Comparison of bioluminescent kinase assays using substrate depletion and product formation.

Methods Development

Abstract

Assays for ATPases have been enabled for high-throughput screening (HTS) by employing firefly luciferase to detect the remaining ATP in the assay. However, for any enzyme assay, measurement of product formation is a more sensitive assay design. Recently, technologies that allow detection of the ADP product from ATPase reactions have been described using fluorescent methods of detection. We describe here the characterization of a bioluminescent assay that employs firefly luciferase in a coupled-enzyme assay format to enable detection of ADP levels from ATPase assays (ADP-Glo, Promega Corp.). We determined the performance of the ADP-Glo assay in 1,536-well microtiter plates using the protein kinase Clk4 and a 1,352 member kinase focused combinatorial library. The ADP-Glo assay was compared to the Clk4 assay performed using a bioluminescence ATP-depletion format (Kinase-Glo, Promega Corp). We performed this analysis using quantitative HTS (qHTS) where we determined potency values for all library members and identified approximately 300 compounds with potencies ranging from as low as 50 nM to >10 microM, yielding a robust dataset for the comparison. Both assay formats showed high performance (Z'-factors approximately 0.9) and showed a similar potency distribution for the actives. We conclude that the bioluminescence ADP detection assay system is a viable generic alternative to the widely used ATP-depletion assay for ATPases and discuss the advantages and disadvantages of both approaches.

Authors

Tanega, Cordelle; Shen, Min; Mott, Bryan T; Thomas, Craig; MacArthur, Ryan; Inglese, James; Auld, Douglas S;

Keywords

  • Adenosine Diphosphate/ analysis
  • Adenosine Diphosphate/ chemistry
  • Chemistry Techniques, Analytical
  • Luminescent Measurements
  • Luminescent Proteins/ analysis
  • Protein Interaction Mapping/ methods
  • Protein-Serine-Threonine Kinases/ analysis
  • Protein-Serine-Threonine Kinases/ chemistry
  • Protein-Tyrosine Kinases/ analysis
  • Protein-Tyrosine Kinases/ chemistry
  • Reproducibility of Results
  • Sensitivity and Specificity

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