AID	Name	Type	Comment
2661	Luminescence Cell-Free Homogenous Primary HTS to Identify Inhibitors of Serine/Threonine Kinase 33 Activity	screening	Primary HTS
2689	Summary of Broad Institute MLPCN Serine Threonine Kinase 33 Inhibitor Project	summary	Project Summary

Keywords: STK33 Kinase, Non-ATP Competitive Inhibitor

Assay Overview: Purified STK33 Kinase is preincubated with potential inhibitors and allowed to phosphorylate MBP at ~ 1.5 x 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

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

1536 well plate assay

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 15 sec integration time on ViewLux plate reader (Perkin Elmer).

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 E 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 full plate. Incubate 60 minutes.

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

Dose Data Analysis

Neutral control wells (NC), positive controls wells (PC) and blank wells were included on every plate.
Active compounds result in decreased readout signal.

Analysis used to determine PubChem Activity Score and Outcome

The raw signal was normalized using the 'Neutral Controls Minus Blanks' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate NC wells was set to a normalized activity value of 0.
The median raw signal of the intraplate blank wells was set to a normalized activity of -100.
The raw signal of each compound well was then scaled to these two reference points to obtain a normalized activity value.

No plate pattern correction algorithm was used.

IC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). IC50 values were extrapolated up to 1 log
over the highest tested concentration.


PubChem Activity Score and Outcome

PUBCHEM_ACTIVITY_SCORE:

Inactive compounds = 0
Active compounds = -10*Log(IC50)

PUBCHEM_ACTIVITY_OUTCOME:

Activity_Outcome = 1 (inactive)
IC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active)
IC50 <= 1 log over the highest tested concentration

Activity_Outcome = 3 (inconclusive)
Curve fitting strategy resulted in a constant fit with activtity >30% but <70%
or
The fit was not valid due to poor fit quality.

Grant Number: 1 R03 MH089854-01