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BioAssay: AID 686947

qHTS for small molecule inhibitors of Yes1 kinase: Primary Screen

The Src family of non-receptor protein tyrosine kinases contains nine members, including Yes1, Src, Fyn, Lyn, and Lyk. These kinases have important roles in a variety of cellular functions, such as cell proliferation, survival, and differentiation. Src, the most well characterized member of this family, has been previously identified as a proto-oncogene. Like Src, Yes1 kinase activity has been more ..
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 Tested Compounds
 Tested Compounds
All(839)
 
 
Active(349)
 
 
Inactive(380)
 
 
Inconclusive(115)
 
 
 Tested Substances
 Tested Substances
All(858)
 
 
Active(361)
 
 
Inactive(381)
 
 
Inconclusive(116)
 
 
AID: 686947
Data Source: NCGC (Yes1-inhibition-p1)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-04-18
Hold-until Date: 2013-07-04
Modify Date: 2013-07-09

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 349
Related Experiments
AIDNameTypeComment
686946qHTS for small molecule inhibitors of Yes1 kinase: SummarySummarydepositor-specified cross reference
686948qHTS for small molecule inhibitors of Yes1 kinase: Confirmatory screen on the cherrypicksConfirmatorysame project related to Summary assay
Description:
The Src family of non-receptor protein tyrosine kinases contains nine members, including Yes1, Src, Fyn, Lyn, and Lyk. These kinases have important roles in a variety of cellular functions, such as cell proliferation, survival, and differentiation. Src, the most well characterized member of this family, has been previously identified as a proto-oncogene. Like Src, Yes1 kinase activity has been shown to be activated in a variety of cancers, including colon carcinomas, melanoma, head and neck, renal, lung, and stomach cancers. In melanoma, it has been shown that Yes1, and not other family members, such as Src, is functionally involved in the malignant phenotype. Additionally, a recent study examining the downregulation of Yes1 by shRNA found significant effects on cell survival/growth for basal-like and Her2-positive breast cancers. Despite the potential for Yes1 to be a target for the above-described cancers, there are very few reports on the identification of potent and selective inhibitors of Yes1 kinase. Antagonists of Yes1 would further elucidate Yes1 biology and help to confirm whether Yes1 is a viable target for therapeutic intervention in a variety of cancers.

A compound that inhibits Yes1 could be important for the treatment of various cancers in which Yes1 is highly expressed and can also be used as a tool compound to help further elucidate Yes1 biology. To that end, we have developed a sensitive HTS biochemical assay for Yes1 activity. This assay was screened against several small compound libraries to look for modulators of activity. Inhibition is the desired output for this assay.

NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: MH084681
Protocol
Two micoliter of Yes1 enzyme (columns 1-2, 5-48; Millipore, Billerica, MA) are dispensed into Greiner, white solid-bottom 1536-well assay plates; columns 3 and 4 receive buffer without Yes1 as a control for maximum inhibition. Compounds (23 nL) are then transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). The library compounds are in columns 5-48 and columns 1-4 serve as controls with DMSO in columns 1, 3 and 4 and 10 mM 1:3 fold serial of dasatinib in column 2. Following addition of compound and a 20 minute incubation, 1 uL of substrate (ATP and poly-E4Y from Sigma-Aldrich, St. Louis, MO) is added, and the biochemical reaction is allowed to progress for 60 minutes at room temperature. The production of ADP is then quantified by the ADP-Glo Kit (Promega, Madison, WI). First, 2 uL of the first kit reagent is added, and the reaction is incubated for 45 minutes; then, 4 uL of the second kit reagent are added and the reaction is incubated for 30 minutes at which time the luminescence is read on a Viewlux plate reader (Perkin-Elmer, Waltham, MA). The final concentration of reagents in the reaction are 4 nM Yes1, 0.1 mM ATP, 100 uM EGTA, 10 mM MgCl2, 0.3 mg/mL poly(E4Y), 50 mM Tris pH 7.5, 1 mM DTT, 150 mM NaCl, 0.01% Brij-35, and 5% glycerol. Data are normalized to DMSO-treated control columns with (maximum signal) and without (minimum signal) enzyme.
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 0.0004350541 uM (0.000435054μM**)% Activity at given concentration.Float%
16Activity at 0.0007425385 uM (0.000742538μM**)% Activity at given concentration.Float%
17Activity at 0.00130 uM (0.00130375μM**)% Activity at given concentration.Float%
18Activity at 0.00257 uM (0.00257418μM**)% Activity at given concentration.Float%
19Activity at 0.00391 uM (0.00390941μM**)% Activity at given concentration.Float%
20Activity at 0.00757 uM (0.00757205μM**)% Activity at given concentration.Float%
21Activity at 0.012 uM (0.0117243μM**)% Activity at given concentration.Float%
22Activity at 0.022 uM (0.0221656μM**)% Activity at given concentration.Float%
23Activity at 0.035 uM (0.0351755μM**)% Activity at given concentration.Float%
24Activity at 0.066 uM (0.0664966μM**)% Activity at given concentration.Float%
25Activity at 0.105 uM (0.105477μM**)% Activity at given concentration.Float%
26Activity at 0.157 uM (0.156972μM**)% Activity at given concentration.Float%
27Activity at 0.315 uM (0.314772μM**)% Activity at given concentration.Float%
28Activity at 0.478 uM (0.477847μM**)% Activity at given concentration.Float%
29Activity at 0.631 uM (0.630579μM**)% Activity at given concentration.Float%
30Activity at 0.951 uM (0.9507μM**)% Activity at given concentration.Float%
31Activity at 1.910 uM (1.91007μM**)% Activity at given concentration.Float%
32Activity at 2.851 uM (2.85132μM**)% Activity at given concentration.Float%
33Activity at 5.567 uM (5.56664μM**)% Activity at given concentration.Float%
34Activity at 8.551 uM (8.5511μM**)% Activity at given concentration.Float%
35Activity at 16.73 uM (16.7259μM**)% Activity at given concentration.Float%
36Activity at 25.64 uM (25.641μM**)% Activity at given concentration.Float%
37Activity at 47.78 uM (47.78μM**)% Activity at given concentration.Float%
38Activity at 76.92 uM (76.9231μM**)% Activity at given concentration.Float%
39Activity at 153.8 uM (153.846μM**)% Activity at given concentration.Float%
40Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH084681

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