A cryptic hydrophobic pocket in the polo-box domain of the polo-like kinase PLK1 regulates substrate recognition and mitotic chromosome segregation

The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Tyr pocket mutations selectively impair PLK1 binding to the kinetochore phosphoprotein substrate PBIP1, but not to the centrosomal substrate NEDD1. Through a structure-guided approach, we develop a small-molecule inhibitor, Polotyrin, which occupies the Tyr pocket. Polotyrin recapitulates the mitotic defects caused by mutations in the Tyr pocket, further evidencing its essential function, and exemplifying a new approach for selective PLK1 inhibition. Thus, our findings support a model wherein substrate discrimination via the Tyr pocket in the human PLK1 PBD regulates mitotic chromosome segregation to preserve genome integrity.


Fluorescence Polarisation assay
Final concentrations of assay components used in binding assays were as follows: TAMRA-labelled PBIP1 phosphopeptide, 5-TAMRA-Glu-Thr-Phe(71)-Asp-Pro-Pro-Leu-His-pThr(78)-Ala-Ile-Tyr-Ala-Asp-Glu-acid 10nM; PLK1 PBD (aa345-603) 42nM (1.25ng/µl). Assays were carried out in PBS (pH 7.4) plus 0.03% tween. DMSO controls were run alongside all experimental compounds and percentage inhibition normalised to these controls. Compounds were titrated 2-fold from a top concentration of 250µM giving a maximum final concentration of DMSO in the assay of 0.25%. The total assay volume per well was 45µl. Experiments were performed in NBS black 384-well microtiter plates (Corning). All assay components were incubated together at 22°C for 20 minutes prior to Fluorescence Polarisation (FP) being read using a BMG PheraStar plate reader with a 540/590/590nm FP module and unbound 10nm TAMRA-labelled peptide set to a FP value of 35mP.

PBD structure determination
PBD (residues 371-594) of human Plk1 was expressed and purified as described in Sledz et al 20 . The purified PBD domain was crystallised in 100-200 mM K/NA Tartrate, 10-20% PEG3350. Crystals were soaked overnight with Polotyrin or 3-iodobenzyl bromide in the presence of 10% DMSO and 10% PEG8000 as cryoprotectant and crystals cryocooled in liquid N2. Diffraction data was collected at Diamond Light Source beamlines i24 and i03, the data was processed with XDS 57 . Structures were solved by molecular replacement using unliganded PBD structure (PDB code 3P2W) as the search model. The structure was refined briefly before electron density evaluated for the presence of clear additional density for the soaked ligand. The resulting complex structures were refined using phenix.refine 58 , with manual rebuilding and validation in Coot 59 . The refined coordinates have been submitted to Protein Data Bank under accession codes 5NEI (complex with Polotyrin) and 5NMM (complex with 3iodobenzyl bromide).

Synthesis and characterisation of Polotyrin: General information
All non-aqueous reactions were performed at room temperature under a constant stream of dry nitrogen using glassware that had been oven-dried overnight unless otherwise stated.
Room temperature (RT) refers to ambient temperature. All temperatures below 0 C were that of the external bath. Temperatures of 0 C were produced and maintained with an ice-water bath. Temperatures below 0 C were produced and maintained using an acetone-dry ice bath.
All reagents and solvents were used as received unless otherwise stated. Where appropriate, reagents and solvents were purified using standard experimental techniques. Ethyl acetate and methanol were distilled under nitrogen with calcium hydride. Tetrahydrofuran was dried over Na wire and distilled, while under nitrogen, from a combination of calcium hydride and lithium aluminium hydride with triphenylmethane as indicator. Pet ether refers to the fraction of light petroleum ether that had a boiling point between 40 and 60 C. Brine refers to a sat. aqueous NaCl solution.
Yields refer to spectroscopically and chromatographically pure compounds unless otherwise stated in the experimental text. Reactions were monitored using thin layer chromatography performed on commercially prepared glass plates pre-coated with Merck silica gel 60 F254 and visualised by quenching of UV fluorescence (max = 254 nm), iodine, potassium permanganate, p-anisaldehyde, vanillin, phosphomolybdic acid, ninhydrin or by liquid chromatography mass spectrometry (LCMS) using a Waters Micromass ZQ spectrometer. Retention factors (R f) are quoted to 0.01. Rf values were not determined for carboxylic acids due to their propensity to stick to the baseline.
Column chromatography was carried out using Merck 9385 Keiselgel 60 SiO2 (230-400 mesh) under a positive pressure of compressed air.
Lyophilisation was achieved by suspending the required residue in a MeCN-H2O (1:1) solution which was cooled to -196 °C with liquid nitrogen. The frozen sample was concentrated using a Scanvac CoolSafe 100-9 Pro freeze dryer overnight.
Infrared spectra were recorded neat on a Perkin-Elmer 1600 FT IR spectrometer. Only absorption maxima (max) of interest are reported in wavenumbers (cm -1 ) with the following abbreviations: w, weak; m, medium; s, strong; br, broad.
Melting points were obtained on a Büchi B-545 melting point apparatus and are uncorrected.
Proton magnetic resonance spectra were recorded using an internal deuterium lock at ambient probe temperatures on the following instruments: Bruker Avance 400 CRYO QNP (400 MHz), Bruker Avance 400 QNP (400 MHz), Bruker Avance 500 CRYO (500 MHz). Chemical shifts (H) are quoted in parts per million (ppm) to the nearest 0.01 ppm downfield of trimethylsilane (H = 0) and are referenced to the residual nondeuterated solvent peak as follows: CDCl3, 7.26 ppm; d6-DMSO, 2.50 ppm. Integration, chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; m, multiplet; br, broad; app, apparent; obs, obscured or a combination of these) and coupling constants (J, measured in Hertz (Hz) and quoted to the nearest 0.5 Hz) were identified using the commercially available iNMR 3.4.7 processor software. Where possible and appropriate, J values have been adjusted to match for coupling nuclei.
Assignment was based on chemical shift, integration, multiplicity, coupling constants and where appropriate, COSY, HMQC and HMBC experiments or by analogy to fully interpreted spectra for related compounds.
Carbon magnetic resonance spectra were recorded by broadband proton spin decoupling at ambient probe temperatures using an internal deuterium lock on the

Diethyl 2-(3-nitrobenzyl)malonate
Adapted from the procedure of Rotthaus et al. 1 Diethyl malonate (4.42 mL, 29.1 mmol, 1 equiv) was dissolved in anhydrous THF (60 mL) and the resulting solution cooled to 0 °C. Sodium hydride (60% dispersion in mineral oil, 1.17 g, 29.1 mmol, 1 equiv) and 3nitrobenzyl chloride (5.00 g, 29.1 mmol, 1 equiv) were added sequentially. The resulting mixture was refluxed o/n, allowed to cool to RT and poured into a sat. aqueous NH4Cl solution. The organic layer was collected and the aqueous extracted with Et2O (× 3). The organic fractions were combined, washed with water and brine, dried (MgSO4) and concentrated in vacuo. The crude product was purified by column chromatography (SiO2; pet ether-EtOAc gradient, 12:1-4:1) to yield the title compound as a light yellow oil

Methyl thiophene-2-carboxylate
To a solution of 2-thiophenecarboxylic acid (10.0 g, 78.0 mmol, 1 equiv) in MeOH (100 mL) was added a concentrated solution of H2SO4 (5 mL). The resulting solution was heated to reflux for 17 hr, allowed to cool to RT and concentrated in vacuo. The residue was dissolved in EtOAc, washed with a sat. aqueous NaHCO3 solution (× 3), dried (MgSO4) and concentrated in vacuo. 111 was isolated as a brown oil (9.86 g, 69.4 mmol, 92%) that was used without further purification.