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BioAssay: AID 651725

qHTS Assay for Inhibitors of the Six1/Eya2 Interaction

The Six1 homeobox gene encodes a transcription factor that is crucial for the development of many organs but is down-regulated after organ development is complete. Its expression is low or undetectable in normal adult breast tissue but the gene is over-expressed in 50% of primary breast tumors and 90% of metastatic lesions. Our analysis of public microarray databases containing 535 breast more ..
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 Tested Compounds
 Tested Compounds
All(364041)
 
 
Active(2025)
 
 
Inactive(354729)
 
 
Inconclusive(7298)
 
 
 Tested Substances
 Tested Substances
All(364407)
 
 
Active(2028)
 
 
Inactive(355075)
 
 
Inconclusive(7304)
 
 
AID: 651725
Data Source: NCGC (Six1100)
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: 2025
Related Experiments
AIDNameTypeComment
651736qHTS Assay for Inhibitors of the Six1/Eya2 InteractionSummarydepositor-specified cross reference
Description:
The Six1 homeobox gene encodes a transcription factor that is crucial for the development of many organs but is down-regulated after organ development is complete. Its expression is low or undetectable in normal adult breast tissue but the gene is over-expressed in 50% of primary breast tumors and 90% of metastatic lesions. Our analysis of public microarray databases containing 535 breast cancer samples demonstrates that Six1 levels correlate significantly with shortened time to relapse, shortened time to metastasis, and decreased survival. Using transgenic and xenograft mouse models, we have demonstrated that mammary specific overexpression of Six1 results in enhanced proliferation, transformation, increased tumor volume, and metastasis. Importantly, RNA interference against Six1 significantly decreases breast cancer associated metastasis. Together these data demonstrate that Six1 is necessary and sufficient to induce breast cancer metastasis. Six1 does not have an intrinsic activation or repression domain and requires the Eya coactivator proteins to activate transcription. Similar to Six1, Eya2 is also not expressed in most normal adult tissues, but is reexpressed in cancers. The Six1-Eya interaction is essential for proliferation during embryonic development, and both Six1 and Eya2 have been independently implicated in the same types of cancer. Because Six1 and Eya2 are embryonic genes with little expression in adult tissues, inhibitors of their expression/activity are likely to have limited side effects. Our goal is to identify inhibitors of the Six1/Eya interaction for potential cancer therapy.

To this end, we have developed a homogenous AlphaScreen assay targeting the Six1/Eya interaction. We performed a large scale HTS of the Molecular Libraries Small Molecule Repository (MLSMR) to identify inhibitors of the Six1/Eya interaction as potential anti-cancer therapeutics. Compounds identified can also be used as chemical probes to better understand the biological functions of Six1 and Eya.

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

MLPCN Grant: DA033147
Assay Submitter (PI): Rui Zhao, University of Colorado Denver
Protocol
The first step of the 5 uL 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 (250 nM GST-Six1 protein + 500 nM His-tagged Eya2 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. 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: DA033147

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