Bookmark and Share
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
_
   
 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 ):           View Active Data    View All Data
Target
BioActive Compounds: 17
Related Experiments
Show more
AIDNameTypeComment
2551qHTS for inhibitors of ROR gamma transcriptional activityConfirmatorydepositor-specified cross reference
2604Quantitative high throughput screen for inhibitors of ROR gamma transcriptional activity: SummarySummarydepositor-specified cross reference
2546VP16 counterscreen qHTS for inhibitors of ROR gamma transcriptional activityConfirmatorysame project related to Summary assay
2762Concentration response confirmation assay for inhibitors of ROR gamma transcriptional activityConfirmatorysame project related to Summary assay
2763Concentration response confirmation VP16 counterscreen for inhibitors of ROR gamma transcriptional activityConfirmatorysame project related to Summary assay
489036VP16 selectivity assay for inhibitors of ROR gamma transcriptional activityConfirmatorysame project related to Summary assay
489037Secondary confirmation assay for inhibitors of ROR gamma transcriptional activityConfirmatorysame project related to Summary assay
489038DHR3 selectivity assay for inhibitors of ROR gamma transcriptional activityConfirmatorysame project related to Summary assay
489039ROR alpha selectivity assay for inhibitors of ROR gamma transcriptional activityConfirmatorysame project related to Summary assay
492962Mouse Th1 T cell differentiation assay for inhibitors of ROR gamma transcriptional activityOthersame project related to Summary assay
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
Show more
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)
Data Table ( Complete ):     View Active Data    View All Data
Classification
PageFrom: