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

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

Abstract

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 

References

  1. 1.
    Knight ZA, Lin H, Shokat KM (2010) Targeting the cancer kinome through polypharmacology, Nat Rev Cancer 10: 130–137PubMedCrossRefGoogle Scholar
  2. 2.
    Liu Y, Gray NS (2006) Rational design of inhibitors that bind to inactive kinase conformations. Nat Chem Biol 2:358–364PubMedCrossRefGoogle Scholar
  3. 3.
    Backes AC, Zech B, Felber B, Klebl B, Müller G (2008) Small-molecule inhibitors binding to protein kinases. Part I: exceptions from the traditional pharmacophore approach of type I inhibition Expert Opin Drug Discovery 3:1409–1425CrossRefGoogle Scholar
  4. 4.
    Backes AC, Zech B, Felber B, Klebl B, Müller G (2008) Small-molecule inhibitors binding to protein kinase. Part II: the novel pharmacophore approach of type II and type III inhibition. Expert Opin Drug Discovery 3:1427–1449CrossRefGoogle Scholar
  5. 5.
    Rabiller M, Getlik M, Kluter S, Richters A, Tuckmantel S, Simard JR, Rauh D (2010) Proteus in the world of proteins: conformational changes in protein kinases. Arch Pharm (Weinheim) 343:193–206CrossRefGoogle Scholar
  6. 6.
    Simard JR, Getlik M, Grutter C, Pawar V, Wulfert S, Rabiller M, Rauh D (2009) Develop­ment of a fluorescent-tagged kinase assay ­system for the detection and characterization of ­allosteric kinase inhibitors. J Am Chem Soc 131:13286–13296PubMedCrossRefGoogle Scholar
  7. 7.
    Simard JR, Kluter S, Grutter C, Getlik M, Rabiller M, Rode HB, Rauh D (2009) A new screening assay for allosteric inhibitors of cSrc, Nat Chem Biol 5: 394–396PubMedCrossRefGoogle Scholar
  8. 8.
    Simard JR, Grutter C, Pawar V et al. (2009) High-throughput screening to identify inhibitors which stabilize inactive kinase conformations in p38alpha. J Am Chem Soc 131:18478–18488PubMedCrossRefGoogle Scholar
  9. 9.
    Simard JR, Getlik M, Grutter C, Schneider R, Wulfert S, Rauh D (2010) Fluorophore labeling of the glycine-rich loop as a method of identifying inhibitors that bind to active and inactive kinase conformations. J Am Chem Soc 132:4152–4160PubMedCrossRefGoogle Scholar
  10. 10.
    de Lorimier RM, Smith JJ, Dwyer MA, et al. (2002) Construction of a fluorescent biosensor family. Protein Sci 11:2655–2675PubMedCrossRefGoogle Scholar
  11. 11.
    Pargellis C, Tong L, Churchill L, et al. (2002) Inhibition of p38 MAP kinase by utilizing a novel allosteric binding site. Nat Struct Biol 9: 268–272PubMedCrossRefGoogle Scholar
  12. 12.
    Simard JR, Kamp F, Hamilton JA (2007) Acrylodan-labeled intestinal fatty acid-­binding protein to measure concentrations of unbound fatty acids. Methods Mol Biol 400:27–43PubMedCrossRefGoogle Scholar

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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