Identification Of A Novel Polo-like Kinase 1 Inhibitor That Specifically Blocks Polo-Box Domain-Mediated Protein-Protein Interactions

Polo-like kinase 1 (Plk1) is a conserved serine/threonine kinase regulating multiple essential steps in mitosis, such as mitotic entry, spindle formation, chromosome segregation and cytokinesis. Overexpression of Plk1 has been reported in many human cancers, which provides a possible basis for selective elimination of cancer cells. Therefore, Plk1 is generally regarded as an anticancer drug target as well as a potential prognostic biomarker for cancer patients. Hence, Plk1 is believed to be an attractive therapeutic target for cancer cells. Compared with the role of Plk1 in cell cycle progression, Plk2 and Plk3 have been identified as tumor suppressors to prevent mitotic catastrophe and preserve genomic integrity. Therefore, specific inhibition of Plk1 kinase activity, but not other Plks, could be important for cancer therapy.Plk1 is characterized by a unique C-terminal noncatalytic region containing two tandem Polo boxes and a highly conserved N-terminal catalytic kinase domain containing an ATP binding pocket. The Polo-Box domain (PBD) of Plk1 has been shown to be crucial for its subcellular localization and mitotic functions by interacting with phosphorylated substrates that contain a consensus S-pS/pT-P/X motif. Due to the highly conserved nature and drug resistance issues of Plk1 inhibitors targeting the kinase domain, the development of Plk1-specific inhibitors remains challenging to date. The unique PBD of Plk1 is a promising target for Plk1 inhibitors, because these inhibitors could show stronger specificity but with less drug resistance for cancer therapy.Poloxin, Thymoquinone (TQ), Poloxipan, Purpurogallin (PPG) and Green Tea Catechins have recently been identified as potent Plk1 inhibitors targeting the Plk1 PBD. Given the limited number of Plk1 PBD inhibitors and their modest selectivity, we performed high-throughput screen for small molecules that specifically inhibit Plk1 PBD.Here we have successfully developed a fluorescence polarization-based high-throughput screening assay, and with this assay, we identified compound T521 (apparent IC50≈1.22±0.13μM) as a specific small molecule inhibitor of Plk1 PBD from the diverse chemical libraries of 20,000 small molecule compounds. To our knowledge, this is the first report for a compound with phenylsulfonyl oxazole scaffold that shows anti-cancer activity as a new chemical entity. Using an in vivo PBD substrate Map205, we further confirmed that compound T521 efficiently blocks the interaction between Plkl PBD and GST-Map205PBM in vitro and displays more desirable specificity profile than Poloxin and TQ. Interestingly, we found that the inhibition of the interaction between Plkl PBD and FITC-Poloboxtide by compound T521 was time and temperature dependent, but the binding mode was a competitive noncovalent binding instead of covalent modification.Compound T521 efficiently inhibited the proliferation of all tested 12 human cancer cell lines in a dose-dependent manner with IC50 ranging from 1 to 5μM. Moreover, the proliferation of three exponentially growing normal cell lines, MRC5, HEK-293 and L02 cells, was also blocked by T521 and IC50 values range from 3 to 8μM, indicating a comparable sensitivity of nontransformed cycling cells to T521. This observation has already been reported with Poloxin and BI2536 treatment. According to HeLa cell cycle arrest analysis by flow cytometry, compound T521 blocked HeLa cells at G2/M phases in synchronized HeLa cells with a significant dose-dependent manner. Prolonged G2/M phase induced by compound T521 was further confirmed by Western Blotting. Strikingly, T521-treated cells exhibited dramatic Plkl subcellular mislocalization, obvious centrosome fragmentation and abnormal chromosome congression. Furthermore, T521 treatment led to a dose-dependent increase of cell population positive for Annexin V staining and PARP cleavage, strongly indicative of massive apoptosis.In this study, we show that compound T521 efficiently inhibits PBD-mediated substrate binding in vitro and HeLa cells treated with this compound exhibit Plkl mislocalization, centrosome fragmentation, chromosome misalignment, spindle assembly defect, mitotic arrest and massive apoptosis, which is likely the consequence of inhibition of Plkl by targeting PBD. Taken together, we identified a novel Plkl inhibitor, which specifically targets Plkl PBD and shows cytotoxicity to various cancer cell lines. Therefore, compound T521 exhibits potential as an anticancer drug by targeting Plkl PBD.