Fluorescence Labels in Kinases: A High-Throughput Kinase Binding Assay for the Identification of DFG-Out Binding Ligands

  • Jeffrey R. Simard
  • Daniel RauhEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 800)


Despite the hundreds of kinase inhibitors currently in discovery and pre-clinical phases, the number of kinase inhibitors which have been approved and are on the market remains low by comparison. This discrepancy reflects the challenges which accompany the development of kinase inhibitors which are relatively specific and less toxic. Targeting protein kinases with ATP-competitive inhibitors has been the classical approach to inhibiting kinase activity, but the highly conserved nature of the ATP-binding site contributes to poor inhibitor selectivity, issues which have particularly hampered the development of novel kinase inhibitors. We developed a high-throughput screening technology that can discriminate for inhibitors which stabilize the inactive “DFG-out” kinase conformation by binding within an allosteric pocket adjacent to the ATP-binding site. Here, we describe how to use this approach to measure the K d of ligands, as well as how to kinetically characterize the binding and dissociation of ligands to the kinase. We also describe how this technology can be used to rapidly screen small molecule libraries at high throughput.

Key words

FLiK Fluorescence Acrylodan DFG-out DFG-in Kinase inhibitors Activation loop High-throughput screening 


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Chemical Genomics Centre of the Max Planck SocietyDortmundGermany
  2. 2.Fakultät Chemie, Chemische BiologieTechnische Universität DortmundDortmundGermany

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