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BioAssay: AID 588756

Counter screen for activity against Aurora B, in dose Measured in Biochemical System Using Scintillation - 2052-06_Inhibitor_Dose_DryPowder_Activity_Set2

Assay Overview: This assay was outsourced to Millipore corporation. The inhibition of hAuroraB kinase activity is measured in response to various concentrations of the compound of interest (STK33 inhibitors). Comparison is made between the IC50s to determine which compounds are specific against STK33. ..more
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 Tested Compounds
 Tested Compounds
All(8)
 
 
Active(5)
 
 
Inactive(4)
 
 
 Tested Substances
 Tested Substances
All(9)
 
 
Active(5)
 
 
Inactive(4)
 
 
AID: 588756
Data Source: Broad Institute (2052-06_Inhibitor_Dose_DryPowder_Activity_Set2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: Assay Vendor
Deposit Date: 2011-10-31
Hold-until Date: 2012-10-27
Modify Date: 2012-10-27

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 5
Related Experiments
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AIDNameTypeProbeComment
2689Summary of Broad Institute MLPCN Serine Threonine Kinase 33 Inhibitor ProjectSummary2 depositor-specified cross reference: Summary assay
2661Luminescence Cell-Free Homogenous Primary HTS to Identify Inhibitors of Serine/Threonine Kinase 33 ActivityScreening same project related to Summary assay
2821Luminescence Cell-Free Homogenous Dose Retest to Identify Inhibitors of Serine/Threonine Kinase 33 ActivityConfirmatory same project related to Summary assay
504583Protein Kinase A Activity Assay Measured in Biochemical System Using Plate Reader - 2052-04_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
588480STK-33 Kinase Inhibition Measured in Biochemical System Using Plate Reader - 2052-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
588629Protein Kinase A Activity Assay Measured in Biochemical System Using Plate Reader - 2052-04_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
588632STK-33 Kinase Inhibition Measured in Biochemical System Using Plate Reader - 2052-02_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
588757Counter screen for activity against Aurora B, in dose Measured in Biochemical System Using Scintillation - 2052-06_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
588808Counter screen for activity against Aurora B, in dose Measured in Biochemical System Using Scintillation - 2052-06_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
602158STK-33 Kinase Inhibition Measured in Biochemical System Using Plate Reader - 2052-02_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
Description:
Keywords: hAuroraB Kinase

Assay Overview: This assay was outsourced to Millipore corporation. The inhibition of hAuroraB kinase activity is measured in response to various concentrations of the compound of interest (STK33 inhibitors). Comparison is made between the IC50s to determine which compounds are specific against STK33.

Expected Outcome: Compounds showing AurB inhibition activity are reported as active in this assay. Compounds which are specific STK33 inhibitors will not inhibit AurB activitiy. Non-specific STK-33 inhibitors will inhibit AurB activity.
Protocol
These assays were performed at [ATP] = Km

Aurora-B (h)
Aurora-B (h) is incubated with 8 mM MOPS pH 7.0,
0.2 mM EDTA, 30 uM AKRRRLSSLRA, 10 mM MgAcetate
and [gamma-32P-ATP] (specific activity approx. 500 cpm/
pmol, concentration as required). The reaction is
initiated by the addition of the MgATP mix. After
incubation for 40 minutes at room temperature, the
reaction is stopped by the addition of a 3% phosphoric
acid solution. 10 microL of the reaction is then spotted
onto a P30 filtermat and washed three times for
5 minutes in 75 mM phosphoric acid and once in
methanol prior to drying and scintillation counting
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (10)(Max_Concentration).

NORMALIZATION:
No normalization was applied to the raw data signals.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4Hill_SlopeThe slope at AC50Float
5S0_(%)The fitted activity value at zero concentrationFloat%
6Sinf_(%)The fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Max_Activity_(%)The maximum activity value observed, based on mean of replicates per concentrationFloat%
9Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
10Max_Concentration_uMMaximum valid test concentrationFloatμM
11Activity_at_0.001uM_(%) (0.001μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.003uM_(%) (0.003μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.01uM_(%) (0.01μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.03uM_(%) (0.03μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.1uM_(%) (0.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.3uM_(%) (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_10uM_(%) (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_1uM_(%) (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_3uM_(%) (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH089854-01

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