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BioAssay: AID 2821

Luminescence Cell-Free Homogenous Dose Retest to Identify Inhibitors of Serine/Threonine Kinase 33 Activity

Assay Overview: Purified STK33 Kinase is preincubated with potential inhibitors and allowed to phosphorylate MBP at ~ 1.5 x Km ATP. Kinase activity is measured using the ADP Glo Kit (Promega) which converts ADP reaction product into a luminescent signal. ..more
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 Tested Compounds
 Tested Compounds
All(224)
 
 
Active(174)
 
 
Inactive(50)
 
 
 Tested Substances
 Tested Substances
All(224)
 
 
Active(174)
 
 
Inactive(50)
 
 
AID: 2821
Data Source: Broad Institute (2052-01_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-05-10

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 174
Related Experiments
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AIDNameTypeProbeComment
2661Luminescence Cell-Free Homogenous Primary HTS to Identify Inhibitors of Serine/Threonine Kinase 33 ActivityScreening depositor-specified cross reference: Primary HTS
2689Summary of Broad Institute MLPCN Serine Threonine Kinase 33 Inhibitor ProjectSummary2 depositor-specified cross reference: Project Summary
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
588756Counter screen for activity against Aurora B, in dose Measured in Biochemical System Using Scintillation - 2052-06_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory 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: STK33 Kinase, Non-ATP Competitive Inhibitor

Assay Overview: Purified STK33 Kinase is preincubated with potential inhibitors and allowed to phosphorylate MBP at ~ 1.5 x Km ATP. Kinase activity is measured using the ADP Glo Kit (Promega) which converts ADP reaction product into a luminescent signal.

Expected Outcome: Inhibitors of STK33 Kinase will cause a decreased luminescent readout.
Protocol
Assay Buffer (AB)
10 mM MOPS pH 7
300 uM EDTA
0.5 % (V/V) Glycerol
0.001% (V/V) Brij-35
0.1 mg/mL BSA
0.01% (V/V) beta mercapto ethanol
ATP/Mg
250 uM ATP
50 mM MgAc2
in AB
STK33/MBP (E)
50 nM STK33
0.5 mg/mL Myelin Basic Protein
in AB
1536 well plate assay
Add 2.0 uL AB to last two columns of 1536 well white Aurora high-base assay plate (containing 2.5 nL 10 mM compound or DMSO) using combi-nL dispenser (Thermo). Add 2.0 uL E to all but last two columns of 1536 ARP (containing 2.5 nL 10 mM compound/well). Incubate 15 minutes at room temperature.
Add 0.5 uL ATP/Mg / well to full plate. Incubate 60 minutes.
Add 2.5 uL / well ADP Glo Reagent I (Promega). Incubate 40 minutes.
Add 5 uL / well ADP Glo Reagent II (Promega). Incubate 30 minutes.
Read luminescence 15 sec integration time on ViewLux plate reader (Perkin Elmer).
384 well plate assay
Add 20 uL AB to last column of 384 well white Aurora assay plate using combi dispenser (Thermo). Add 20 uL E to all but last column. Using a pintool, transfer 25 nL compound in DMSO stock solution into assay plate. Incubate 15 minutes at room temperature.
Add 5 uL ATP/Mg / well to full plate. Incubate 60 minutes.
Add 25 uL / well ADP Glo Reagent I (Promega). Incubate 40 minutes.
Add 50 uL / well ADP Glo Reagent II (Promega). Incubate 30 minutes.
Read luminescence 0.1 sec integration time on Envision plate reader (Perkin Elmer).
Comment
Dose Data Analysis
Neutral control wells (NC), positive controls wells (PC) and blank wells were included on every plate.
Active compounds result in decreased readout signal.
Analysis used to determine PubChem Activity Score and Outcome
The raw signal was normalized using the 'Neutral Controls Minus Blanks' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate NC wells was set to a normalized activity value of 0.
The median raw signal of the intraplate blank wells was set to a normalized activity of -100.
The raw signal of each compound well was then scaled to these two reference points to obtain a normalized activity value.
No plate pattern correction algorithm was used.
IC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). IC50 values were extrapolated up to 1 log
over the highest tested concentration.
PubChem Activity Score and Outcome
PUBCHEM_ACTIVITY_SCORE:
Inactive compounds = 0
Active compounds = -10*Log(IC50)
PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive)
IC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active)
IC50 <= 1 log over the highest tested concentration
Activity_Outcome = 3 (inconclusive)
Curve fitting strategy resulted in a constant fit with activtity >30% but <70%
or
The fit was not valid due to poor fit quality.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50_Qualifier'>', '=', or '<'String
2EC50_uM*the concentration whereupon activity reaches 50% of the maximumFloatμM
3Log_EC50_Mthe log of the EC50 (molar)Float
4Log_EC50_M_Standard_Errorthe standard error for the log of the EC50 valueFloatM
5S0the fitted activity level at zero concentrationFloat%
6SInfthe fitted activity level at infinite concentrationFloat%
7Hill_Slopethe slope at EC50Float
8Num_Pointsthe number of data points included in the plotInteger
9Max_Activitythe maximum activity value observedFloat%
10Activity_at_0.1uM (0.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity_at_0.195uM (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.38uM (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.8uM (0.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_1.6uM (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_3uM (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_6uM (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_12uM (12μM**)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
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