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BioAssay: AID 588855

qHTS for Inhibitors of TGF-b

TGF-b is a main component in the TGF-b signaling pathway which plays diverse roles in cellular and development pathways. TGF-b is the mediated by the transcription factors Smads. Although the Smad-dependent pathway is the primary canonical TGF-b signaling node, TGF-b1 can also activate alternative signaling pathways, including those involving MAPK (ERK, JNK and p38). This interaction may mediate more ..
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 Tested Compounds
 Tested Compounds
All(403336)
 
 
Active(4970)
 
 
Inactive(348070)
 
 
Inconclusive(51267)
 
 
 Tested Substances
 Tested Substances
All(407820)
 
 
Active(4995)
 
 
Inactive(351223)
 
 
Inconclusive(51602)
 
 
AID: 588855
Data Source: NCGC (SMAD3101)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-12-06

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 4970
Depositor Specified Assays
AIDNameTypeComment
588856qHTS for Inhibitors of TGF-b: Cytotox Counterscreenconfirmatory
588860qHTS for Inhibitors of TGF-b: Summarysummary
720534qHTS for Inhibitors of TGF-b: Confirmation of Cherry Picksconfirmatory
720535qHTS for Inhibitors of TGF-b: Cytotox Counterscreen for Cherry Picksconfirmatory
720537qHTS for Inhibitors of TGF-b: Hit Validation in HepG2 Cells using COP promoterconfirmatory
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 the mediated by the transcription factors Smads. Although the Smad-dependent pathway is the primary canonical TGF-b signaling node, 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.

A high-throughput assay was developed to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR). This is a cell based assay, where TGF-b is tagged with GFP. A counterscreen is run that looks at cytotoxicity (please see summary AID).

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
5Curve_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
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_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
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.018 uM (0.0184μM**)% Activity at given concentration.Float%
15Activity at 0.037 uM (0.0369μM**)% Activity at given concentration.Float%
16Activity at 0.074 uM (0.073789μM**)% Activity at given concentration.Float%
17Activity at 0.164 uM (0.164463μM**)% Activity at given concentration.Float%
18Activity at 0.369 uM (0.369μM**)% Activity at given concentration.Float%
19Activity at 0.461 uM (0.461μM**)% Activity at given concentration.Float%
20Activity at 0.737 uM (0.737μM**)% Activity at given concentration.Float%
21Activity at 0.922 uM (0.922μM**)% Activity at given concentration.Float%
22Activity at 1.840 uM (1.84μM**)% Activity at given concentration.Float%
23Activity at 2.300 uM (2.3μM**)% Activity at given concentration.Float%
24Activity at 3.690 uM (3.69μM**)% Activity at given concentration.Float%
25Activity at 4.610 uM (4.61μM**)% Activity at given concentration.Float%
26Activity at 9.231 uM (9.23099μM**)% Activity at given concentration.Float%
27Activity at 20.57 uM (20.5718μM**)% Activity at given concentration.Float%
28Activity at 46.10 uM (46.1μM**)% Activity at given concentration.Float%
29Activity at 92.20 uM (92.2μM**)% Activity at given concentration.Float%
30Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

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

Data Table (Concise)
Classification
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