Abstract 5782: Probing monomer - dimer and conformational transitions of PLK1 using catalytic and polo-box domain inhibitors
Authors: Sanhaji Mourad , Raab Monika , Merhej George , Rao Chintada Nageswara , Babic Ivan , Nurmemmedov Elmar , Strebhardt Klaus , Wyatt MIchael , McInnes Campbell 
Published: March 2024

Polo Like Kinase 1 (PLK1), a sought-after target for drug discovery, is an essential mitotic kinase consisting of a catalytic domain (KD) and a polobox domain (PBD), the latter of which plays roles in substrate recognition, subcellular localization and further acting as an autoinhibitory regulatory domain. PLK1 can exist either in an inhibited closed conformation or an open active state allowing interaction with and phosphorylation of substrates. PLK1 has also been shown to be regulated by interchange between monomeric and dimeric forms through binding to the Bora protein and by activation loop phosphorylation. We have described PBD-binding molecules termed abbapolins that inhibit cellular phosphorylation of a PLK1 substrate and induce loss of intracellular PLK1. In addition, due to their engagement of the cryptic pocket (ligand induced) of the PBD, we proposed that abbapolins would interfere with formation of PLK1 dimers. Here we provide evidence further supporting the ability of abbapolins to block PLK1 cellular dimerization and that this is related to their mechanism of anti-proliferative activity. In a separate study, using a cellular thermal shift assay, KD inhibitors were shown to decrease soluble PLK1, indicating that catalytic-site binding results in less stable PLK1 suggestive of an open conformation. We have extended these observations using a sensitive readout of cellular target engagement employing a Microtag overexpressed PLK1. In this format, we observe heat-induced destabilization of PLK1 upon nanomolar engagement by two lead KD inhibitors, BI2536 and onvansertib, currently being clinically evaluated. Furthermore, cyclapolin 9, a low molecular weight kinase inhibitor that, unlike the other two KD inhibitors does not protrude from the active site, did not induce significant destabilization. We have also carried out experiments using a fluorescently labeled version of BI2536 to probe the conformational effects of PBD binding upon the KD. These data solidify the observations that relief of autoinhibited PLK1 is induced by certain KD binders and highlight the conformational perturbations from KD versus PBD binding. These observations have implications for the development of ATP-competitive PLK1 inhibitors because catalytic inhibitors may conversely promote PLK1 non-catalytic functions, which may explain their lack of clinical efficacy to date. Furthermore, the demonstration that the abbapolins are mechanistically unique in terms of blocking the dimerization of PLK through the PBD provides impetus for their development as next generation PLK1 inhibitors.

Citation Format: Mourad Sanhaji, Monika Raab, George Merhej, Chintada Nageswara Rao, Ivan Babic, Elmar Nurmemmedov, Klaus Strebhardt, MIchael Wyatt, Campbell McInnes. Probing monomer - dimer and conformational transitions of PLK1 using catalytic and polo-box domain inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5782.