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
BioActive Compounds: 235
Robert Gould,Broad Institute,Cambridge, MA,email@example.com,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.
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
250 uM ATP
50 mM MgAc2
50 nM STK33
0.5 mg/mL Myelin Basic Protein
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).
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)
Summary of compound activity counts:
Unique Pubchem SIDs screened: 321908
** Test Concentration.
Data Table (Concise)