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BioAssay: AID 2661

Luminescence Cell-Free Homogenous Primary HTS to Identify Inhibitors of Serine/Threonine Kinase 33 Activity

Assay Overview: Purified STK33 Kinase is pre-incubated with potential inhibitors and allowed to phosphorylate MBP at ~ 1.5 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(321812)
 
 
Active(235)
 
 
Inactive(321578)
 
 
 Tested Substances
 Tested Substances
All(321908)
 
 
Active(235)
 
 
Inactive(321673)
 
 
AID: 2661
Data Source: Broad Institute (2052-01_INHIBITORS_SINGLE-POINT_MLPCN-HTS)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-24

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 235
Depositor Specified Assays
AIDNameTypeComment
2689Summary of Broad Institute MLPCN Serine Threonine Kinase 33 Inhibitor Projectsummary
2821Luminescence Cell-Free Homogenous Dose Retest to Identify Inhibitors of Serine/Threonine Kinase 33 Activityconfirmatory
Description:
Primary Collaborators:
Robert Gould,Broad Institute,Cambridge, MA,rgould@broainstitute.org,617.714.7220,

Keywords: STK33 Kinase, Non-ATP Competitive Inhibitor

Assay Overview: Purified STK33 Kinase is pre-incubated with potential inhibitors and allowed to phosphorylate MBP at ~ 1.5 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

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 on ViewLux plate reader (Perkin Elmer).
Comment
HTS Data Analysis:
Negative control wells (DMSO) were included on every plate.
Active inhibitor compounds result in decreased readout signal.

Raw luminescent signal was normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Therefore a negative activity value is expected when signal decreases relative to NC.

The plate pattern correction algorithm 'Assay Pattern (Additive)' in Genedata was applied to the normalized plate data to correct for a modest edge effect.

This screen was run in singletons, except for a small number of compounds (635) which were run as duplicates--in these cases, the most active replicate value was used as a final score for the compound.

The compound activity scores were multiplied by -1 to convert Genedata negative percent inhibition values to Pubchem positive percent activity values.
The final PUBCHEM_ACTIVITY_SCORE was set as equal to the most active of the replicates' scores.

The PUBCHEM_ACTIVITY_OUTCOME class was assigned as described below, based on an activity threshold of 25%:

Activity_Outcome = 1 (inactive)
when PubChem_Activity_Score < 25

Activity_Outcome = 2 (active)
when PubChem_Activity_Score >= 25

Activity_Outcome = 3 (inconclusive)
None

Summary of compound activity counts:
Unique Pubchem SIDs screened: 321908
Actives: 235
Inconclusives: 0
Inactives: 321673
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the "replicate vector" (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was InvalidFloat
2BROAD_SCREENING_RUNIDSThis is the unique internal ID given to each screening run at the Broad Institute, corresponding to replicates C & D.String
3REPLICATE_A_ACTIVITY_SCORE (10μM**)The calculated activity score for the sample A, a NULL here denotes data was not used or was not producedFloat%
4REPLICATE_B_ACTIVITY_SCORE (10μM**)The calculated activity score for the sample B, a NULL here denotes data was not used or was not producedFloat%
5DATE_REPORTEDThe date the data was internally reportedString

** Test Concentration.
Additional Information
Grant Number: 1 R03 MH089854-01

Data Table (Concise)
Classification
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