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BioAssay: AID 588632

STK-33 Kinase Inhibition Measured in Biochemical System Using Plate Reader - 2052-02_Inhibitor_Dose_DryPowder_Activity

Assay Overview: Purified STK33 Kinase is preincubated with potential inhibitors and then allowed to phosphorylate MBP at [ATP] = Km, which can be compared to other [ATP] experiments to determine competitive or non-competitive mechanism of action. 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(55)
 
 
Active(28)
 
 
Inactive(23)
 
 
Inconclusive(4)
 
 
 Tested Substances
 Tested Substances
All(56)
 
 
Active(29)
 
 
Inactive(23)
 
 
Inconclusive(4)
 
 
AID: 588632
Data Source: Broad Institute (2052-02_Inhibitor_Dose_DryPowder_Activity)
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: 28
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
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: STK-33 Kinase

Assay Overview: Purified STK33 Kinase is preincubated with potential inhibitors and then allowed to phosphorylate MBP at [ATP] = Km, which can be compared to other [ATP] experiments to determine competitive or non-competitive mechanism of action. Kinase activity is measured using the ADP Glo Kit (Promega) which converts ADP reaction product into a luminescent signal.

Expected outcome: Inhibitors of the STK33 will reduce the production of ADP, resulting in a decreased signal from the ADP Glo Kit.
Protocol
Protocol:

Buffer Stock - 10 mL 1 M MOPS-Na pH 7.0 + 0.6 mL 500 mM EDTA + 10 mL 50% glycerol + 100 uL 10% Brij-35 -> 1L 0.2 u filter

10X BSA/BME - 50 mL Buffer + 50 mg BSA + 50 uL BME, aliquot 4X 10 mL -20C

Assay Buffer (AB) = 10 mL 10X BSA/BME + 90 mL Buffer Stock

STK33/MBP = 19 uL 26.5 uM STK33 + 78 uL 64 mg/mL MBP -> 10 mL w/ AB
ATP/Mg = 150 10 mM ATP + 500 uL 1M MgAc2 -> 10 ml w/ AB to a final conc of 0.15 mM
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 STK33/MBP 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 all wells of plate to initiate kinase reaction. Incubate 60 minutes.

Add 25 uL / well ADP Glo Reagent I (Promega). Incubate 40-60 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
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 (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.

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.
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_0.038uM_(%)_TEST1 (0.038μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.08uM_(%)_TEST1 (0.08μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.16uM_(%)_TEST1 (0.16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.3uM_(%)_TEST1 (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.6uM_(%)_TEST1 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_1.2uM_(%)_TEST1 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_2.6uM_(%)_TEST1 (2.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_5uM_(%)_TEST1 (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_10uM_(%)_TEST1 (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26ACTIVITY_OUTCOME_TEST2The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
27ACTIVITY_SCORE_TEST2The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
28ASSAYDATA_COMMENT_TEST2Comments relevant to the subset of data included in the indicated Test number.String
29AC50_Qualifier_TEST2'>','=', or '<'String
30AC50_uM_TEST2The concentration at which activity reaches 50% of the maximumFloatμM
31pAC50_M_TEST2Equal to -1*log10(AC50)String
32Hill_Slope_TEST2The slope at AC50Float
33S0_(%)_TEST2The fitted activity value at zero concentrationFloat%
34Sinf_(%)_TEST2The fitted activity value at infinite concentrationFloat%
35Num_Points_TEST2The number of data points used to generate the plotInteger
36Max_Activity_(%)_TEST2The maximum activity value observed, based on mean of replicates per concentrationFloat%
37Max_Activity_Conc_uM_TEST2The concentration at which the maximum activity is observedFloatμM
38Max_Concentration_uM_TEST2Maximum valid test concentrationFloatμM
39Activity_at_0.038uM_(%)_TEST2 (0.038μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
40Activity_at_0.08uM_(%)_TEST2 (0.08μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
41Activity_at_0.16uM_(%)_TEST2 (0.16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
42Activity_at_0.3uM_(%)_TEST2 (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43Activity_at_0.6uM_(%)_TEST2 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
44Activity_at_1.2uM_(%)_TEST2 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
45Activity_at_2.6uM_(%)_TEST2 (2.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
46Activity_at_5uM_(%)_TEST2 (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
47Activity_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
Classification
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