Cell-Free Homogeneous Primary HTS to Identify Inhibitors of GSK3beta Activity
The glycogen synthase kinase-3 beta (GSK-3b) is a known master regulator for several cellular pathways and plays a critical role in metabolism, transcription, development, cell survival, and neuronal functions. The overall objective is to identify one or multiple series of inhibitors of GSK-3beta with micromolar potency. Such compounds will become probe(s) with demonstrated kinase-selectivity more ..
BioActive Compounds: 116
Keywords: GSK3beta, kinase, inhibition, HTS
The glycogen synthase kinase-3 beta (GSK-3b) is a known master regulator for several cellular pathways and plays a critical role in metabolism, transcription, development, cell survival, and neuronal functions. The overall objective is to identify one or multiple series of inhibitors of GSK-3beta with micromolar potency. Such compounds will become probe(s) with demonstrated kinase-selectivity (≥10-fold IC50) and activation of Wnt signaling (EC50 < 10uM) and desired effect on neuronal morphology. Structural series that demonstrates non-ATP competitive mechanism of action will be strongly preferred. For primary screen, activity of 1.6 ng of GSK3beta (as a GST fusion from BPS Bioscience) was incubated with 10 uM of compounds in the presence of 25 uM of ATP (specific reaction conditions see Protocol) for 60 minutes at ambient temperature in 1536 plates (Aurora 29847). The kinase activity was measured with ADP-Glo (Promega V9103) and signals were read with Viewlux (PerkinElmer). Positive control (GW8510 at 20 uM) was included in each plate and used to scale the data in conjunction with in-plate DMSO controls (details see Data Analysis or Comments section).
Inhibitors for GSK3beta activity will show as loss of luminescence signal.
1) Dispense 1 uL/well of CABPE, 0.5 uL of ATP, and 1 uL of positive control GW8510 or AB in respective wells according to plate design to 1536-well assay ready plates (Aurora 29847) that contain 2.5 nL/well of 10 mM compound using BioRAPTR (Beckman) to start the reaction. Incubate at room temperature for 60 minutes.
2) Add 2.5 uL/well of ADP-glo (Promega, V9103) with BioRAPTR, incubate at room temperature for 40 minutes
3) Add 5 uL/well of ADP-glo (Promega, V9103) with Combi nL (Thermo), incubate at room temperature for 30 minutes
4) Read on Viewlux (PerkinElmer) for luminescence
25 mM Tris7.5
10 mM MgCl2
GW8510 (in AB, Sigma G7791)
50 uM GW8510
CABPE (in AB):
12.5 mM DTT (Sigma 43816)
0.25 mg/ml BSA (Sigma A4503)
0.5 U/ml Heparin (Baxter NDC 0641-2440-41)
8 uM Peptide (American Peptide)
23 nM GSK3beta (BPS Biosciences)
ATP (in AB, Promega V9103 component):
125 uM ATP
HTS Data Analysis
Each compound was tested in singlicate in 1536-well plates; a small percentage of compounds were duplicated.
128 neutral control wells (DMSO) and 128 positive control wells were included on every plate.
Active inhibitor compounds result in decreased signal.
Analysis used to determine PubChem Activity Score and Outcome
The raw luminescent signal of the DMSO control wells was normalized using the 'Neutral Controls Minus Inhibitors' 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.
The median raw signal of the intraplate positive (active) controls (AC) is set to a normalized activity value of -100.
This normalization was then applied to all compound treated wells, giving an activity score as percent change in luminescence signal relative to the intraplate controls.
A plate correction matrix was applied using the 'Run-wise Multiplicative Correction' in Genedata Assay Analyzer (v7.0.3)
The final PUBCHEM_ACTIVITY_SCORE was calculated by multiplying the mean of all valid replicate values (which can be described in units of negative percent activity) by -1, resulting in a score between 0 and 100 (in units of 'percent inhibition'). Most wells were not tested in replicate, so the mean of the well activity value was the same as the well value itself.
The PUBCHEM_ACTIVITY_OUTCOME class was assigned as described below, based on an activity threshold of -50%:
Activity_Outcome = 1 (inactive)
0% of replicates fall outside threshold.
Activity_Outcome = 2 (active)
>50% of replicates fall outside threshold.
Activity_Outcome = 3 (inconclusive)
>0% and <= 50% of replicates fall outside threshold. (None for singlicates.)
Data Table (Concise)