A Coiled-Coil Enabled Split-Luciferase Three-Hybrid System: Applied Toward Profiling Inhibitors of Protein Kinases
- 29 July 2010
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Chemical Society
- Vol. 132 (33), 11727-11735
- https://doi.org/10.1021/ja104491h
Abstract
The 518 protein kinases encoded in the human genome are exquisitely regulated and their aberrant function(s) are often associated with human disease. Thus, in order to advance therapeutics and to probe signal transduction cascades, there is considerable interest in the development of inhibitors that can selectively target protein kinases. However, identifying specific compounds against such a large array of protein kinases is difficult to routinely achieve utilizing traditional activity assays, where purified protein kinases are necessary. Toward a simple, rapid, and practical method for identifying specific inhibitors, we describe the development and application of a split-protein methodology utilizing a coiled-coil-assisted three-hybrid system. In this approach, a protein kinase of interest is attached to the C-terminal fragment of split-firefly luciferase and the coiled-coil Fos, which is specific for the coiled-coil Jun, is attached to the N-terminal fragment. Upon addition of Jun conjugated to a pan-kinase inhibitor such as staurosporine, a three-hybrid complex is established with concomitant reassembly of the split-luciferase enzyme. An inhibitor can be potentially identified by the commensurate loss in split-luciferase activity by displacement of the modified staurosporine. We demonstrate that this new three-hybrid approach is potentially general by testing protein kinases from the different kinase families. To interrogate whether this method allows for screening inhibitors, we tested six different protein kinases against a library of 80 known protein kinase inhibitors. Finally, we demonstrate that this three-hybrid system can potentially provide a rapid method for structure/function analysis as well as aid in the identification of allosteric inhibitors.Keywords
This publication has 79 references indexed in Scilit:
- Targeting the cancer kinome through polypharmacologyNature Reviews Cancer, 2010
- Biochemical Mechanisms of Resistance to Small-Molecule Protein Kinase InhibitorsACS Chemical Biology, 2010
- An Autoinhibited Coiled-Coil Design Strategy for Split-Protein Protease SensorsJournal of the American Chemical Society, 2009
- Staurosporine tethered peptide ligands that target cAMP-dependent protein kinase (PKA): Optimization and selectivity profilingBioorganic & Medicinal Chemistry, 2009
- Design and Applications of Bifunctional Small Molecules: Why Two Heads Are Better Than OneACS Chemical Biology, 2008
- Structural specificity in coiled-coil interactionsCurrent Opinion in Structural Biology, 2008
- The selectivity of protein kinase inhibitors: a further updateBiochemical Journal, 2007
- Structural basis for AMP binding to mammalian AMP-activated protein kinaseNature, 2007
- Inflammation and metabolic disordersNature, 2006
- Specificity and mechanism of action of some commonly used protein kinase inhibitorsBiochemical Journal, 2000