A homogeneous and nonisotopic assay for phosphatidylinositol 4-kinases

Anal Biochem. 2011 Oct 1;417(1):97-102. doi: 10.1016/j.ab.2011.05.046. Epub 2011 Jun 7.


Phosphatidylinositol 4-kinases (PI 4-kinases) catalyze the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate (PtdIns4P). The four known mammalian PI 4-kinases, PI4KA, PI4KB, PI4K2A, and PI4K2B have roles in intracellular lipid and protein trafficking. PI4KA and PI4KB also assist in the replication of several positive-sense RNA viruses. The identification of selective inhibitors of these kinases would be facilitated by assays suitable for high-throughput screening. We describe a homogeneous and nonisotopic assay for PI 4-kinase activity based on the bioluminescent detection of the ADP produced by kinase reactions. We have evaluated this assay with known nonselective inhibitors of PI 4-kinases and show that it performs similar to radiometric assay formats previously described in the literature. In addition, this assay generates Z-factor values of >0.7 for PI4KA in a 384-well format, demonstrating its suitability for high-throughput screening applications.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Phosphatidylinositol 4-Kinase / antagonists & inhibitors
  • 1-Phosphatidylinositol 4-Kinase / isolation & purification
  • 1-Phosphatidylinositol 4-Kinase / metabolism*
  • Adenosine / metabolism
  • Adenosine Diphosphate / analysis
  • Adenosine Triphosphate / metabolism
  • Androstadienes / pharmacology
  • Animals
  • Cattle
  • Cell Line
  • Chromatography, Affinity
  • Dimethyl Sulfoxide / pharmacology
  • Enzyme Assays / methods*
  • High-Throughput Screening Assays
  • Humans
  • Isotope Labeling
  • Kinetics
  • Protein Kinase Inhibitors / pharmacology
  • Radiometry
  • Wortmannin


  • Androstadienes
  • Protein Kinase Inhibitors
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • 1-Phosphatidylinositol 4-Kinase
  • Adenosine
  • Wortmannin
  • Dimethyl Sulfoxide