Abstract 4712: Selective polo-like kinase 1 PBD inhibitor
Authors: Chapagai Danda P. , Baxter Merissa , Ramamoorthy Gurusankar , McInnes Campbell , Wyatt Michael 
Published: August 2019

Polo Like Kinase 1 (PLK1) is only expressed in dividing cells and plays a critical role in several stages of mitosis. PLK1 is highly expressed in tumors of various origins while its expression is largely absent in normal tissues. PLK1 inhibition selectively kills cancer cells because cancer cells utterly depend on the mitotic functions of PLK1 overexpression. PLK1 consists of a highly conserved N-terminal catalytic kinase domain and a unique, functionally essential C-terminal Polo Box Domain (PBD). The PBD of each PLK is a phospho-peptide binding motif that determines substrate recognition and sub-cellular localization. PLK1 catalytic inhibitorshave advanced to clinical trials but they inhibit other kinases including family members PLK2 and PLK3. This is problematic because PLK3 is a tumor suppressor and partial inhibition of PLK3 could lead to a long-term malignant progression. Moreover, a single point mutation (C67V) in the PLK1 kinase domain confers substantial cancer cell resistance against PLK1 catalytic inhibitors. An alternative approach to developing potent and selective PLK1 inhibitors is to allosterically target the PBD.

We generated fragment ligated inhibitory peptides (FLIPs) through a strategy called REPLACE. This is a computational and synthetic approach that uses structure activity relationships of peptide inhibitors to generate pharmaceutically acceptable lead molecules. Fragments are docked into the crystal structure of a truncated peptide/receptor complex, then prioritized for synthesis with the peptide and subsequently tested in an in vitro binding assay. PBD-interacting protein (PBIP) and Cdc25c are natural substrates of PLK1. We replaced individual amino acid residues in the PBIP (PLHSpTAI) and Cdc25C PBD substrate peptides (LLCSpTPNGL) with benzoic acid fragments. Here we show PLK1 specificity for PBD inhibitors in vitro. A fluorescent polarization (FP) competitive binding assay to the PBD of PLK1 was used to determine binding specificity. A PLK3 counter screen was used to determine the binding selectivity of our PBD inhibitors. To measure the PLK1 inhibitory activity of the FLIPs in cells, phosphorylation of mitotic proteins was measured. Candidate FLIPs induce mitotic arrest, block PLK1 mediated phosphorylation, and inhibit growth of human cancer cells. To make more drug-like molecules, we modified FLIPs to Small Molecule Inhibitors (SMIs). Further, we are optimizing SMIs to improve biochemical selectivity. SMIs bind to the PLK1 PBD as determined by FP assay. Also, SMIs inhibit cancer cell growth as determined by viability measurements and live cell imaging. Overall these results highlight that targeting the PBD of PLK1 has promise as an antitumor strategy.

Citation Format: Danda P. Chapagai, Merissa Baxter, Gurusankar Ramamoorthy, Campbell McInnes, Michael Wyatt. Selective polo-like kinase 1 PBD inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4712.