PLK1, polo-like kinase 1, is a central player regulating mitosis. Inhibition of the subcellular localization and kinase activity of PLK1 through the PBD, polo-box domain, is a viable alternative to ATP-competitive inhibitors, for which the development of resistance and inhibition of related PLK family members are concerns. We describe novel nonpeptidic PBD-binding inhibitors, termed abbapolins, identified through successful application of the REPLACE strategy and demonstrate their potent antiproliferative activity in prostate tumors and other cell lines. Furthermore, abbapolins show PLK1specific binding and inhibitory activity, as measured by a cellular thermal shift assay and an ability to block phosphorylation of TCTP, a validated target of PLK1-mediated kinase activity. Additional evidence for engagement of PLK1 was obtained through the unique observation that abbapolins induce PLK1 degradation in a manner that closely matches antiproliferative activity. Moreover, abbapolins demonstrate antiproliferative activity in cells that are dramatically resistant to ATP-competitive PLK1 inhibitors.

The polo‐box domain (PBD) of PLK1 determines mitotic substrate recognition and subcellular localization. Compounds that target PLK1 selectively are required due to the tumor‐suppressor roles of PLK3. A structure‐activity analysis of the PBD phosphopeptide binding motif has identified potent peptides that delineate the determinants required for mimicry by nonpeptidic inhibitors and provide insights into the structural basis for the selectivity of inhibitors for the PLK1 PBD. Fragment‐ligated inhibitory peptides (FLIPs) obtained through REPLACE have been optimized to enhance in vitro binding and a systematic analysis of selectivity for PLK1 vs PLK3 has been carried out for peptides and peptidomimetics. Furthermore, these more drug‐like non‐ATP‐competitive inhibitors had on‐target engagement in a cellular context, as evidenced by stabilization of PLK1 in a thermal‐shift assay and by inhibition of the phosphorylation of TCTP, a target of PLK1. Investigation in cells expressing a mutant PLK1 showed that these cells are sensitive to PBD inhibitors but dramatically resistant to clinically investigated ATP‐competitive compounds. These results further validate targeting the PBD binding site in the move towards PLK1 inhibitors that are active against tumors resistant to ATP inhibitors.