Luminescence Cell-Free Homogeneous Dose Retest to Identify Inhibitors of Glycogen Synthase Kinase-3 beta 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. We had conducted a primary screen and now this assay is to determine the more ..
BioActive Compounds: 581
Keywords: GSK3beta, dose response, 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. We had conducted a primary screen and now this assay is to determine the potency of the screen activities by testing these actives in doses. Basically, 1.6 ng of GSK3beta (as a GST fusion from BPS Bioscience) was incubated with compounds in doses 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) generated by acoustic transfer using Labcyte Echo that contain 7.5 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 Tris 7.5
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)
9 nM GSK3beta (BPS Biosciences)
ATP (in AB, Promega V9103 component):
125 uM ATP
Dose Data Analysis
Neutral control wells (NC) and positive controls wells (PC) were included on every plate.
Active compounds result in decreased readout signal.
Normalization and Pattern Correction
The raw signal was normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate PC wells is set to a normalized activity value of -100.
The median raw signal of the intraplate NC wells is set to a normalized activity of 0.
The raw signal of each compound well is then scaled to these two reference points to obtain a normalized activity value.
No plate pattern correction algorithm method was applied.
EC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). EC50 values were extrapolated up to 1 log
over the highest tested concentration.
PubChem Activity Score and Outcome
Inactive compounds = 0
Active compounds = -10*Log(EC50)
Activity_Outcome = 1 (inactive) when
EC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active) when
EC50 <= 1 log over the highest tested concentration
Activity_Outcome = 3 (inconclusive) when
Curve fitting strategy resulted in a constant fit with activity >30% but <70%
The fit was not valid due to poor fit quality.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)