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BioAssay: AID 524

Primary biochemical high-throughput screening assay for inhibitors of protein kinase A (PKA) activity

Primary biochemical high-throughput screening assay for inhibitors of protein kinase A (PKA) activity ..more
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AID: 524
Data Source: The Scripps Research Institute Molecular Screening Center (PKA_INH_Lumi_1536_%INH)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2006-11-07
Modify Date: 2007-04-09

Data Table ( Complete ):           Active    All
BioActive Compounds: 94
Depositor Specified Assays
524Primary biochemical high-throughput screening assay for inhibitors of protein kinase A (PKA) activityscreening
548Dose-response biochemical assay for inhibitors of protein kinase A (PKA) activityconfirmatory
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

External Assay ID: PKA_INH_Lumi_1536_%INH


Primary biochemical high-throughput screening 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 [1], transcription [2], cell cycle and apoptosis [3]. 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 Staurosoporine was used as a positive control.


[1].#Skalhegg, B.S., et al., Protein kinase A (PKA) - a potential target for therapeutic intervention of dysfunctional immune cells. Curr Drug Targets, 2005. 6(6): p. 655-64.

[2].#Ofir, R., et al., CREB represses transcription of fos promoter: role of phosphorylation. Gene Expr, 1991. 1(1): p. 55-60.

[3].#Vermeulen, K., Z.N. Berneman, and D.R. Van Bockstaele, Cell cycle and apoptosis. Cell Prolif, 2003. 36(3): p. 165-75.


PKA, kinase, Protein kinase A, luciferase, luminescence, apoptosis, immune response, cAMP dependant enzyme
Assay Overview:
The 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.

The primary HTS campaign was conducted in 1536 well plate format. All compounds were tested once at a 6 micromolar final

Protocol Summary:
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 or positive and negative control (50 micromolar Staurosporine and DMSO, respectively) were then added to the appropriate wells. 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, 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.

Prior to inhibition calculations, background signal (i.e. signal from wells containing all reagents except enzyme) was subtracted from test compound and control well values. To calculate percent inhibition the test compound well signal was divided by the median signal from the 100 percent inhibition positive control wells (i.e. wells containing staurosporine) and multiplied by 100. A mathematical algorithm was used to determine nominally inhibitory compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater %inhibition than the cutoff parameter was declared active.

The reported Pubchem_Activity_Score has been normalized to 100% of the highest observed primary inhibition. Negative % inhibition values are reported as activity score zero.
All data reported were normalized on a per-plate basis.

Possible artifacts of this assay can include, but are not limited to: toxic compounds, compounds that inhibit luciferase activity, compounds that non-specifically inhibit or activate transcriptional activity.
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition_Primary at 6 uM%Inhibition in the primary screen at 6 micromolar test compound concentration;
relative to PKA in the presence of 13 micromolar Y-27632.

Data Table (Concise)