A homogeneous fluorescence polarization assay adaptable for a range of protein serine/threonine and tyrosine kinases

J Biomol Screen. 2003 Apr;8(2):164-75. doi: 10.1177/1087057103252309.

Abstract

Recently, a new technology for high-throughput screening has been developed, called IMAP(patent pending). IMAP technology has previously been implemented in an assay for cyclic nucleotide phosphodiesterases (PDE). The authors describe the development of a homogeneous, non-antibody-based fluorescence polarization (FP) assay for a variety of protein kinases. In this assay, fluorescently labeled peptide substrate phosphorylated by the kinase is captured on modified nanoparticles through interactions with immobilized metal (M(III)) coordination complexes, resulting in a change from low to high polarization values. This assay is applicable to protein kinases that phosphorylate serine, threonine, or tyrosine residues. The IMAP platform is very compatible with high-throughput robotics and can be applied to the 1536-well format. As there are hundreds of different kinases coded for in the human genome, the assay platform described in this report is a valuable new tool in drug discovery.

Publication types

  • Evaluation Study

MeSH terms

  • Biological Assay / methods*
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / metabolism
  • Fluorescence Polarization / methods*
  • Humans
  • Protein Serine-Threonine Kinases / chemistry
  • Protein Serine-Threonine Kinases / metabolism*
  • Receptor Protein-Tyrosine Kinases / chemistry
  • Receptor Protein-Tyrosine Kinases / metabolism*
  • Staurosporine / metabolism

Substances

  • Enzyme Inhibitors
  • Receptor Protein-Tyrosine Kinases
  • Protein Serine-Threonine Kinases
  • Staurosporine