Fluorescence polarization-based biochemical primary high throughput screening assay to identify activators of the Protein Kinase A-R2B (PKA-R2B) complex
Name: Fluorescence polarization-based biochemical primary high throughput screening assay to identify activators of the Protein Kinase A-R2B (PKA-R2B) complex. ..more
BioActive Compounds: 273
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Susan Taylor, University of California, San Diego (UCSD)
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: R01 GM34921
Grant Proposal PI: Susan Taylor, University of California, San Diego (UCSD)
External Assay ID: PKAR2B_ACT_FP_1536_1X%ACT PRUN
Name: Fluorescence polarization-based biochemical primary high throughput screening assay to identify activators of the Protein Kinase A-R2B (PKA-R2B) complex.
Protein phosphorylation is one of the most important mechanisms for regulation in mammalian cells, and cAMP is an ancient second messenger signaling molecule. Protein kinase A (PKA) is a ubiquitous cAMP-dependent serine/threonine protein kinase that phosphorylates intracellular protein substrates in response to the secondary messenger, adenosine 3',5'-cyclic monophosphate (cAMP). It has several functions in the cell, including regulation of immune response (1), transcription (2), cell cycle and apoptosis (3). PKA exists in its native inactive form as a 4 subunit enzyme with two regulatory and two catalytic subunits. The regulatory R-subunits are the primary receptors for cAMP, while the catalytic (C) subunit serves as the prototype for the protein kinase superfamily. In the absence of cAMP the R-subunit binds with high affinity to the catalytic (PKAC) subunit and renders it inactive. Cooperative binding of cAMP to the R-subunit induces significant conformational changes, to release the inhibitory binding and thus unleash the enzyme activity (4). Thus PKA serves to couple protein phosphorylation and cAMP second messenger signaling pathways (5-6). Crystal structures of regulatory subunits isoforms R1A, R2A and R2B and their complexes with the PKAC reveal that novel pockets are formed in the complexes that are unique from the binding sites in the cAMP bound form (7- 9). As a result the identification of novel small molecules targeting the PKA holoenzymes will provide insight into cAMP signaling (10). Abundant studies have linked deregulation of PKA to several diseases. For example, Carney complex (CNC) is an inherited tumor predisposition disease caused by mutations in R1A gene (11). R1A has also been shown to be a bone tumor suppressor; deletion of R1A gene in mouse caused osteosarcoma (12), the second highest cause of cancer-related death in the pediatric age group. Identifying compounds targeting specifically to R-subunit isoforms will hopefully open up new avenues for the cure of some of these diseases.
1. 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.
4. Johnson DA, Akamine P, Radzio-Andzelm E, Madhusudan M, Taylor SS. Dynamics of cAMP-dependent protein kinase. Chem Rev. 2001 Aug;101(8):2243-70.
5. Garren LD, Gill GN, Walton GM. The isolation of a receptor for adenosine 3',5'-cyclic monophosphate (cAMP) from the adrenal cortex: the role of the receptor in the mechanism of action of cAMP. Ann N Y Acad Sci. 1971 Dec 30;185:210-26.
6. Francis SH, Corbin JD. Structure and function of cyclic nucleotide-dependent protein kinases. Annu Rev Physiol. 1994;56:237-72.
7. Kim C, Cheng CY, Saldanha SA, Taylor SS. PKA-I holoenzyme structure reveals a mechanism for cAMP-dependent activation. Cell. 2007 Sep 21;130(6):1032-43.
8. Wu J, Brown SH, von Daake S, Taylor SS. PKA type IIalpha holoenzyme reveals a combinatoR1Al strategy for isoform diversity. Science. 2007 Oct 12;318(5848):274-9.
9. Brown SH, Wu J, Kim C, Alberto K, Taylor SS. Novel isoform-specific interfaces revealed by PKA R2Beta holoenzyme structures. J Mol Biol. 2009 Nov 13;393(5):1070-82.
10. Saldanha SA, Kaler G, Cottam HB, Abagyan R, Taylor SS. Assay principle for modulators of protein-protein interactions and its application to non-ATP-competitive ligands targeting protein kinase A. Anal Chem. 2006 Dec 15;78(24):8265-72.
11. Boikos SA, Stratakis CA. Carney complex: the first 20 years. Curr Opin Oncol. 2007 Jan;19(1):24-9.
12. Molyneux SD, Di Grappa MA, Beristain AG, McKee TD, Wai DH, Paderova J, Kashyap M, Hu P, Maiuri T, Narala SR, Stambolic V, Squire J, Penninger J, Sanchez O, Triche TJ, Wood GA, Kirschner LS, Khokha R. Prkar1a is an osteosarcoma tumor suppressor that defines a molecular subclass in mice. J Clin Invest. 2010 Sep 1;120(9):3310-25.
PRKACB, PKA, protein kinase, protein kinase A, kinase, enzyme, PKAc, cAMP, cAMP-dependent, catalytic, beta, cAMP-dependent, regulatory, subunit, type II, PRKAR2, RII-BETA, PRKAR2B, biochemical, R2B, FP, fluorescence, fluorescent, FP, fluorescence polarization, enzyme, IP20, Cy5, Cy5-IP20, holoenzyme, polarization, agonist, agonism, activator, activate, activation, increase, HTS, primary, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this biochemical assay is to identify compounds that induce the dissociation of the PKA-R2B complex. This fluorescent polarization-based assay monitors activation of PKA holoenzyme (Saldanha et al, 2006). This assay employs a 20-residue peptide (IP20) ligand conjugated at the N-terminus with Cy5 (Cy5-IP20) that binds the PKA catalytic subunit (PKAc) with high affinity. The sample is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value. As designed, compounds that activate the PKA-R2B holoenzyme, will induce PKAc dissociation from the R2B subunit, causing the free PKAc subunit to bind to the labeled Cy5-IP20, increasing the Cy5-IP20 effective molecular weight, slowing its rotation rate, and increasing the polarization signal of the sample. Compounds are tested in singlicate at a final nominal concentration of 11 uM.
Protein Mix containing assay buffer (40 mM HEPES (pH 7.4), 2 mM ATP, 0.02% BSA, 0.01% Triton-X100, 10 mM Magnesium sulfate, 1 mM DTT and 10 nM Cy5-IP20) with 45nM PKAc and 56 nM R2B protein was prepared. Next, 4.0 uL of Protein Mix was dispensed into 1536-well microtiter plates. The was assay started by dispensing 46 nL of test compound in DMSO or DMSO alone (1% final concentration) to the appropR1Ate wells. Plates were then incubated for 1 hour at room temperature.
Fluorescence polarization was read on an Envision microplate reader (PerkinElmer, Turku, Finland) using a Cy5 FP filter set (Excitation = 620 nm, Emission = 688 nm) and a Cy5 dichroic mirror.
The well Fluorescence Polarization value (P) was calculated from the parallel (S) and perpendicular (P) polarization values using the following formula:
P = (S * P) / (S * P)
The percent activation for each compound was calculated as follows:
%_Activation = ( ( Test_Compound_P - median_Low_Control_P ) / ( median_High_Control_P - median_High_Control_P ) ) *100
Test_Compound is defined as wells containing Protein mix in the presence of test compound.
Low_Control is defined as wells containing Protein mix.
High_Control is defined as wells containing Protein mix and 113 uM cGMP.
A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation 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 % activation than the cutoff parameter was declared active.
PubChem Activity Outcome and Score:
The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-17, and for inactive compounds 17-0.
List of Reagents:
PKAc and R2B(108-402)(S112A) (provided by Prof. Susan Taylor)
Cy5-IP20 [20-residue PKAc inhibitory peptide IP20 (sequence Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-H- is-Asp) conjugated at the C-terminus with Cy5] (Anaspec custom synthesis)
Guanosine 3',5'-cyclic monophosphate (EMD chemicals, cGMP, part 370656)
Adenosine 3',5'-cyclic monophosphate (EMD chemicals, cAMP, part 116801)
HEPES (Invitrogen, part 15630-080)
ATP (MP Biomedicals, part ICN10000801)
BSA (Calbiochem, part 126609)
Triton-X100 (Fisher, part BP151-100)
Magnesium sulfate (Sigma, part M2773)
DTT (Sigma, part D9779)
1536-well plates (Corning, part 7261)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)