Bioluminescence Methods for Assaying Kinases in Quantitative High-Throughput Screening (qHTS) Format Applied to Yes1 Tyrosine Kinase, Glucokinase, and PI5P4Kα Lipid Kinase

  • Mindy I. Davis
  • Douglas S. Auld
  • James Inglese
Part of the Methods in Molecular Biology book series (MIMB, volume 1360)


Assays in which the detection of a biological phenomenon is coupled to the production of bioluminescence by luciferase have gained widespread use. As firefly luciferases (FLuc) and kinases share a common substrate (ATP), coupling of a kinase to FLuc allows for the amount of ATP remaining following a kinase reaction to be assessed by quantitating the amount of luminescence produced. Alternatively, the amount of ADP produced by the kinase reaction can be coupled to FLuc through a two-step process. This chapter describes the bioluminescent assays that were developed for three classes of kinases (lipid, protein, and metabolic kinases) and miniaturized to 1536-well format, enabling their use for quantitative high-throughput (qHTS) of small-molecule libraries.

Key words

Quantitative high-throughput screening (qHTS) Yes1 Glucokinase PI5P4Kα Kinase Bioluminescence Luciferase ADP-Glo 



This work was supported by the Molecular Libraries Initiative of the NIH Roadmap for Medical Research. The content of this publication does not necessarily reflect the views of policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.


  1. 1.
    Cohen P, Alessi DR (2012) Kinase drug discovery—what’s next in the field? ACS Chem Biol 8:96–104PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Walsh MJ, Brimacombe KR, Veith H et al (2011) 2-Oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamides as activators of the tumor cell specific M2 isoform of pyruvate kinase. Bioorg Med Chem Lett 21:6322–6327PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Harbert C, Marshall J, Soh S, Steger K (2008) Development of a HTRF kinase assay for determination of Syk activity. Curr Chem Genomics 1:20–26PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Fabian MA, Biggs WH 3rd, Treiber DK et al (2005) A small molecule-kinase interaction map for clinical kinase inhibitors. Nat Biotechnol 23:329–336CrossRefPubMedGoogle Scholar
  5. 5.
    Kleman-Leyer KM, Klink TA, Kopp AL et al (2009) Characterization and optimization of a red-shifted fluorescence polarization ADP detection assay. Assay Drug Dev Technol 7:56–67PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Hastie CJ, McLauchlan HJ, Cohen P (2006) Assay of protein kinases using radiolabeled ATP: a protocol. Nat Protoc 1:968–971CrossRefPubMedGoogle Scholar
  7. 7.
    Tanega C, Shen M, Mott BT, Thomas CJ, MacArthur R, Inglese J, Auld DS (2009) Comparison of bioluminescent kinase assays using substrate depletion and product formation. Assay Drug Dev Technol 7:606–614PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Somberg R, Goueli SA (2004) Method for detecting transferase enzymatic activity. In US 2004/0101922 A1 (USPTO ed., Promega Corporation, USAGoogle Scholar
  9. 9.
    Wu G, Yuan Y, Hodge CN (2003) Determining appropriate substrate conversion for enzymatic assays in high-throughput screening. J Biomol Screen 8:694–700CrossRefPubMedGoogle Scholar
  10. 10.
    Segel IH (1993) Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems. John Wiley and Sons, New YorkGoogle Scholar
  11. 11.
    Li H, Totoritis RD, Lor LA, Schwartz B, Caprioli P, Jurewicz AJ, Zhang G (2009) Evaluation of an antibody-free ADP detection assay: ADP-Glo. Assay Drug Dev Technol 7:598–605CrossRefPubMedGoogle Scholar
  12. 12.
    Sanghera J, Li R, Yan J (2009) Comparison of the luminescent ADP-Glo assay to a standard radiometric assay for measurement of protein kinase activity. Assay Drug Dev Technol 7:615–622CrossRefPubMedGoogle Scholar
  13. 13.
    Zegzouti H, Goueli SA (2010) ADP Detection based luminescent phosphotransferase or ATP hydrolase assay. In US 2010/0075350 A1 (USPTO ed., Promega Corporation, USAGoogle Scholar
  14. 14.
    Auld DS, Southall NT, Jadhav A, Johnson RL, Diller DJ, Simeonov A, Austin CP, Inglese J (2008) Characterization of chemical libraries for luciferase inhibitory activity. J Med Chem 51:2372–2386CrossRefPubMedGoogle Scholar
  15. 15.
    Auld DS, Zhang YQ, Southall NT, Rai G, Landsman M, MacLure J, Langevin D, Thomas CJ, Austin CP, Inglese J (2009) A basis for reduced chemical library inhibition of firefly luciferase obtained from directed evolution. J Med Chem 52:1450–1458PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Thorne N, Shen M, Lea WA, Simeonov A, Lovell S, Auld DS, Inglese J (2012) Firefly luciferase in chemical biology: a compendium of inhibitors, mechanistic evaluation of chemotypes, and suggested use as a reporter. Chem Biol 19:1060–1072PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Dranchak P, MacArthur R, Guha R, Zuercher WJ, Drewry DH, Auld DS, Inglese J (2013) Profile of the GSK published protein kinase inhibitor set across ATP-dependent and-independent luciferases: implications for reporter-gene assays. PLoS One 8, e57888PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Davis MI, Sasaki AT, Shen M, Emerling BM et al (2013) A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation. PLoS One 8, e54127PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Patel PR, Sun H, Li SQ, Shen M, Khan J, Thomas CJ, Davis MI (2013) Identification of potent Yes1 kinase inhibitors using a library screening approach. Bioorg Med Chem Lett 23:4398–4403PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Rees MG, Davis MI, Shen M, Titus S, Raimondo A, Barrett A, Gloyn AL, Collins FS, Simeonov A (2014) A panel of diverse assays to interrogate the interaction between glucokinase and glucokinase regulatory protein, two vital proteins in human disease. PLoS One 9, e89335PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Inglese J, Auld DS, Jadhav A, Johnson RL, Simeonov A, Yasgar A, Zheng W, Austin CP (2006) Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci U S A 103:11473–11478PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Yasgar A, Shinn P, Jadhav A, Auld D, Michael S, Zheng W, Austin CP, Inglese J, Simeonov A (2008) Compound management for quantitative high-throughput screening. JALA Charlottesv Va 13:79–89PubMedCentralPubMedGoogle Scholar
  23. 23.
    Rix U, Hantschel O, Durnberger G, Remsing Rix LL et al (2007) Chemical proteomic profiles of the BCR-ABL inhibitors imatinib, nilotinib, and dasatinib reveal novel kinase and nonkinase targets. Blood 110:4055–4063CrossRefPubMedGoogle Scholar
  24. 24.
    Copeland RA (2005) Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists. Methods Biochem Anal 46:1–265PubMedGoogle Scholar
  25. 25.
    Jenkins WT (1991) The pyruvate kinase-coupled assay for ATPases: a critical analysis. Anal Biochem 194:136–139CrossRefPubMedGoogle Scholar
  26. 26.
    Demian DJ, Clugston SL, Foster MM, Rameh L, Sarkes D, Townson SA, Yang L, Zhang M, Charlton ME (2009) High-throughput, cell-free, liposome-based approach for assessing in vitro activity of lipid kinases. J Biomol Screen 14:838–844CrossRefPubMedGoogle Scholar
  27. 27.
    Lloyd DJ, St Jean DJ Jr, Kurzeja RJ, Wahl RC et al (2013) Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors. Nature 504:437–440CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Mindy I. Davis
    • 1
  • Douglas S. Auld
    • 2
  • James Inglese
    • 1
  1. 1.Division of Preclinical Innovation, National Center for Advancing Translational SciencesNational Institutes of HealthRockvilleUSA
  2. 2.Center for Proteomic ChemistryNovartis Institutes for Biomedical ResearchCambridgeUSA

Personalised recommendations