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BioAssay: AID 651724

qHTS Assay for Inhibitors of the CtBP/E1A Interaction

Carboxyl-terminal binding protein (CtBP) is a co-repressor for many transcription factors. CtBP was initially recognized as an adenoviral E1A-binding protein and its over-activation, in combination with mutated Ras, leads to tumorigenesis and metastasis, suggesting CtBP plays a critical role in oncogenesis. Our studies have shown that CtBP is overexpressed in multiple human cancers, including more ..
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 Tested Compounds
 Tested Compounds
All(335201)
 
 
Active(1652)
 
 
Inactive(328062)
 
 
Inconclusive(5500)
 
 
 Tested Substances
 Tested Substances
All(335532)
 
 
Active(1654)
 
 
Inactive(328376)
 
 
Inconclusive(5502)
 
 
AID: 651724
Data Source: NCGC (CTBP100)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-10-29

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1652
Related Experiments
AIDNameTypeComment
651737qHTS Assay for Inhibitors of the CtBP/E1A InteractionSummarydepositor-specified cross reference
Description:
Carboxyl-terminal binding protein (CtBP) is a co-repressor for many transcription factors. CtBP was initially recognized as an adenoviral E1A-binding protein and its over-activation, in combination with mutated Ras, leads to tumorigenesis and metastasis, suggesting CtBP plays a critical role in oncogenesis. Our studies have shown that CtBP is overexpressed in multiple human cancers, including lung, breast, and head and neck cancers. In particular, it is over-expressed in 50% of primary lung cancer and 90% of metastatic lesions. CtBP suppresses epithelial-specific genes as well as genes critical for apoptosis, providing the underlying molecular mechanism for its role in tumorigenesis. Moreover, we found that CtBP-knockdown in lung cancer cells induced p53-independent apoptosis, and suppressed human tumor growth in mouse model. Based on the above data, we hypothesize that 1) CtBP suppresses the pro-apoptotic and epithelial genes, therefore overexpressed CtBP in human cancer cells increases EMT and cancer survival; 2) decreasing CtBP's function represents a targeted therapy for lung cancer and potentially multiple cancer types. As a co-repressor, CtBP binds to E1A and other transcription factors through a conserved peptide motif to carry out its action. Inhibiting this interaction using small molecules can potentially serve as effective cancer therapeutics.

To this end, we have developed a homogenous AlphaScreen assay targeting the CtBP/E1A interaction. We performed a large scale HTS of the Molecular Libraries Small Molecule Repository (MLSMR) to identify inhibitors of the CtBP/E1A interaction as potential anti-cancer therapeutics. Compounds identified can also be used as chemical probes to better understand the biological functions of CtBP.

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

MLPCN Grant: DA033982
Assay Submitter (PI): Rui Zhao, University of Colorado Denver
Protocol
The first step of the 5uL AlphaScreen assay is to dispense assay buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 0.05% BSA, 0.02% Tween 20, 1 mM TCEP) into a Greiner 1536 white solid bottom microtiter plate (789175-F). Five microliters is added to Column 1, 4 uL is added to Column 2 and 3 uL is added to Columns 3 through 48. A 5X protein solution (125 nM GST-E1A protein + 125 nM His-tagged CtBP protein) is mixed and 1 uL is dispensed to Columns 3-48. Using a 1536 pintool, 23 nL of compound is transferred to the assay plate and the plate is incubated with a lid at 37 deg C in the dark for two hours. A solution of 5X AlphaScreen beads is mixed (50 ug/mL Glutathione linked donor beads + Nickel chelated acceptor beads) and 1 uL is dispensed to Columns 2-48. The assay plate is incubated for one hour, with a lid, at 37 deg C in the dark. The plate is read using a Perkin Elmer Envision using an AlphaScreen protocol.
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
2PotencyConcentration 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%
15Percent of ActivityFloat%
16Compound QCString
Additional Information
Grant Number: DA033982

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