A high-throughput assay based on fluorescence polarization for inhibitors of the polo-box domain of polo-like kinase 1

Anal Biochem. 2008 Dec 15;383(2):205-9. doi: 10.1016/j.ab.2008.08.014. Epub 2008 Aug 22.


The serine/threonine kinase polo-like kinase 1 (Plk1) is critically involved in multiple mitotic processes and has been established as an adverse prognostic marker for tumor patients. Plk1 localizes to its substrates and its intracellular anchoring sites via its polo-box domain (PBD), which is unique to the family of polo-like kinases. Therefore, inhibition of the Plk1 PBD has been suggested as an approach to the inhibition of Plk1 that circumvents specificity problems associated with the inhibition of the conserved adenosine triphosphate (ATP) binding pocket. Here we report on the development of a high-throughput assay based on fluorescence polarization that allows the discovery of small-molecule inhibitors of the Plk1 PBD. The assay is based on binding of the Plk1 PBD to a phosphothreonine-containing peptide comprising its optimal binding motif with a K(d) of 26+/-2 nM. It is stable with regard to dimethyl sulfoxide (DMSO) and time, and it has a Z' value of 0.73+/-0.06 in a 384-well format.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Cycle Proteins / antagonists & inhibitors*
  • Cell Cycle Proteins / chemistry*
  • Cell Cycle Proteins / metabolism
  • Drug Evaluation, Preclinical / methods*
  • Enzyme Inhibitors / analysis*
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Fluoresceins / metabolism
  • Fluorescence Polarization / methods*
  • Humans
  • Molecular Sequence Data
  • Peptides / chemistry
  • Peptides / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors*
  • Protein-Serine-Threonine Kinases / chemistry*
  • Protein-Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins / antagonists & inhibitors*
  • Proto-Oncogene Proteins / chemistry*
  • Proto-Oncogene Proteins / metabolism


  • Cell Cycle Proteins
  • Enzyme Inhibitors
  • Fluoresceins
  • Peptides
  • Proto-Oncogene Proteins
  • 4-carboxyfluorescein
  • Protein-Serine-Threonine Kinases
  • polo-like kinase 1