AID	Name	Type	Comment
588856	qHTS for Inhibitors of TGF-b: Cytotox Counterscreen	confirmatory
588860	qHTS for Inhibitors of TGF-b: Summary	summary

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

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.

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.

Grant Number: MH087449