qHTS for Inhibitors of TGF-b: Cytotox Counterscreen
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 by 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 more ..
BioActive Compounds: 3100
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 by 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.
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. This is a counterscreen that evaluates the cytotoxicity of the compounds that were screened in the primary cell-based screen (AID 588855).
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. Two uL of CellTiter Glo (Promega), a luminescence-based viability reagent, was dispensed, followed by a 10 minute room temperature incubation. The plates were then measured on a PerkinElmer ViewLux plate reader for luminescence (clear filter) using a 5 second exposure. The %Activity was determined from the corrected luminescence values. Wells containing media only (no cells) were used to normalize %Activity of identified toxic compounds; media-only wells corresponded to 100%Activity (complete cell-killing), while DMSO-dosed cell controls were used to normalize 0%Activity (no toxicity).
Concentration-response curves were fitted to the signals arising from the resulting luminescence. 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. Toxic compounds showed concentration-dependent decreases in luminescence, concordant with a decrease in intracellular ATP concentration (CellTiter Glo's marker of viability), and thus a decrease in the number of viable cells. Inactive (non-toxic) compounds showed no effect on luminescence signal. Active (toxic) compounds showed concentration dependent decrease in luminescence.
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".
2. For all inactive (non-toxic) compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active (toxic) compounds, a score range was given for each curve class type given above. Active (toxic) 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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)