A high-throughput assay based on fluorescence polarization for inhibitors of the polo-box domain of polo-like kinase 1.
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.
Reindl
Wolfgang
W
Department of Molecular Biology, Max Planck Institute of Biochemistry, Munich Center for Integrated Protein Science (CIPSM), Am Klopferspitz 18, 82152 Martinsried, Germany.
Strebhardt
Klaus
K
Berg
Thorsten
T
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology*; D005452:Fluoresceins / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D000097869:Polo-Like Kinase 1
D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D010455:Peptides; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D000097869:Polo-Like Kinase 1; C045132:4-carboxyfluorescein