Abstract 2572: A novel strategy for PLK1 kinase inhibition by allosterically targeting the Polo Box Domain
Authors: Chapagai Danda , Baxter Merissa , Craig Sandra , Ramamoorthy Guru , Stafford Jessy , Vitali Sikirzhytski , Nurmemmedov Elmar , McInnes Campbell , Wyatt Michael D. 
Published: June 2022

Polo Like Kinase 1 (PLK1) is only expressed in dividing cells and plays a critical role in several stages of mitosis. PLK1 is overexpressed in many tumor types. PLK1 inhibition selectively kills cancer cells because they are dependent on the mitotic functions of PLK1. PLK1 consists of a highly conserved N-terminal catalytic kinase domain and a unique, functionally essential C-terminal Polo Box Domain (PBD). The PBD is a phospho-peptide binding motif that determines substrate recognition and sub-cellular localization. PLK1 catalytic inhibitors have advanced to clinical trials but not demonstrated convincing efficacy. Targeting the PBD offers an attractive alternative to pursue PLK1 inhibition. An iterative strategy called REPLACE, involving computational and synthetic approaches, was utilized to generate fragment-ligated inhibitory peptides and further application of REPLACE resulted in non-peptidic compounds named abbapolins. In recently published studies, abbapolins were found to specifically bind to the PBD of PLK1 in biochemical and cellular assays. The phosphorylation of TCTP, a specific PLK1 substrate, was measured in abbapolin treated cancer cells. Abbapolins produced a dose dependent reduction in p-TCTP. We also made a novel observation that abbapolins upon binding to PLK1 induced its intracellular loss in a mechanism at least partially dependent on the proteasome. Spurred on by this unique mechanism of action, we initiated studies on the interactions of catalytic inhibitors and PBD-binding abbapolins with PLK1 in vitro and in cellular contexts. Biochemical assays were performed with full length PLK1 in vitro and intracellular PLK1 binding was measured by a Cellular Thermal Shift Assay (CETSA). We report novel findings during mitosis inferred from our collective data, namely that catalytic binding by BI2536 or volasertib unexpectedly decreased soluble PLK1 as determined by CETSA, inferring induction of a conformational change in intracellular PLK1. In contrast, abbapolins produced the expected right shift in the melting curve of PLK1. Intriguingly, these differential effects on PLK1 thermal stability have opposing impacts on the fate of intracellular PLK1. Binding by catalytic inhibitors cause accumulation of PLK1, whereas PBD binding by abbapolins ultimately lead to its loss. Results from quantifying intracellular PLK1 were also supported in vitro by evidence of cooperative binding between catalytic inhibitors and abbapolins in the context of the FL protein and suggest that conformational changes induced by binding to the catalytic site increase affinity of abbapolins for the PDB. Collectively, the results shed further insight into the unique mechanism of action for abbapolins potentially due to their engagement of a cryptic hydrophobic pocket of the PBD. Abbapolins are thus a compelling alternative to catalytic-based inhibitors for the development of novel therapeutics targeting PLK1.

Citation Format: Danda Chapagai, Merissa Baxter, Sandra Craig, Guru Ramamoorthy, Jessy Stafford, Sikirzhytski Vitali, Elmar Nurmemmedov, Campbell McInnes, Michael D. Wyatt. A novel strategy for PLK1 kinase inhibition by allosterically targeting the Polo Box Domain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2572.