Inhibition of Human GSK-3 alpha Activity Measured in Biochemical System Using Microfluidics - 2063-06_Inhibitor_Dose_DryPowder_Activity_Set2
Keywords: Glycogen synthase kinase-3 alpha (GSK3alpha), kinase, acute myeloblastic leukemia (AML), multiple myeloma (MM), cancer, high-throughput screening (HTS) ..more
BioActive Compounds: 24
Keywords: Glycogen synthase kinase-3 alpha (GSK3alpha), kinase, acute myeloblastic leukemia (AML), multiple myeloma (MM), cancer, high-throughput screening (HTS)
GSK-3 alpha has emerged as a target for acute myeloblastic leukemia (AML) and multiple myeloma (MM) in both RNAi-based and chemical genomic-based high-throughput screening. The overall objective is to identify one or more specific inhibitors of GSK-3 alpha with micromolar potency. We had conducted a primary screen and identified active compounds inhibiting GSK3alpha activity. Now we measured the potency of these compounds under the following conditions: 3.2 nM of GSK-3 alpha (as a GST fusion from BPS Bioscience) was incubated with active compounds in doses in the presence of 4.3 uM of ATP (for specific reaction conditions see Protocol) for 60 minutes at ambient temperature in 384-well plates (Seahorse Bioscience, MA). The kinase activity was measured with EZ Reader II (Caliper Life Sciences, MA) as percent conversion. Positive control (GW8510 at 20 uM; CID 6539118) was included in each plate and used to scale the data in conjunction with in-plate DMSO controls (details see Data Analysis in Comments section).
Expected Outcome: Inhibitors of GSK-3 alpha activity will show as loss of signal.
Protocol:1) Dispense 20 uL/well of CBPE (Combi, Thermo)
2) Pin 100 nL/well of compounds (Cybi Well, Cybio)
3) Dispense 10 uL/well of ATP (Combi, Thermo)
4) Incubate at room temperature for 60 minutes.
5) Dispense 10 uL/well of 80 mM EDTA (Combi, Thermo)
6) Store the plates at -20oC until they are read in Caliper EZ ReaderII (Caliper).
Hepes 7.5100mMBrij-350.003%Tween200.004%MgCl210mMDTT (1M)3.8mMPeptide 15 (Caliper)12.3uMGSK3a64.2nM
PRESENCE OF CONTROLS: Neutral control wells (NC; n=60) and positive control wells (PC; n=12) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (3)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)