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BioAssay: AID 652025

qHTS of IL-2 Activators

The adaptive immune response against any infectious agent begins with the activation of an antigen specific T cell. This relatively rare and inert ("naive") T cell undergoes a massive proliferative expansion and differentiation process to generate sufficient number of potent pathogen-specific effector T cells. The effector T cells fight the infection by making cytokines and helping other cells more ..
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 Tested Compounds
 Tested Compounds
All(364605)
 
 
Active(238)
 
 
Inactive(364167)
 
 
Inconclusive(200)
 
 
 Tested Substances
 Tested Substances
All(364943)
 
 
Active(238)
 
 
Inactive(364505)
 
 
Inconclusive(200)
 
 
AID: 652025
Data Source: NCGC (IL2100)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-02-12

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 238
Depositor Specified Assays
AIDNameTypeComment
652024qHTS of IL-2 Activators: Summarysummary
Description:
The adaptive immune response against any infectious agent begins with the activation of an antigen specific T cell. This relatively rare and inert ("naive") T cell undergoes a massive proliferative expansion and differentiation process to generate sufficient number of potent pathogen-specific effector T cells. The effector T cells fight the infection by making cytokines and helping other cells to become fully armed as well. If any naive T cell has antigen receptors (TCRs) that can be activated by self peptides, then a variety of regulatory mechanisms kick in to prevent this cell from performing to its full potential. These processes collectively constitute the phenomenon of immunological tolerance and their failure leads to autoimmunity. One arm of tolerance involves generation of biochemical negative feedback downstream of the TCR (tuning). This is especially relevant to self antigens present in the body over long periods of time (chronic), which form the bulk of our proteome. The negative feedback serves to tune the TCR signaling, such that the ambient antigenic stimulation is nullified intracellularly. Unfortunately, this critical machinery serves as a target for chronic viral, bacterial and parasitic infections (as well as tumors), which can evade a robust immune response, simply by allowing T cells to tune down their signaling to pathogen or malignancy derived antigens. As such, disrupting the tuning in such cases would allow the tolerant T cell to function again and deliver a robust effector response.
If a therapeutic can restore responsiveness to the tuned down T cell, even for a short time, they could combat the pathogen without any additional immunization. To that end, we have developed a sensitive HTS T cell based assay based on IL-2. This assay will be screened against the Molecular Libraries Small Molecule Repository (MLSMR) to find small molecules that could elicit this response.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: MH096572
Assay Submitter (PI): Ron Schwartz, NIAID
Protocol
4 microL (750 cells/well) of unstimulated transgenic mouse T cells (columns 1-3,5-48) are dispensed into Aurora LoBase, black clear-bottom 1536-well assay plates; column 4 receives an identical concentration of transgenic memory T cells as a non-tolerant control. Compounds are then transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). Following addition of compound and a 60 minute incubation, 1 microL of bead stimulation solution ((0.04 micron Fluospheres (Life technologies, CA) coated with 10ug/ml each anti-CD3 and anti-CD4 (clones 2C11 and GK1.5, BD Biosciences, CA) and mixed with 1ug/ml anti-CD28 (clone PV-1, BD biosciences, CA)). columns 2-48) is added to stimulate the IL-2 response pathway; media is instead added to column 1 as an unstimulated control, while PMA/ionomycin (Sigma Aldrich, MO) is added to column 3 as a control for IL-2 tolerance bypass. The plates are then incubated at 37 degrees C for 18 hours. EGFP and DsRed signal are then detected using an Acumen eX3 Explorer fluorescent plate cytometer. All cells are dispensed in cell media containing EHAA (Quality Biologicals, MD), RPMI (Quality Biologicals, MD), Glutamine (Life Technologies, CA), Penicillin/Streptomycin (Quality Biologicals, MD), 2-ME (Sigma Aldrich, MO) and 10% FBS (Gemini Bioproducts, CA) .
Comment
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 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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*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
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Activity_ScoreActivity score.Integer
6Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.0007804147 uM (0.000780415μM**)% Activity at given concentration.Float%
16Activity at 0.00234 uM (0.00234124μM**)% Activity at given concentration.Float%
17Activity at 0.00702 uM (0.00702378μM**)% Activity at given concentration.Float%
18Activity at 0.021 uM (0.0210713μM**)% Activity at given concentration.Float%
19Activity at 0.063 uM (0.0632139μM**)% Activity at given concentration.Float%
20Activity at 0.190 uM (0.189642μM**)% Activity at given concentration.Float%
21Activity at 0.569 uM (0.568925μM**)% Activity at given concentration.Float%
22Activity at 1.707 uM (1.70678μM**)% Activity at given concentration.Float%
23Activity at 5.120 uM (5.12033μM**)% Activity at given concentration.Float%
24Activity at 9.220 uM (9.22μM**)% Activity at given concentration.Float%
25Activity at 15.36 uM (15.361μM**)% Activity at given concentration.Float%
26Activity at 46.08 uM (46.0829μM**)% Activity at given concentration.Float%
27Activity at 46.10 uM (46.1μM**)% Activity at given concentration.Float%
28Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH096572

Data Table (Concise)
Classification
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