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.
Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.
Mindy I
MI
Davis
Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
Douglas S
DS
Auld
Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA. jinglese@mail.nih.gov.
James
J
Inglese
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000032:analysis; D000255:Adenosine Triphosphate / Q000032:analysis; D005941:Glucokinase / Q000032:analysis* / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D049409:Luciferases, Firefly / Q000378:metabolism; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis*; D011485:Protein Binding; D051575:Proto-Oncogene Proteins c-yes / Q000032:analysis*; D013379:Substrate Specificity
D048868:Adaptor Proteins, Signal Transducing; C485100:GCKR protein, human; D007202:Indicators and Reagents; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D049409:Luciferases, Firefly; C514871:PIP4K2A protein, human; D017853:Phosphotransferases (Alcohol Group Acceptor); D005941:Glucokinase; D051575:Proto-Oncogene Proteins c-yes; C489834:YES1 protein, human
ADP-Glo; Bioluminescence; Glucokinase; Kinase; Luciferase; PI5P4Kα; Quantitative high-throughput screening (qHTS); Yes1