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BioAssay: AID 623998

GSK3beta Measured in Biochemical System Using Plate Reader - 2046-01_Inhibitor_Dose_DryPowder_Activity

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. The overall objective is to identify one or multiple series of inhibitors of GSK-3beta with micromolar potency. Such compounds will become probe(s) with demonstrated kinase-selectivity more ..
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 Tested Compounds
 Tested Compounds
All(87)
 
 
Active(78)
 
 
Inactive(3)
 
 
Inconclusive(6)
 
 
 Tested Substances
 Tested Substances
All(90)
 
 
Active(81)
 
 
Inactive(3)
 
 
Inconclusive(6)
 
 
AID: 623998
Data Source: Broad Institute (2046-01_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-04-04
Hold-until Date: 2013-04-04
Modify Date: 2013-04-04

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 78
Related Experiments
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AIDNameTypeComment
2119Summary of Probe Development Efforts to Identify Inhibitors of Glycogen Synthase Kinase-3 beta (GSK-3b)Summarydepositor-specified cross reference: Summary assay
2097Cell-Free Homogeneous Primary HTS to Identify Inhibitors of GSK3beta ActivityScreeningsame project related to Summary assay
434947Luminescence Cell-Free Homogeneous Counter Screen to Identify Inhibitors of ADP-glo ReagentsConfirmatorysame project related to Summary assay
434954Luminescence Cell-Free Homogeneous Dose Retest to Identify Inhibitors of Glycogen Synthase Kinase-3 beta ActivityConfirmatorysame project related to Summary assay
435010Fluorescence Cell-Free Homogeneous Dose Retest to Identify Inhibitors of RecA-Intein Splicing 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
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
624076Kinome profiling single dose point Measured in Biochemical System Using Plate Reader - 2133-09_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
624086TCF/LEF Reporter Assay Measured in Cell-Based System Using Plate Reader - 2133-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624088PathHunter bCatenin (U2-OS) Measured in Cell-Based System Using Plate Reader - 2133-06_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624091Dose responses of selected compounds against GSK3b Measured in Biochemical System Using Plate Reader - 2133-10_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
624108GSK3beta Surface Plasmon Resonance (SPR) Assay Measured in Biochemical System Using Microfluidics - 2133-08_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
Description:
Keywords: 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. The overall objective is to identify one or multiple series of inhibitors of GSK-3beta with micromolar potency. Such compounds will become probe(s) with demonstrated kinase-selectivity and activation of Wnt signaling and desired effect on neuronal morphology. Structural series that demonstrates non-ATP competitive mechanism of action will be strongly preferred. After initial characterization of potency and selectivity, dry powders of promising compounds and their analogues were ordered or synthesized. This assay measures potency of these compounds. 4.7 ng of GSK3beta (as a GST fusion from BPS Bioscience) was incubated with varied doses of compounds (specific reaction conditions see Protocol) for 60 minutes at ambient temperature in 384 plates. The kinase activity was measured with ADP-Glo (Promega V9103) and signals were read with Viewlux (PerkinElmer). Positive control (GW8510 at 20 uM) was included in each plate and used to scale the data in conjunction with in-plate DMSO controls (details see Data Analysis or Comments section).

Expected Outcome:
Inhibitors for GSK3beta activity will show as loss of luminescence signal.
Protocol
Protocol:
1) Dispense 20 uL/well of KBAPE onto 384-well plate(s) with Combi
2) Pin 100 nL/well of compounds
3) Dispense 1 uL/well of ATP Combi NL
4) Incubate at room temperature for 60 minutes.
5) Add 20uL/well of ADP-glo (Promega, V9103) with Combi, incubate at room temperature for 40 minutes
6) Add 40 uL/well of ADP-glo (Promega, V9103) with Combi, incubate at room temperature for 30 minutes
7) Read on Envision for luminescence
Solutions:
KBAPE: 4.7nM GSK3b and 7uM peptide in KBA (250 mM HEPES (pH 7.5), 50 mM MgCl2, 5 mM EGTA, 0.05% BRIJ-35)
ATP: 140 uM ATP in KBA
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=168) and positive control wells (PC; n=18) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
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 (2)(Max_Concentration).
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
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.
DATA AGGREGATION:
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
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
4ACTIVITY_OUTCOME_TEST1The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
5ACTIVITY_SCORE_TEST1The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
6ASSAYDATA_COMMENT_TEST1Comments relevant to the subset of data included in the indicated Test number.String
7AC50_Qualifier_TEST1'>','=', or '<'String
8AC50_uM_TEST1The concentration at which activity reaches 50% of the maximumFloatμM
9pAC50_M_TEST1Equal to -1*log10(AC50)String
10Hill_Slope_TEST1The slope at AC50Float
11S0_(%)_TEST1The fitted activity value at zero concentrationFloat%
12Sinf_(%)_TEST1The fitted activity value at infinite concentrationFloat%
13Num_Points_TEST1The number of data points used to generate the plotInteger
14Max_Activity_(%)_TEST1The maximum activity value observed, based on mean of replicates per concentrationFloat%
15Max_Activity_Conc_uM_TEST1The concentration at which the maximum activity is observedFloatμM
16Max_Concentration_uM_TEST1Maximum valid test concentrationFloatμM
17Activity_at_0.00075uM_(%)_TEST1 (0.00075μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.0021uM_(%)_TEST1 (0.0021μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.0068uM_(%)_TEST1 (0.0068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.0075uM_(%)_TEST1 (0.0075μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.0195uM_(%)_TEST1 (0.0195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.021uM_(%)_TEST1 (0.021μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.06uM_(%)_TEST1 (0.06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.068uM_(%)_TEST1 (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_0.18uM_(%)_TEST1 (0.18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_0.195uM_(%)_TEST1 (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_0.5uM_(%)_TEST1 (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_0.6uM_(%)_TEST1 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_1.6uM_(%)_TEST1 (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_1.8uM_(%)_TEST1 (1.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
31Activity_at_4.6uM_(%)_TEST1 (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
32Activity_at_5uM_(%)_TEST1 (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
33Activity_at_16uM_(%)_TEST1 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
34Activity_at_46uM_(%)_TEST1 (46μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
35ACTIVITY_OUTCOME_TEST2The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
36ACTIVITY_SCORE_TEST2The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
37ASSAYDATA_COMMENT_TEST2Comments relevant to the subset of data included in the indicated Test number.String
38AC50_Qualifier_TEST2'>','=', or '<'String
39AC50_uM_TEST2The concentration at which activity reaches 50% of the maximumFloatμM
40pAC50_M_TEST2Equal to -1*log10(AC50)String
41Hill_Slope_TEST2The slope at AC50Float
42S0_(%)_TEST2The fitted activity value at zero concentrationFloat%
43Sinf_(%)_TEST2The fitted activity value at infinite concentrationFloat%
44Num_Points_TEST2The number of data points used to generate the plotInteger
45Max_Activity_(%)_TEST2The maximum activity value observed, based on mean of replicates per concentrationFloat%
46Max_Activity_Conc_uM_TEST2The concentration at which the maximum activity is observedFloatμM
47Max_Concentration_uM_TEST2Maximum valid test concentrationFloatμM
48Activity_at_0.00075uM_(%)_TEST2 (0.00075μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
49Activity_at_0.0021uM_(%)_TEST2 (0.0021μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
50Activity_at_0.0068uM_(%)_TEST2 (0.0068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
51Activity_at_0.0075uM_(%)_TEST2 (0.0075μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
52Activity_at_0.0195uM_(%)_TEST2 (0.0195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
53Activity_at_0.021uM_(%)_TEST2 (0.021μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
54Activity_at_0.06uM_(%)_TEST2 (0.06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
55Activity_at_0.068uM_(%)_TEST2 (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
56Activity_at_0.18uM_(%)_TEST2 (0.18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
57Activity_at_0.195uM_(%)_TEST2 (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
58Activity_at_0.5uM_(%)_TEST2 (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
59Activity_at_0.6uM_(%)_TEST2 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
60Activity_at_1.6uM_(%)_TEST2 (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
61Activity_at_1.8uM_(%)_TEST2 (1.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
62Activity_at_4.6uM_(%)_TEST2 (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
63Activity_at_5uM_(%)_TEST2 (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
64Activity_at_16uM_(%)_TEST2 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
65Activity_at_46uM_(%)_TEST2 (46μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
66ACTIVITY_OUTCOME_TEST3The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
67ACTIVITY_SCORE_TEST3The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
68ASSAYDATA_COMMENT_TEST3Comments relevant to the subset of data included in the indicated Test number.String
69AC50_Qualifier_TEST3'>','=', or '<'String
70AC50_uM_TEST3The concentration at which activity reaches 50% of the maximumFloatμM
71pAC50_M_TEST3Equal to -1*log10(AC50)String
72Hill_Slope_TEST3The slope at AC50Float
73S0_(%)_TEST3The fitted activity value at zero concentrationFloat%
74Sinf_(%)_TEST3The fitted activity value at infinite concentrationFloat%
75Num_Points_TEST3The number of data points used to generate the plotInteger
76Max_Activity_(%)_TEST3The maximum activity value observed, based on mean of replicates per concentrationFloat%
77Max_Activity_Conc_uM_TEST3The concentration at which the maximum activity is observedFloatμM
78Max_Concentration_uM_TEST3Maximum valid test concentrationFloatμM
79Activity_at_0.00075uM_(%)_TEST3 (0.00075μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
80Activity_at_0.0021uM_(%)_TEST3 (0.0021μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
81Activity_at_0.0068uM_(%)_TEST3 (0.0068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
82Activity_at_0.0075uM_(%)_TEST3 (0.0075μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
83Activity_at_0.0195uM_(%)_TEST3 (0.0195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
84Activity_at_0.021uM_(%)_TEST3 (0.021μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
85Activity_at_0.06uM_(%)_TEST3 (0.06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
86Activity_at_0.068uM_(%)_TEST3 (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
87Activity_at_0.18uM_(%)_TEST3 (0.18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
88Activity_at_0.195uM_(%)_TEST3 (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
89Activity_at_0.5uM_(%)_TEST3 (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
90Activity_at_0.6uM_(%)_TEST3 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
91Activity_at_1.6uM_(%)_TEST3 (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
92Activity_at_1.8uM_(%)_TEST3 (1.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
93Activity_at_4.6uM_(%)_TEST3 (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
94Activity_at_5uM_(%)_TEST3 (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
95Activity_at_16uM_(%)_TEST3 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
96Activity_at_46uM_(%)_TEST3 (46μM**)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
Classification
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