Bioluminescence Methods for Assaying Kinases in Quantitative High-Throughput Screening (qHTS) Format Applied to Yes1 Tyrosine Kinase, Glucokinase, and PI5P4Kα Lipid Kinase
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 wordsQuantitative 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.
- 8.Somberg R, Goueli SA (2004) Method for detecting transferase enzymatic activity. In US 2004/0101922 A1 (USPTO ed., Promega Corporation, USAGoogle Scholar
- 10.Segel IH (1993) Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems. John Wiley and Sons, New YorkGoogle Scholar
- 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
- 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.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