Identification of Polo-like Kinase 1 inhibitors through phenotype-based approaches

A234 Polo-like Kinase 1 (PLK1) plays a key role in progression of mitosis. Currently, a lot of interest exists in identifying potent and selective inhibitors of PLK1 and developing them into cancer therapeutics. Down-regulation of Plk1 activity with siRNA or small molecules is characterized by inhibition of proliferation, cell cycle arrest in mitotis, apoptosis, and the so-called 9dumbbell9 morphology, in which post-mitotic cells have failed to separate via cytokinesis.
 We have screened a subset of Rigel compound library using an image-based high content screening (HCS) assay in double thymidine-synchronized tumor cells, and looked for the compounds that arrest cells at mitosis. Morphological information such as the pattern of DNA arrangement in mitosis-arrested cells, nuclear fragmentation (due to apoptosis or mitotic catastrophe) and “dumbbell” nuclei was assessed by visual inspection. We also monitored Plk1 activity in cells by Western blot and Phospho-flow assays based on detecting Cdc25C Ser-198 phosphorylation, a Plk1 specific target site.
 Combining image-based HCS with Plk1 cell-based assays we identified several hits. The anti-proliferative activity of these molecules in cells corresponds to the EC50 of PLK1 inhibition in cells (60-120nM). Phenotypes characteristic of PLK1 inhibition, including circular DNA arrangement in mitosis-arrested cells, apoptosis and dumbbell-shaped nuclei were observed upon incubation of cells with these compounds. In addition, in vitro kinase studies showed that at 100 nM PLK1 was fully inhibited by these compounds.
 Thus, a series of potent PLK1 inhibitors have been identified using a phenotype-based approach. SAR is currently being optimized to further improve potency and selectivity of lead molecules.