Structure-activity and mechanistic studies of non-peptidic inhibitors of the PLK1 polo box domain identified through REPLACE.

Polo-like kinase 1 (PLK1) is a serine/threonine-protein kinase involved in cell cycle regulation and mitotic progression. Studies have shown that PLK1 is upregulated in many tumors and high levels are adversely related to a poor prognosis. Knocking down or inhibiting PLK1 results in synthetic lethality in PTEN deficient prostate tumors and Kras mutant colorectal tumors, further validating PLK1 as an oncotarget. Substrate recognition by PLK1 occurs through the Polo-Box Domain (PBD), which is a phospho-peptide binding site also responsible for subcellular localization. Much effort has been directed to target this kinase therapeutically through the ATP-binding site, and a few such inhibitors have advanced to clinical trials however with limited clinical efficacy. Moreover, it has been shown that a point mutation in PLK1 (C67V) confers dramatic cellular resistance to catalytic site inhibitors. An alternative approach to target PLK1 potently and selectively is through the PBD to block its protein-protein interactions. Through the REPLACE strategy, for converting peptide inhibitors into more drug-like non peptidic compounds, a PBD targeting compound series ("ABBAs"), has been identified and the key determinants of potency and selectivity elucidated through structure-activity relationship studies. In cellular experiments, the ABBAs were shown to lead to profound effects on the cell cycle, to inhibit tumor proliferation and overcome resistance of cells expressing the PLK1 C67V mutant to ATP-based inhibitors. These non-ATP competitive inhibitors of PLK1 were also used chemical biology probes to investigate the gene regulatory effects of PLK1, known to act on transcription factors such as p53.

Peptidomimetic Polo-Box-Targeted Inhibitors that Engage PLK1 in Tumor Cells and Are Selective against the PLK3 Tumor Suppressor.

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.

Targeting subcellular localization through the polo-box domain: non-ATP competitive inhibitors recapitulate a PLK1 phenotype.

The polo-box domain (PBD) has critical roles in the mitotic functions of polo-like kinase 1 (PLK1). The replacement with partial ligand alternative through computational enrichment (REPLACE) strategy to develop inhibitors of protein-protein interactions has identified alternatives for the N-terminal tripeptide of a Cdc25C substrate. In addition, a peptide structure-activity relationship described key determinants and novel information useful for drug design. Fragment-ligated inhibitory peptides (FLIP) were generated with comparable affinity to peptide PBD inhibitors and possessed antiproliferative phenotypes in cells consistent with the observed decrease in PLK1 centrosomal localization. These FLIPs showed evidence of enhanced PLK1 inhibition in cells relative to peptides and induced monopolar and multipolar spindles, which stands in contrast to previously reported small-molecule PBD inhibitors that display phenotypes only partially representative of PLK1 knockdown. Progress obtained applying REPLACE validates this approach for identifying fragment alternatives for determinants of the Cdc25C-binding motif and extends its applicability of the strategy for discovering protein-protein interaction inhibitors. In addition, the described PBD inhibitors retain high specificity for PLK1 over PLK3 and therefore show promise as isotype selective, non-ATP competitive kinase inhibitors that provide new impetus for the development of PLK1-selective antitumor therapeutics.