Abstract
Protein–protein interactions (PPIs) are highly specific and diverse. Their selective inhibition with peptides, peptidomimetics, or small molecules allows determination of functions of individual PPIs. Moreover, inhibition of disease-associated PPIs may lead to new concepts for the treatment of diseases with an unmet medical need. Protein kinase A (PKA) is an ubiquitously expressed protein kinase that controls a plethora of cellular functions. A-kinase anchoring proteins (AKAPs) are multivalent scaffolding proteins that directly interact with PKA. AKAPs spatially and temporally restrict PKA activity to defined cellular compartments and thereby contribute to the specificity of PKA signaling. However, it is largely unknown which of the plethora of PKA-dependent signaling events involve interactions of PKA with AKAPs. Moreover, AKAP–PKA interactions appear to play a role in a variety of cardiovascular, neuronal, and inflammatory diseases, but it is unclear whether these interactions are suitable drug targets. Here we describe an enzyme-linked immunosorbent assay (ELISA) for the screening of small molecule libraries for inhibitors of AKAP–PKA interactions. In addition, we describe a homogenous time-resolved fluorescence (HTRF) assay for use in secondary validation screens. Small molecule inhibitors are invaluable molecular tools for elucidating the functions of AKAP–PKA interactions and may eventually lead to new concepts for the treatment of diseases where AKAP–PKA interactions represent potential drug targets.
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References
Scott JD, Dessauer CW, Tasken K (2013) Creating order from chaos: cellular regulation by kinase anchoring. Annu Rev Pharmacol Toxicol 53:187–210
Skroblin P, Grossmann S, Schafer G et al (2010) Mechanisms of protein kinase a anchoring. Int Rev Cell Mol Biol 283:235–330
Szaszak M, Christian F, Rosenthal W et al (2008) Compartmentalized cAMP signalling in regulated exocytic processes in non-neuronal cells. Cell Signal 20:590–601
Diviani D, Dodge-Kafka KL, Li J et al (2011) A-kinase anchoring proteins: scaffolding proteins in the heart. Am J Physiol Heart Circ Physiol 301:H1742–H1753
Esseltine JL, Scott JD (2013) AKAP signaling complexes: pointing towards the next generation of therapeutic targets? Trends Pharmacol Sci 34:648–655
Hundsrucker C, Klussmann E (2008) Direct AKAP-mediated protein-protein interactions as potential drug targets. Handb Exp Pharmacol 186:483–503
Hundsrucker C, Skroblin P, Christian F et al (2010) Glycogen synthase kinase 3beta interaction protein functions as an A-kinase anchoring protein. J Biol Chem 285:5507–5521
Carr DW, Hausken ZE, Fraser ID et al (1992) Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain. J Biol Chem 267:13376–13382
Carr DW, Stofko-Hahn RE, Fraser ID et al (1991) Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif. J Biol Chem 266:14188–14192
Hundsrucker C, Krause G, Beyermann M et al (2006) High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides. Biochem J 396:297–306
Alto NM, Soderling SH, Hoshi N et al (2003) Bioinformatic design of A-kinase anchoring protein-in silico: a potent and selective peptide antagonist of type II protein kinase A anchoring. Proc Natl Acad Sci U S A 100:4445–4450
Craik DJ, Fairlie DP, Liras S et al (2013) The future of peptide-based drugs. Chem Biol Drug Des 81:136–147
Troger J, Moutty MC, Skroblin P et al (2012) A-kinase anchoring proteins as potential drug targets. Br J Pharmacol 166:420–433
Schafer G, Milic J, Eldahshan A et al (2013) Highly functionalized terpyridines as competitive inhibitors of AKAP-PKA interactions. Angew Chem Int Ed Engl 52:12187–12191
Christian F, Szaszak M, Friedl S et al (2011) Small molecule AKAP-protein kinase A (PKA) interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes. J Biol Chem 286:9079–9096
Herman B, Krishnan RV, Centonze VE (2004) Microscopic analysis of fluorescence resonance energy transfer (FRET). Methods Mol Biol 261:351–370
Benicchi T, Iozzi S, Svahn A et al (2012) A homogeneous HTRF assay for the identification of inhibitors of the TWEAK-Fn14 protein interaction. J Biomol Screen 17:933–945
Acknowledgements
We thank Sylvia Niquet for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (DFG KL1415/4-2), the Else Kröner-Fresenius-Stiftung (2013_A145), and the German-Israeli Foundation (I-1210-286.13/2012).
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Schächterle, C., Christian, F., Fernandes, J.M.P., Klussmann, E. (2015). Screening for Small Molecule Disruptors of AKAP–PKA Interactions. In: Zaccolo, M. (eds) cAMP Signaling. Methods in Molecular Biology, vol 1294. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2537-7_12
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DOI: https://doi.org/10.1007/978-1-4939-2537-7_12
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