Abstract 3240: Development of non-ATP competitive, PLK1 selective inhibitors
Authors: Estes Kara , Yang Zhengguan , McInnes Campbell , Wyatt Michael D. 
Published: June 2012

Through a better understanding of the molecular mechanisms that control normal and cancer cell proliferation, new therapeutic targets that regulate the cell cycle have been identified. One of these, Polo like kinase 1 (PLK1) is a member of a conserved family of serine/threonine kinases. Its activity and cellular levels play an important role in mitotic entry, DNA-damage checkpoint activation, and centrosome duplication and maturation. Because other PLK family members such as PLK3 act as putative tumor suppressors, it is imperative that a PLK1 inhibitor be exclusive for PLK1, a potential draw back for ATP competitive inhibitors. The polo box domain (PBD) of PLK1 plays an important role in sub-cellular localization and mitotic functions of PLK1. Importantly, it has been shown that inhibiting the PBD site leads to selective PLK1 inhibition and can lead to mitotic arrest and inhibit proliferation of tumor cells. The structure-guided discovery strategy called REPLACE can be used to target protein-protein interactions otherwise considered undruggable. REPLACE uses structure activity relationships of peptide inhibitors to generate a pharmaceutically acceptable lead molecule by replacing individual amino acid residues with non-peptide fragments. REPLACE has been used to identify fragment mimetics for determinants from the endogenous phosphopeptide sequence, LLCSpTPNGL (CDC25c), known to specifically bind to the PBD of PLK1. Using this approach, FLIPs (Fragment Ligated Inhibitory Peptides) were generated and tested in a fluorescence polarization (FP) assay to determine binding potential for the PBD. Small molecule fragment alternatives for the N-terminal tripeptide (LLC) were identified through this approach and shown to possess increased binding relative to the truncated peptide. Modeling and amino acid replacement was also used to elucidate essential binding sites and specificity determinants needed by the endogenous peptide to maintain affinity and selectivity for the PBD. This was confirmed through comparisons of binding for peptide analogs in the FP assay relative to that of the endogenous peptide. As part of the development process, peptides have been examined for anti-proliferative activity in cancer cells and to determine if a PLK1 inhibitory phenotype is observed. Mitotic events were evaluated by flow cytometry and by direct visualization, utilizing cells expressing GFP-labeled histone H2B. Conditions to transduce peptides have been established and these will be used to characterize FLIPs with sufficient affinity for the PBD. By targeting the PBD of PLK1 using REPLACE, we believe that we can develop a non-ATP competitive inhibitor that is not only PLK1 specific but is less toxic to normal cells and safer than current drugs on the market.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3240. doi:10.1158/1538-7445.AM2011-3240