Dose-response biochemical assay for inhibitors of protein kinase A (PKA) activity
PKA is an ubiquitous serine/threonine protein kinase and belongs to the AGC kinase family. It has several functions in the cell, including regulation of immune response , transcription , cell cycle and apoptosis . PKA is a cAMP dependent enzyme that exists in its native inactive form as a 4 subunit enzyme with two regulatory and two catalytic subunits. Binding of cAMP to the regulatory subunit leads to the disassembly of the complex and release of now active catalytic subunits. ..more
BioActive Compounds: 62
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Scripps Florida
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: NA
PKA is an ubiquitous serine/threonine protein kinase and belongs to the AGC kinase family. It has several functions in the cell, including regulation of immune response , transcription , cell cycle and apoptosis . PKA is a cAMP dependent enzyme that exists in its native inactive form as a 4 subunit enzyme with two regulatory and two catalytic subunits. Binding of cAMP to the regulatory subunit leads to the disassembly of the complex and release of now active catalytic subunits.
This screen is designed to identify inhibitors of PKA. The known PKA inhibitor Staurosporine was used as a positive control.
PKA, kinase, Protein kinase A, luciferase, luminescence, apoptosis, immune response, cAMP dependant enzyme, Scripps, titration, IC50, dose response
Compounds identified from a previously described set of experiments entitled "Primary high-throughput assay for chemical inhibitors of protein kinase A (PKA) activity" (PubChem AID= 524) were selected for testing in this assay. Out of 94 compounds identified during the primary screen, all 94 compounds were assessed in dose response experiments in 10 point, 1:3 serial dilutions starting at a nominal test concentration of 60 micromolar.As with the primary screen, this assay is based on ability of PKA kinase to phosphorylate a Kemptide peptide sequence. PKA uses ATP as a donor of phosphate for the
phosphorylation of the substrate, which leads to the depletion of ATP in the reaction mix. An assay kit (Kinase-Glo, Promega) was used to quantify enzyme activity. Residual amounts of ATP are measured by a secondary enzymatic reaction, during which luciferase utilizes the remaining ATP to produce luminescence. In this assay, the luminescent signal is directly proportional to the amount of ATP and inversely proportional to PKA activity. Each concentration was tested nominally in triplicate. The dose response assay was conducted in 1536 well plate format.
1.25 microliters of substrate solution containing 20 micromolar ATP and 60 micromolar Kemptide peptide (substrate) in assay buffer (50 millimolar HEPES pH 7.3, 10 millimolar MgCl2, 0.1% BSA, 2 millimolar DTT) were dispensed into a 1536 microtiter plate. 15 nanoliters of test compound, positive or negative control (50 micromolar Staurosporine and DMSO, respectively) were then added to the appropriate wells. Each compound dilution was assayed in triplicate, for a nominal total of 30 data points per dose response curve.
The experiment was started by dispensing 1.25 microliters of 0.5 nanomolar PKA in assay buffer (50 millimolar HEPES pH 7.3, 10 millimolar MgCl2, 0.1% BSA, 2 millimolar DTT).
After 2 hours of incubation at 25 degrees Celsius, 2.5 microliters of Kinase Glo reagent (Promega Corporation, Madison, WI) was added to each well. Plates were incubated for 10 minutes and luminescence was read on Perkin-Elmer Viewlux for 60 seconds.
The percent inhibition for each well has been calculated as follows:
%inhibition = (test_compound - median_positive_control)/(median_positive_control - median_negative_control)*100
where positive control is Staurosporine (Alexis Corporation, 300 nanomolar) and negative control is DMSO only.
For each compound, percentage inhibitions were plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported IC50 values were generated from fitted curves by solving for X-intercept at the 50% inhibition level of Y-intercept. In cases where the highest concentration tested (i.e. 60 micromolar) did not result in greater than 50% inhibition, the IC50 was determined
manually as greater than 60 micromolar. Compounds with IC50 of greater than 60 micromolar were considered inactive; compounds with IC50 of equal to or less than 60 micromolar were considered active.
The activity score was calculated based on pIC50 values for compounds for which an exact IC50 value was calculated and based on the observed pIC50 range, specifically the maximum lower limit of the pIC50 value as calculated from the lowest concentration for which greater than 50% inhibition is observed. This results in a conservative estimate of the activity score for compounds for which no exact IC50 value is given while maintaining a reasonable rank order of all compounds tested.
All data reported were normalized on a per-plate basis.
Possible artifacts of this assay can include, but are not limited to: compounds that inhibit luciferase activity.
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Data Table (Concise)