Development of a Novel Cell-Permeable Protein-Protein Interaction Inhibitor for the Polo-box Domain of Polo-like Kinase 1.

David J Huggins,Ashok R Venkitaraman,Alex T Crooks,David W Walker,Meredith Roberts-Thomson,Grahame J Mckenzie,Robert G Boyle,David R Spring,Richard Boyce,Ana J Narvaez,Fengzhi Zhang,Pooja Sharma,Bryn S Hardwick,Amy Emery,Natalia Mateu,Luca Laraia

doi:10.1021/acsomega.9b03626

Abstract

Polo-like kinase 1 (PLK1) is a key regulator of mitosis and a recognized drug target for cancer therapy. Inhibiting the polo-box domain of PLK1 offers potential advantages of increased selectivity and subsequently reduced toxicity compared with targeting the kinase domain. However, many if not all existing polo-box domain inhibitors have been shown to be unsuitable for further development. In this paper, we describe a novel compound series, which inhibits the protein–protein interactions of PLK1 via the polo-box domain. We combine high throughput screening with molecular modeling and computer-aided design, synthetic chemistry, and cell biology to address some of the common problems with protein–protein interaction inhibitors, such as solubility and potency. We use molecular modeling to improve the solubility of a hit series with initially poor physicochemical properties, enabling biophysical and biochemical characterization. We isolate and characterize enantiomers to improve potency and demonstrate on-target activity in both cell-free and cell-based assays, entirely consistent with the proposed binding model. The resulting compound series represents a promising starting point for further progression along the drug discovery pipeline and a new tool compound to study kinase-independent PLK functions.

Highlights

Polo-like kinase (PLK1) is a key regulator of cell cycle progression and is a member of a family of closely related multifunctional kinases comprising Polo-like kinase 1 (PLK1), PLK2, PLK3, and PLK4
A fluorescence polarization (FP) assay was developed to identify compounds that inhibit the binding of a consensus phosphopeptide to the polo-box domain (PBD) domain of PLK1
Using a previously described HeLa cell line with a doxycycline-inducible overexpression of GFP-tagged PLK1,15 we looked at localization of the fluorescently tagged protein in mitosis

Summary

Introduction

Polo-like kinase (PLK1) is a key regulator of cell cycle progression and is a member of a family of closely related multifunctional kinases comprising PLK1, PLK2, PLK3, and PLK4. PLK1 is composed of two main structural elements: a kinase domain, which phosphorylates a great many proteins during mitosis, and a polo-box domain (PBD), comprising two polo-box units, which provides spatio-temporal regulation through interaction with its substrates.[9,10] These two domains are joined by a linker of approximately 50 residues. The kinase domain has been crystallized in the presence and absence of bound inhibitors and shows similarity to many other kinases.[11] The PBD has been crystallized in the presence and absence of bound phosphopeptides, and, in contrast, it has a unique structure, shared only with other members of the PLK family.[12] The PBD offers both the phosphopeptide binding groove and the more recently described tyrosine pocket[10,13−15] as possible sites for small-molecule inhibition and potentially a means to overcome some of the problems of kinase inhibitors. In the last few years, studies on a number of small-molecule inhibitors of the PBD of PLK1 have been published.[16−21] recent work has raised questions over the mechanism of action for some of these inhibitors, casting doubt over the suitability for Received: October 28, 2019 Accepted: December 13, 2019 Published: December 24, 2019

Methods

Results

Conclusion