Discovery and Mechanistic Study of Novel PBD-Targeted Inhibitors of PLK1 for Prostate Cancer Treatment

• Main Author: Chapagai, Danda Pani
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01.01.2022
• Subjects: Biochemistry; Cellular biology; Pharmaceutical sciences
• ISBN: 9798802708392

Polo-like kinase 1 (PLK1) is a central player in regulating entry into and progression through mitosis. Inhibition of sub-cellular localization and kinase activity of PLK1 through the Polo-box domain (PBD) is emerging as a viable alternative to ATP binding site directed drugs for which the development of resistant mutants and inhibition of closely related members of the PLK family (tumor suppressor roles) are primary concerns. I describe related novel non-peptidic PBD binding inhibitors, termed abbapolins, identified through successful application of the REPLACE strategy and demonstrate their potent anti-proliferative activity in prostate tumors and other cell lines. Furthermore, the abbapolins show PLK1-specific binding and inhibitory activity as measured by a cellular thermal denaturation assay and their ability to block phosphorylation of TCTP, a key marker of PLK1 mediated kinase activity. I 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. The therapeutic potential of these compounds was further indicated through their antiproliferative activity on a cell line (C67V PLK1 mutation) which is dramatically resistant to ATP competitive PLK1 inhibitors. I report novel findings during mitosis inferred from our collective data, namely that catalytic site binding by BI2536 or volasertib unexpectedly decreased the stability of PLK1 as determined by thermal stability assay, suggesting an induction of a conformational change in intracellular PLK1. In contrast, abbapolins produced the expected right shift in the melting curve of PLK1 indicating binding and stabilization. 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 in cells. 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. Furthermore in vivo pharmacokinetic studies showed that optimized abbapolins have promising oral bioavailability and inhibited the growth of prostate tumors in a mouse xenograft experiment. Abbapolins are thus a compelling alternative to catalytic-based inhibitors as the basis for the development of novel therapeutics targeting PLK1.