Identification of a novel Polo-like kinase 1 inhibitor that specifically blocks the functions of Polo-Box domain

Abstract

Polo-like kinase 1 (Plk1) is a promising target for cancer therapy due to its essential role in cell division. In addition to a highly conserved kinase domain, Plk1 also contains a Polo-Box domain (PBD), which is essential for Plk1's subcellular localization and mitotic functions. We adopted a fluorescence polarization assay and identified a new Plk1 PBD inhibitor T521 from a small-molecule compound library. T521 specifically inhibits the PBD of Plk1, but not those of Plk2-3. T521 exhibits covalent binding to some lysine residues of Plk1 PBD, which causes significant changes in the secondary structure of Plk1 PBD. Using a cell-based assay, we showed that T521 impedes the interaction between Plk1 and Bub1, a mitotic checkpoint protein. Moreover, HeLa cells treated with T521 exhibited dramatic mitotic defects. Importantly, T521 suppresses the growth of A549 cells in xenograft nude mice. Taken together, we have identified a novel Plk1 inhibitor that specifically disrupts the functions of Plk1 PBD and shows anticancer activity.

Keywords: Polo-Box domain; Polo-like kinase 1 inhibitor; cancer therapy; fluorescence polarization; protein-protein interactions.

Figure 1. Identification of a potent small molecule inhibitor of Plk1 PBD

A. The chemical structure of T521: 5-(ethylsulfonyl)-2-(4-fluorophenyl)-4-(phenylsulfonyl) oxazole. B, C, D. The inhibitory effect of T521, Poloxin and TQ on the binding of the PBDs of Plk1, 2 and 3 to the FITC-labeled phosphopeptide was analyzed by FP assay. Briefly, T521, Poloxin or TQ was incubated with Plk1-3 PBD for 1 hr at room temperature (RT) prior to addition of FITC-labeled phosphopeptide. The inhibitory effect was calculated as described in the Materials and Methods. Error bars represent SD. E. ELISA-based PBD binding inhibition assay to determine the inhibitory effect of T521 on the interaction between Plk1 PBD and its binding target Map205^PBM. His-tagged PBD of Plk1 and different concentrations of T521 were added into GST-Map205^PBM-coated plates. After incubation, the plate was washed and then probed with mouse anti-His primary antibody, followed by goat anti-mouse IgG-HRP secondary antibody. After washing, the plate was incubated with TMB solution and the OD₄₅₀ was measured. Detailed procedure was described in Methods and Materials. DMSO was used as control. F. T521 inhibits Bub1-Plk1 interaction in vivo using Co-immunoprecipitation (IP) assay. HeLa cells synchronized by double-thymidine block were released into medium containing DMSO or T521 at indicated concentrations for 10 hrs, and then lysed and subject to Bub1 IP. The proteins in the precipitates were separated by 10% SDS-PAGE and probed with the anti-hPlk1 and anti-Bub1 antibodies. Moreover, the Bub1 and Plk1 levels were also determined in the lysates of HeLa cells before IP. β-actin served as the loading control.

Figure 2. The binding mode of T521 to Plk1 PBD

A. The inhibition of Plk1 PBD by T521 at different temperatures. Certain concentrations of T521 were incubated with Plk1 PBD for 1 hr at indicated temperatures, and then FITC-Poloboxtide was added into the mixture. FP assay was performed after 15 min incubation. Here shows the average of three independent experiments. Error bars represent SD. B. Time-dependent inhibition of Plk1 PBD by T521. Certain concentrations of T521 were incubated with Plk1 PBD for indicated times at RT, and then FITC-Poloboxtide was added into the mixture. FP assay was performed after 15 min incubation. Here shows the average of three independent experiments and error bars represent SD. C. Indirect analysis of the T521/Plk1 PBD binding kinetics using the FP assay. Plk1 PBD was incubated with T521 (2, 4, and 8 μM) for the indicated times and then FITC-Poloboxtide was added to the mixture for further incubation (15 min). Then FP assay was performed to analyze the binding between PBD and FITC-Poloboxtide, which indirectly reflects the occupancy of PBD by T521. Detailed protocol was described in Methods and Materials.

Figure 3. T521 induces mitotic arrest in HeLa cells

A. HeLa cells (1.0×10⁵ cell/well) were synchronized at the G₁/S boundary by double-thymidine block, and then released into medium with or without T521 (10 μM). Cell cycle progression was analyzed for DNA content by flow cytometry. DNA contents of 2C (left) and 4C (right) are indicated by triangles in the image. B. The G₂/M arrest induced by different concentrations of T521 after release from double-thymidine block for 16 hrs. Cell cycle analysis was performed as described in A. C. Quantitative analysis of cell cycle distribution of DMSO or T521-treated HeLa cells in panel B (n=3). Error bars represent SD. D. Protein levels of mitotic markers in T521-treated HeLa cells. Synchronized HeLa cells were released into medium containing T521 at the indicated concentrations or nocodazole (50 ng/mL) for additional 16 hrs. Cellular extracts were prepared and the protein levels of Plk1, Cyclin B1 and pHH-3 were analyzed by western blotting. Nocodazole-treated cells were used as a control for mitotic arrest. DMSO-treated cells and cells synchronized with thymidine (2 mM) were also used as controls. GAPDH was used as the loading control.

Figure 4. T521 induces defects in centrosome integrity, chromosome alignment and spindle assembly

A. The distribution of mitotic phases within the total mitotic cells treated with or without T521. The percentage of mitotic cells with lagging chromosomes (metaphase with defects) or multiple centrosomes (>2) are also shown. Approximately 50 mitotic cells were analyzed each time and the results are the average from three independent experiments. Error bars represent SD. B. Representative Hela cells in metaphase treated with or without T521. HeLa cells were synchronized at the G₁/S boundary by double-thymidine block and then released into fresh medium with DMSO or T521 (5 μM) for 10 hrs. Cells were fixed and stained for DNA (blue), α-tubulin (green) and centrosome (γ-tubulin, red). The images were taken using an Olympus FV1000 confocal microscopy. Here shows an untreated cell (DMSO) in metaphase as well as a T521-treated cell with condensed but randomly distributed chromosomes, a disorganized spindle, and multiple γ-tubulin foci. Scale bars represent 5 μm.

Figure 5. T521 induces apoptosis in HeLa cells

A. Apoptosis caused by T521 treatment. Exponentially growing HeLa cells were treated with T521 at the indicated concentrations for 24 hrs and AnnexinV/PI dual-staining was performed. Apoptotic HeLa cells were analyzed by FACS. R1, AnnexinV-negative and PI-positive, dead cells; R2, double-positive, late apoptotic cells; R3, AnnexinV-positive and PI-negative, early apoptotic cells; R4, double-negative, live cells. B. Quantitative analysis of apoptotic cells in panel A (n=3). Error bars represent SD. C. PARP cleavage in T521-treated HeLa cells. HeLa cells were treated as described in A. Cell extracts were prepared and PARP cleavage was analyzed by western blotting. GAPDH served as the loading control.

Figure 6. T521 suppresses tumor growth

A. Nude mice bearing established xenografts of A549 (n=10 for each group) were intratumorally treated with DMSO, T521 (100 and 50 mg/kg) on Wednesdays and Fridays for 5 weeks. The tumor size over time is displayed. **p<0.01 compared to DMSO. p values were calculated using t test. Error bars represent SD. B. The total body weight of individual mice in the three groups was determined twice per week and the average body weights are plotted.