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BioAssay: AID 434947

Luminescence Cell-Free Homogeneous Counter Screen to Identify Inhibitors of ADP-glo Reagents

The glycogen synthase kinase-3 beta (GSK-3b) is a known master regulator for several cellular pathways and plays a critical role in metabolism, transcription, development, cell survival, and neuronal functions. We previously screened the MLPCN library for inhibitors of GSK-3b using ADP-Glo (Promega V9103). Screen actives were picked. In this assay, the actives were tested in more ..
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 Tested Compounds
 Tested Compounds
All(2198)
 
 
Active(13)
 
 
Inactive(2181)
 
 
Unspecified(4)
 
 
 Tested Substances
 Tested Substances
All(2198)
 
 
Active(13)
 
 
Inactive(2181)
 
 
Unspecified(4)
 
 
 Related BioAssays
 Related BioAssays
AID: 434947
Data Source: Broad Institute (2046-03_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-05-26
Hold-until Date: 2010-05-28
Modify Date: 2010-05-28

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 13
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434954Luminescence Cell-Free Homogeneous Dose Retest to Identify Inhibitors of Glycogen Synthase Kinase-3 beta ActivityConfirmatorysame project related to Summary assay
623978Inhibition of Human GSK3 alpha Activity Measured in Biochemical System Using Microfluidics - 2046-11_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623979Inhibition of Human CDK5 Activity Measured in Biochemical System Using Microfluidics - 2046-12_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623989Inhibition of Human GSK3 beta Activity Measured in Biochemical System Using Microfluidics - 2046-10_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623998GSK3beta Measured in Biochemical System Using Plate Reader - 2046-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
623999CDK5 Measured in Biochemical System Using Plate Reader - 2046-05_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624027GSK3beta Assay at Varied ATP concentrations Measured in Biochemical System Using Plate Reader - 2046-04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624057Human p-Tau and Total Tau ELISA Measured in Cell-Based System Using Plate Reader - 2133-07_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
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Description:
Keywords: ADP-glo, counter screen, GSK3beta, kinase, inhibition, HTS

Assay Overview:
The glycogen synthase kinase-3 beta (GSK-3b) is a known master regulator for several cellular pathways and plays a critical role in metabolism, transcription, development, cell survival, and neuronal functions. We previously screened the MLPCN library for inhibitors of GSK-3b using ADP-Glo (Promega V9103). Screen actives were picked. In this assay, the actives were tested in concentration-response for their ability to inhibit the detection components of the ADP-glo reagents to rule out false positives. Specifically, the ability of the compounds to inhibit signal generated from 5 uM ADP was measured with Viewlux (PerkinElmer).
Positive control wells, in which no ADP was added, were included in each plate and used to scale the data in conjunction with in-plate DMSO controls (for details see Data Analysis section.)

Expected Outcome:
Inhibitors for the ADP-glo reagents will show as loss of luminescence signal.
Protocol
1) Dispense 2.5 uL of 5 uM ADP or buffer AB into wells on 1536-well assay ready plates (Aurora 29847) generated by acoustic transfer using Labcyte Echo that contain 7.5 nL/well of 10 mM compound using Combi nL (Thermo).

2) Add 2.5 uL/well of ADP-glo (Promega, V9103) with Combi nL (Thermo), incubate at room temperature for 40 minutes

3) Add 5 uL/well of ADP-glo (Promega, V9103) with Combi nL (Thermo), incubate at room temperature for 35 minutes

4) Read on Viewlux (PerkinElmer) for luminescence

Solutions:
AB:
25 nM Tris 7.5
10 mM MgCl2

ADP (in AB, Promega V9103 component):
5 um ADP
Comment
Dose Data Analysis

Neutral control wells (NC) and positive controls wells (PC) were included on every plate.
Active compounds result in decreased readout signal.


Normalization and Pattern Correction

The raw signal was normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate PC wells is set to a normalized activity value of -100.
The median raw signal of the intraplate NC wells is set to a normalized activity of 0.
The raw signal of each compound well is then scaled to these two reference points to obtain a normalized activity value.

No plate pattern correction algorithm method was applied.

EC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). EC50 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(EC50)

PUBCHEM_ACTIVITY_OUTCOME:

Activity_Outcome = 1 (inactive) when
EC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active) when
EC50 <= 1 log over the highest tested concentration

Activity_Outcome = 3 (inconclusive) when
Curve fitting strategy resulted in a constant fit with activity >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 at which 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 valueFloat
5Hill_Slopethe slope at EC50Float
6S0the fitted activity level at zero concentrationFloat%
7SInfthe fitted activity level at infinite concentrationFloat%
8Num_Pointsthe number of data points included in the plotInteger
9Max_Activitythe maximum activity value observedFloat%
10Activity_at_13.5nM (0.0135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity_at_0.042uM (0.042μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.12uM (0.12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.38uM (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_1.1uM (1.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_3.5uM (3.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_10uM (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_30uM (30μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH087442-01

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