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BioAssay: AID 588629

Protein Kinase A Activity Assay Measured in Biochemical System Using Plate Reader - 2052-04_Inhibitor_Dose_DryPowder_Activity_Set2

Assay Overview: Inhibition of protein kinase A (PKA) activity is measured using recombinant His tagged PKA catalytic fragment and a peptide substrate in an end-point readout assay. Purified PKA is mixed together with ATP, Mg ions, fluorescently labeled peptide substrate and various concentrations of compounds of interest. The enzymatic reaction is allowed to proceed and then stopped by the addition of the metal chelator EDTA. Conversion of substrate to product is measured using a Caliper Life Sciences capillary electrophoresis lab on chip device. ..more
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 Tested Compounds
 Tested Compounds
All(80)
 
 
Active(6)
 
 
Inactive(68)
 
 
Inconclusive(6)
 
 
 Tested Substances
 Tested Substances
All(82)
 
 
Active(6)
 
 
Inactive(70)
 
 
Inconclusive(6)
 
 
AID: 588629
Data Source: Broad Institute (2052-04_Inhibitor_Dose_DryPowder_Activity_Set2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-10-24
Hold-until Date: 2012-10-21
Modify Date: 2012-10-21

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 6
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
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: PKA, protein kinase A, cAMP dependent Kinase

Assay Overview: Inhibition of protein kinase A (PKA) activity is measured using recombinant His tagged PKA catalytic fragment and a peptide substrate in an end-point readout assay. Purified PKA is mixed together with ATP, Mg ions, fluorescently labeled peptide substrate and various concentrations of compounds of interest. The enzymatic reaction is allowed to proceed and then stopped by the addition of the metal chelator EDTA. Conversion of substrate to product is measured using a Caliper Life Sciences capillary electrophoresis lab on chip device.

Expected Outcome: Non-selective STK33 inhibitors will also inhibit PKA while selective ones will not. IC50s for compounds of interest are determined based on %conversion vs compound concentration and are used to decide which compounds are selective for STK33 or inhibit both kinases.
Protocol
Assay Buffer (AB)
10 mM MOPS pH 7
300 uM EDTA
0.5 % (V/V) Glycerol
0.001% (V/V) Brij-35

In a 384 well polypropylene plate
+ 20 uL / well 15 uM ATP, 15 mM MgAc2 in AB
+ 25 nL compound dilutions in DMSO using a pintool
+ 5 uL 5 nM PKA, 15 uM Peptide 21 in AB
incubate 1 hr RT
1.5 nM PKA, 3 uM Peptide 21 (Fluorescent Peptide Substrate), 12 uM ATP (final conditions)
stop enzyme reaction
+ 10 uL 75 mM EDTA in AB
Determine % conversion of peptide to phospho-peptide using Caliper capillary electrophoresis instrument at the conditions specified by the manufacturer for Peptide 21.

Reported Km ap (ATP) 6 uM
Measure Km ap (Pep21) 4.6 uM

A high ATP concentration assay may be run identically to above save with final concentrations of 0.3 nM PKA, 120 uM ATP as a preliminary test of whether or not an inhibitor is ATP competitive.
Comment
PRESENCE OF CONTROLS: 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 (1)(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.

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_0.000019uM_(%)_TEST1 (1.9e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.000038uM_(%)_TEST1 (3.8e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.000075uM_(%)_TEST1 (7.5e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.00015uM_(%)_TEST1 (0.00015μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.0003uM_(%)_TEST1 (0.0003μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.0006uM_(%)_TEST1 (0.0006μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.0012uM_(%)_TEST1 (0.0012μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.00235uM_(%)_TEST1 (0.00235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_0.005uM_(%)_TEST1 (0.005μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_0.01uM_(%)_TEST1 (0.01μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_0.0195uM_(%)_TEST1 (0.0195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_0.038uM_(%)_TEST1 (0.038μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_0.08uM_(%)_TEST1 (0.08μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_0.16uM_(%)_TEST1 (0.16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
31Activity_at_0.3uM_(%)_TEST1 (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
32Activity_at_0.6uM_(%)_TEST1 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
33Activity_at_1.2uM_(%)_TEST1 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
34Activity_at_2.6uM_(%)_TEST1 (2.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
35Activity_at_5uM_(%)_TEST1 (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
36Activity_at_10uM_(%)_TEST1 (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
37ACTIVITY_OUTCOME_TEST2The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
38ACTIVITY_SCORE_TEST2The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
39ASSAYDATA_COMMENT_TEST2Comments relevant to the subset of data included in the indicated Test number.String
40AC50_Qualifier_TEST2'>','=', or '<'String
41AC50_uM_TEST2The concentration at which activity reaches 50% of the maximumFloatμM
42pAC50_M_TEST2Equal to -1*log10(AC50)String
43Hill_Slope_TEST2The slope at AC50Float
44S0_(%)_TEST2The fitted activity value at zero concentrationFloat%
45Sinf_(%)_TEST2The fitted activity value at infinite concentrationFloat%
46Num_Points_TEST2The number of data points used to generate the plotInteger
47Max_Activity_(%)_TEST2The maximum activity value observed, based on mean of replicates per concentrationFloat%
48Max_Activity_Conc_uM_TEST2The concentration at which the maximum activity is observedFloatμM
49Max_Concentration_uM_TEST2Maximum valid test concentrationFloatμM
50Activity_at_0.000019uM_(%)_TEST2 (1.9e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
51Activity_at_0.000038uM_(%)_TEST2 (3.8e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
52Activity_at_0.000075uM_(%)_TEST2 (7.5e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
53Activity_at_0.00015uM_(%)_TEST2 (0.00015μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
54Activity_at_0.0003uM_(%)_TEST2 (0.0003μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
55Activity_at_0.0006uM_(%)_TEST2 (0.0006μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
56Activity_at_0.0012uM_(%)_TEST2 (0.0012μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
57Activity_at_0.00235uM_(%)_TEST2 (0.00235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
58Activity_at_0.005uM_(%)_TEST2 (0.005μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
59Activity_at_0.01uM_(%)_TEST2 (0.01μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
60Activity_at_0.0195uM_(%)_TEST2 (0.0195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
61Activity_at_0.038uM_(%)_TEST2 (0.038μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
62Activity_at_0.08uM_(%)_TEST2 (0.08μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
63Activity_at_0.16uM_(%)_TEST2 (0.16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
64Activity_at_0.3uM_(%)_TEST2 (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
65Activity_at_0.6uM_(%)_TEST2 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
66Activity_at_1.2uM_(%)_TEST2 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
67Activity_at_2.6uM_(%)_TEST2 (2.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
68Activity_at_5uM_(%)_TEST2 (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
69Activity_at_10uM_(%)_TEST2 (10μM**)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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