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BioAssay: AID 720541

qHTS for Inhibitors of the Phosphatase Activity of Eya2: Carboxyl-terminal binding protein (CtBP) Counterscreen for Cherry-picked Compounds

The Eya protein family is an essential co-activator of the Six1 transcription factor. Six1 is a developmental gene that is abnormally re-expressed in a large percentage of breast cancers. This over-expression plays a causal role in the initiation and metastatic development of breast cancers. The Eya family of protein is also found to contain a unique HAD phosphatase domain with protein tyrosine more ..
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 Tested Compounds
 Tested Compounds
All(1279)
 
 
Active(416)
 
 
Inactive(454)
 
 
Inconclusive(411)
 
 
 Tested Substances
 Tested Substances
All(1282)
 
 
Active(417)
 
 
Inactive(454)
 
 
Inconclusive(411)
 
 
 Related BioAssays
 Related BioAssays
AID: 720541
Data Source: NCGC (Eya2-six1-f2-CtBP)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-07-20

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 416
Related Experiments
AIDNameTypeComment
488837qHTS Assay for Inhibitors of the Phosphatase Activity of Eya2Confirmatorydepositor-specified cross reference
488939qHTS Assay for Inhibitors of the Phosphatase Activity of Eya2: SummarySummarydepositor-specified cross reference
504895Inhibitors of Eya2ED: Hit Confirmation in Primary ScreenConfirmatorydepositor-specified cross reference
540357Inhibitors of Eya2ED: Hit Confirmation in Malachite Green Secondary AssayOthersame project related to Summary assay
720540qHTS for Inhibitors of the Phosphatase Activity of Eya2: Confirmatory Assay for Cherry-picked CompoundsConfirmatorysame project related to Summary assay
Description:
The Eya protein family is an essential co-activator of the Six1 transcription factor. Six1 is a developmental gene that is abnormally re-expressed in a large percentage of breast cancers. This over-expression plays a causal role in the initiation and metastatic development of breast cancers. The Eya family of protein is also found to contain a unique HAD phosphatase domain with protein tyrosine phosphatase activity which can potentially play a role in Six1-mediated breast tumorigenesis. Recently, Eya2 is found to dephosphorylate the histone variant H2AX and direct cells towards DNA repair instead of apoptosis in the event of DNA damage.

A 1536 well format, fluorescence based qHTS assay was developed and used to target the phosphatase activity of Eya2 (AID 488837). In addition, a CtBP counterscreen was developed using the AlphaScreen technology to evaluate compound selectivity to target Eya1. The data herein reports the activity of the cherry-picked compounds against the CtBP AlphaScreen. Compounds that are active or have > 100 fold selectivity vs. the Eya2 activity were excluded for further characterization. Inactivity is the desired outcome for the CtBP counterscreen.

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

MLPCN Grant: DA030559
Assay Submitter (PI): Rui Zhao, University of Colorado Denver
Protocol
Four microliters of reagent (25 nM GST-E1A, 25 nM CtBP-6x HIS) was dispensed in a 1536 well Greiner plates. Twenty-three nanoliter of test compounds (final concentration of 2.5 nM - 46 uM) and positive control E1A peptide inhibitor (final concentration of 0.14 nM - 4.6 uM) were added to the plates using the Kalypsys pintool equipped with a 1536 pin head. The plates was then incubated at room temperature for 2 hours followed by addition of 1 uL Alpha bead donor / acceptor mix (20 ug/mL final concentration). The assay plates were incubated for additional 1 hour at room temperature and protected from light. The AlphaScreen signal was obtained using the PerkinElmer EnVision plate reader using the 680 nm excitation and 520 - 620 nm emission filters. The data was normalized to the E1A peptide inhibitor for maximum inhibition and DMSO treated wells as negative control.
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
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%
15Activity at 0.00276 uM (0.00276μM**)% Activity at given concentration.Float%
16Activity at 0.015 uM (0.0147μM**)% Activity at given concentration.Float%
17Activity at 0.074 uM (0.0737μM**)% Activity at given concentration.Float%
18Activity at 0.369 uM (0.369μM**)% Activity at given concentration.Float%
19Activity at 1.840 uM (1.84μM**)% Activity at given concentration.Float%
20Activity at 9.220 uM (9.22μM**)% Activity at given concentration.Float%
21Activity at 46.10 uM (46.1μM**)% Activity at given concentration.Float%
22Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: DA030559

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