purified as TCRb+CD8- DAPI- CD19- CD4+CD25- CD62L+CD44low/Int by cell sorting on a FACSAria (BD). Cells were cultivated in an incubator at 37 C and 5% CO2 in RPMI 1640 medium (Invitrogen) supplemented with 10% (vol/vol) heat-inactivated FBS (Hyclone), penicillin-streptomycin, 2 mM glutamine and 0.1 mM nonessential amino acids. Cells were seeded on day 0 at a density of 0.4x105 cells per ml in 96-well plates coated with anti-CD3e (5 ug/ml) and anti-CD28 (10 ug/ml). Cells were cultured for 4-5 days in Th17 (TGFb (0.3 ng/ml), IL6 (20 ng/ml), anti-IFN-g, and anti-IL-4) condition. At day 1, compounds dissolved in DMSO were added. GFP expression was assayed on day 4 or 5 with DAPI- and PECy7-conjugated CD4+ cells. An LSR II (BD Biosciences) and FlowJo software (Tree Star) were used for flow cytometry.

Keywords: NIH Roadmap, MLPCN, MLI, MLSMR, qHTS, NCGC, qHTS, nuclear hormone receptor, retinoic acid-related orphan receptor, ROR gamma
Comment
Compound Ranking:

For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. Active compounds are assigned a PUBCHEM_ACTIVITY_SCORE between 50 and 100 based on compound potency. Inconclusive compounds are assigned a PUBCHEM_ACTIVITY_SCORE of 25.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Potency (uM)*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
2Activity at 0.1 uM (0.1μM**)% Activity at given concentration.Float%
3Activity at 0.2 uM (0.2μM**)% Activity at given concentration.Float%
4Activity at 0.7 uM (0.7μM**)% Activity at given concentration.Float%
5Activity at 2.2 uM (2.2μM**)% Activity at given concentration.Float%
6Activity at 6.7 uM (6.7μM**)% Activity at given concentration.Float%
7Activity at 20 uM (20μM**)% Activity at given concentration.Float%
8Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R03 DA026211-01

Data Table (Concise)
Classification
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