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BioAssay: AID 720534

qHTS for Inhibitors of TGF-b: Confirmation of Cherry Picks

TGF-b is a main component in the TGF-b signaling pathway which plays diverse roles in cellular and development pathways. TGF-b is mediated through transcription factors called "Smads". Although the Smad-dependent pathway is the primary canonical TGF-b signaling mode, TGF-b1 can also activate alternative signaling pathways, including those involving MAPK (ERK, JNK and p38). This interaction may more ..
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 Tested Compounds
 Tested Compounds
All(1571)
 
 
Active(985)
 
 
Inactive(119)
 
 
Inconclusive(467)
 
 
 Tested Substances
 Tested Substances
All(1572)
 
 
Active(986)
 
 
Inactive(119)
 
 
Inconclusive(467)
 
 
AID: 720534
Data Source: NCGC (tgfb-smad3-f1-cherrypick-hepg2-caga-gfp)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-07-20
Modify Date: 2013-07-25

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 985
Related Experiments
AIDNameTypeComment
588855qHTS for Inhibitors of TGF-bConfirmatorydepositor-specified cross reference
588860qHTS for Inhibitors of TGF-b: SummarySummarydepositor-specified cross reference
588856qHTS for Inhibitors of TGF-b: Cytotox CounterscreenConfirmatorysame project related to Summary assay
720535qHTS for Inhibitors of TGF-b: Cytotox Counterscreen for Cherry PicksConfirmatorysame project related to Summary assay
720536qHTS for Inhibitors of TGF-b: CCL64 Cells Orthogonal Assay for Cherry PicksConfirmatorysame project related to Summary assay
720537qHTS for Inhibitors of TGF-b: Hit Validation in HepG2 Cells using COP promoterConfirmatorysame project related to Summary assay
Description:
TGF-b is a main component in the TGF-b signaling pathway which plays diverse roles in cellular and development pathways. TGF-b is mediated through transcription factors called "Smads". Although the Smad-dependent pathway is the primary canonical TGF-b signaling mode, TGF-b1 can also activate alternative signaling pathways, including those involving MAPK (ERK, JNK and p38). This interaction may mediate or enhance Smad-dependent responses, or can exert Smad-independent effects. The complexity of this signaling cascade allows the TGF-b superfamily to perform unique, overlapping or redundant functions. We believe that targeting the TGF-beta pathway at the Smad-transcription factor level may eliminate the consequences of disrupting the entire pathway and offer specificity without affecting other signaling pathways. Smad3 is the primary transducer of TGF-b's signals and Smad3 regulates many functions attributed to TGF-b signaling. We hypothesize that Smad3 inhibitors will selectively eliminate Smad3-specific TGF-beta signals without undesired off-target effects. We aim to identify Smad3-small molecule antagonists using a quantitative high throughput screening (qHTS) approach.

In this assay, hits identified from the primary screen were re-tested.

MH087449NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: MH087449
Assay Submitter (PI): Sushil Rane, National Institute of Diabetes and Kidney Diseases
Protocol
Suspensions of trypsinized HEPG2 CAGA-GFP cells were dispensed into white, tissue culture-treated, solid 1536-well plates at 5uL/well (1000 cells/well final concentration) in DMEM medium supplemented with 1% FBS. Plates were incubated at 37 degrees C for 2 hours, after which 23 nL of compounds or DMSO were delivered to each well using a pin tool. One uL of recombinant TGF-beta in DMEM (1% FBS) was then dispensed (500 pg/mL final concentration), and plates were incubated at 37 degrees C for 18 hours. The plates were measured on an Acumen eX3 Explorer plate reader for GFP fluorescence (ex488/em500-530). GFP values were calculated by determining the mean GFP fluorescence of individual cells, and compiling these values for each well to determine a total well GFP signal. The %Activity was determined from the corrected fluorescence values. A titration of the known TGF-B inhibitor SB431542 was included to monitor plate performance, while unstimulated HEPG2 (-TGF-B) control wells were used to normalize %Activity of identified inhibitors; unstimulated wells corresponded to 100%Activity (full inhibition), while stimulated cell controls (+DMSO) were used to normalize 0%Activity (no inhibition).

Concentration-response curves were fitted to the signals arising from the resulting fluorescence. The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity, and compounds were subsequently categorized based on their curve class. Active inhibitors showed concentration-dependent decreases in GFP fluorescence, concordant with a decrease in TGF-B/SMAD3-driven GFP expression. Inactive compounds showed no effect on fluorescence signal.
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 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.
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 0.00295 uM (0.00295μM**)% Activity at given concentration.Float%
16Activity at 0.015 uM (0.0147μM**)% Activity at given concentration.Float%
17Activity at 0.074 uM (0.0737μM**)% Activity at given concentration.Float%
18Activity at 0.369 uM (0.369μM**)% Activity at given concentration.Float%
19Activity at 1.840 uM (1.84μM**)% Activity at given concentration.Float%
20Activity at 9.220 uM (9.22μM**)% Activity at given concentration.Float%
21Activity at 46.10 uM (46.1μM**)% Activity at given concentration.Float%
22Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH087449

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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