Skip to main content
SpringerLink
Log in

Screening for Inhibitors of Kinase Autophosphorylation

  • Protocol
  • First Online:
High Throughput Screening

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1439))

Abstract

Autophosphorylation of kinases influences their conformational state and can also regulate enzymatic activity. Recently, this has become an area of interest for drug discovery. Using Alk2 as an example, we present two protocols — one based on phosphate-binding Alphascreen beads, the other on coupled luminescence measurements of ADP formation — that can be used to screen for inhibitors of autophosphorylation.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 139.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Klumpp M, Boettcher A, Becker D, Meder G, Blank J, Leder L, Forstner M, Ottl J, Mayr LM (2006) Readout technologies for highly miniaturized kinase assays applicable to high-throughput screening in a 1536-well format. J Biomol Screen 11(6):617–633. doi:10.1177/108705106288444

    Article  CAS  Google Scholar 

  2. Drueckes P (2016) Protein kinase selectivity profiling using microfluidic mobility shift assays. In: Janzen WP (ed) High throughput screening: methods and protocols, 3rd edn. Methods Mol Biol 1439

    Google Scholar 

  3. Cohen P, Alessi DR (2013) Kinase drug discovery—what’s next in the field? ACS Chem Biol 8(1):96–104. doi:10.1021/cb300610s

    Article  CAS  Google Scholar 

  4. Newbatt Y, Hardcastle A, McAndrew PC, Strover JA, Mirza A, Morgan GJ, Burke R, Davies FE, Collins I, van Montfort RL (2013) Identification of autophosphorylation inhibitors of the inositol-requiring enzyme 1 alpha (IRE1alpha) by high-throughput screening using a DELFIA assay. J Biomol Screen 18(3):298–308. doi:10.1177/1087057112465647

    Article  Google Scholar 

  5. Liu Z, Galemmo RA Jr, Fraser KB, Moehle MS, Sen S, Volpicelli-Daley LA, DeLucas LJ, Ross LJ, Valiyaveettil J, Moukha-Chafiq O, Pathak AK, Ananthan S, Kezar H, White EL, Gupta V, Maddry JA, Suto MJ, West AB (2014) Unique functional and structural properties of the LRRK2 ATP-binding pocket. J Biol Chem 289:32937–32951. doi:10.1074/jbc.M114.602318

    Article  CAS  Google Scholar 

  6. Wang JH, Stull JT, Huang TS, Krebs EG (1976) A study on the autoactivation of rabbit muscle phosphorylase kinase. J Biol Chem 251(15):4521–4527

    CAS  Google Scholar 

  7. Nakayama GR, Parandoosh Z (1999) An immunoassay for assessment of receptor tyrosine kinase autophosphorylation. J Immunol Methods 225(1–2):67–74

    Article  CAS  Google Scholar 

  8. Deacon SW, Beeser A, Fukui JA, Rennefahrt UE, Myers C, Chernoff J, Peterson JR (2008) An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase. Chem Biol 15(4):322–331. doi:10.1016/j.chembiol.2008.03.005

    Article  CAS  Google Scholar 

  9. Fabbro D, Cowan-Jacob SW, Mobitz H, Martiny-Baron G (2012) Targeting cancer with small-molecular-weight kinase inhibitors. Methods Mol Biol 795:1–34. doi:10.1007/978-1-61779-337-0_1

    Article  CAS  Google Scholar 

  10. Ohmi N, Wingfield JM, Yazawa H, Inagaki O (2000) Development of a homogeneous time-resolved fluorescence assay for high throughput screening to identify Lck inhibitors: comparison with scintillation proximity assay and streptavidin-coated plate assay. J Biomol Screen 5(6):463–470

    Article  CAS  Google Scholar 

  11. Zimmerman CM, Mathews LS (1996) Activin receptors: cellular signalling by receptor serine kinases. Biochem Soc Symp 62:25–38

    CAS  Google Scholar 

  12. Huse M, Muir TW, Xu L, Chen YG, Kuriyan J, Massague J (2001) The TGF beta receptor activation process: an inhibitor- to substrate-binding switch. Mol Cell 8(3):671–682

    Article  CAS  Google Scholar 

  13. Eglen RM, Reisine T, Roby P, Rouleau N, Illy C, Bosse R, Bielefeld M (2008) The use of AlphaScreen technology in HTS: current status. Curr Chem Genomics 1:2–10. doi:10.2174/1875397300801010002

    Article  CAS  Google Scholar 

  14. Pope AJ (1999) Introduction LANCEtrade mark vs. HTRF(R) technologies (or vice versa). J Biomol Screen 4(6):301–302

    Article  Google Scholar 

  15. Seethala R (2000) Fluorescence polarization competition immunoassay for tyrosine kinases. Methods 22(1):61–70. doi:10.1006/meth.2000.1037

    Article  CAS  Google Scholar 

  16. Delom F, Chevet E (2006) Phosphoprotein analysis: from proteins to proteomes. Proteome Sci 4:15. doi:10.1186/1477-5956-4-15

    Article  Google Scholar 

  17. Fernando SA, Wilson GS (1992) Studies of the ‘hook’ effect in the one-step sandwich immunoassay. J Immunol Methods 151(1–2):47–66

    Article  CAS  Google Scholar 

  18. Briggs GE, Haldane JB (1925) A note on the kinetics of enzyme action. Biochem J 19(2):338–339

    Article  CAS  Google Scholar 

  19. Hong CC, Yu PB (2009) Applications of small molecule BMP inhibitors in physiology and disease. Cytokine Growth Factor Rev 20(5–6):409–418. doi:10.1016/j.cytogfr.2009.10.021

    Article  CAS  Google Scholar 

  20. Chen YG, Liu F, Massague J (1997) Mechanism of TGFbeta receptor inhibition by FKBP12. EMBO J 16(13):3866–3876. doi:10.1093/emboj/16.13.3866

    Article  CAS  Google Scholar 

  21. Zegzouti H, Zdanovskaia M, Hsiao K, Goueli SA (2009) ADP-Glo: a bioluminescent and homogeneous ADP monitoring assay for kinases. Assay Drug Dev Technol 7(6):560–572. doi:10.1089/adt.2009.0222

    Article  CAS  Google Scholar 

  22. Macias-Silva M, Hoodless PA, Tang SJ, Buchwald M, Wrana JL (1998) Specific activation of Smad1 signaling pathways by the BMP7 type I receptor, ALK2. J Biol Chem 273(40):25628–25636

    Article  CAS  Google Scholar 

  23. Huse M, Chen YG, Massague J, Kuriyan J (1999) Crystal structure of the cytoplasmic domain of the type I TGF beta receptor in complex with FKBP12. Cell 96(3):425–436

    Article  CAS  Google Scholar 

  24. Leder L (2015) Site-specific protein labeling in the pharmaceutical industry: experiences from novartis drug discovery. Methods Mol Biol 1266:7–27. doi:10.1007/978-1-4939-2272-7_2

    Article  CAS  Google Scholar 

  25. Bouchecareilh M, Caruso ME, Roby P, Parent S, Rouleau N, Taouji S, Pluquet O, Bosse R, Moenner M, Chevet E (2010) AlphaScreen-based characterization of the bifunctional kinase/RNase IRE1alpha: a novel and atypical drug target. J Biomol Screen 15(4):406–417. doi:10.1177/1087057110363823

    Article  CAS  Google Scholar 

  26. Huse M, Kuriyan J (2002) The conformational plasticity of protein kinases. Cell 109(3):275–282

    Article  CAS  Google Scholar 

  27. Iwasaki Y, Nishiyama H, Suzuki K, Koizumi S (1997) Sequential cis/trans autophosphorylation in TrkB tyrosine kinase. Biochemistry 36(9):2694–2700. doi:10.1021/bi962057x

    Article  CAS  Google Scholar 

  28. Schwarz JK, Lovly CM, Piwnica-Worms H (2003) Regulation of the Chk2 protein kinase by oligomerization-mediated cis- and trans-phosphorylation. Mol Cancer Res 1(8):598–609

    CAS  Google Scholar 

  29. Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4(2):67–73

    Article  Google Scholar 

  30. Senisterra G, Chau I, Vedadi M (2012) Thermal denaturation assays in chemical biology. Assay Drug Dev Technol 10(2):128–136. doi:10.1089/adt.2011.0390

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We highly appreciate the supply of recombinant Alk2 and its substrate Smad1-MH2 by Sonia Faut and Myriam Duckely as well as protein characterization through LC-MS by Peggy Lefeuvre and Francis Bitsch. The support from Geoffrey Cutler in implementing the miniaturized ADP formation assay on an automated screening system was also very helpful. The whole Alk2 project team is acknowledged for input and stimulating discussions. Last but not least, we would like to thank Joerg Trappe for suggesting and encouraging the drafting of this manuscript.

Author information

Authors and Affiliations

  1. Center for Proteomic Chemistry, Novartis Institute for Biomedical Research Basel, Novartis Pharma AG, Postfach, CH-4002, Basel, Switzerland

    Bianca Heedmann & Martin Klumpp

Authors
  1. Bianca Heedmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Martin Klumpp
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Martin Klumpp .

Editor information

Editors and Affiliations

  1. Epizyme, Inc., Cambridge, Massachusetts, USA

    William P. Janzen

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Heedmann, B., Klumpp, M. (2016). Screening for Inhibitors of Kinase Autophosphorylation. In: Janzen, W. (eds) High Throughput Screening. Methods in Molecular Biology, vol 1439. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3673-1_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3673-1_10

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3671-7

  • Online ISBN: 978-1-4939-3673-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation