Development of high-throughput assays based on fluorescence polarization for inhibitors of the polo-box domains of polo-like kinases 2 and 3

Abstract

The serine/threonine kinases Plk1, Plk2, and Plk3 harbor a protein–protein interaction domain dubbed polo-box domain (PBD). Recently, the inhibition of the PBD of the cancer target Plk1 has been successfully explored as an alternative to the inhibition of the kinase by ATP-competitive ligands. However, because the PBDs of Plk1, Plk2, and Plk3 have very similar optimal binding motifs, absolute specificity for the PBD of Plk1 over the PBDs of Plk2 and Plk3 may also represent a big challenge for a small molecule. To aid in the activity profiling of Plk PBD inhibitors, and to identify selective small molecules that will reveal the cellular consequences of inhibiting the PBDs of Plk2 and Plk3, we have developed high-throughput assays based on fluorescence polarization against the PBDs of Plk2 and Plk3. The assays are stable with regard to time and 10% dimethyl sulfoxide and have Z′ values ⩾0.7, making them well-suited for high-throughput screening. Moreover, our data provide insights into the binding preferences of the PBDs of Plk2 and Plk3.