A Fluorescence Polarization Assay for the Discovery of Inhibitors of the Polo-Box Domain of Polo-Like Kinase 1

Polo-like kinase 1 (Plk1) is a key player in mitosis and has been widely recognized as a therapeutic target for many human cancer types. Apart from its kinase domain, Plk1 harbors a protein–protein interaction domain dubbed “polo-box domain” (PBD), by which the enzyme binds to its intracellular anchorage sites and to at least a fraction of its substrates. Recent evidence indicates that the inhibition of the PBD by small molecules is feasible and might allow for the discovery of highly specific inhibitors of the enzyme. This chapter details the practical work necessary to set up an assay based on fluorescence polarization for the discovery of inhibitors of the Plk1 PBD, which can be used for high-throughput screening in a 384-well format.