Designed inhibitor for nuclear localization signal of polo-like kinase 1 induces mitotic arrest.

Polo-like kinase 1 (Plk1), a member of polo-like kinase family, regulates multiple essential steps of the cell cycle progression. Plk1 is overexpressed in multiple cancer cell lines and considered to be a prime anticancer target. Plk1 accumulates in the nucleus during S and G2 phases by its bipartite nuclear localization signal (NLS) sequence, which is crucial for Plk1 regulation during normal cell cycle progression. Here, through combined computational and experimental studies, we identified compound D110, which inhibits Plk1 kinase activity with an IC50 of 85 nm and blocks the nuclear localization of Plk1 during S and G2 phases. D110-treated cancer cells were arrested at mitosis with monopolar spindle, indicating the inhibition of the Plk1 kinase activity in cell. As D110 interacts with both the ATP site and the NLS in Plk1, it demonstrates good selectivity toward Plk2 and Plk3. The strategy of simultaneously inhibiting kinase activity and its subcellular translocations offers a novel approach for selective kinase inhibitor design.

Discovery of Novel Polo-Like Kinase 1 Polo-Box Domain Inhibitors to Induce Mitotic Arrest in Tumor Cells.

Polo-like kinase 1(Plk1) is vital for cell mitosis and has been identified as anticancer target. Its polo-box domain (PBD) mediates substrate binding, blocking of which may offer selective Plk1 inhibition compared to kinase domain inhibitors. Although several PBD inhibitors were reported, most of them suffer from low cell activity. Here, we report the discovery of novel inhibitors to induce mitotic arrest in HeLa cells by virtual screening with Plk1 PBD and cellular activity testing. Of the 81 compounds tested in the cell assay, 10 molecules with diverse chemical scaffolds are potent to induce mitotic arrest of HeLa at low micromolar concentrations. The best compound induces mitotic arrest of HeLa cells with an EC50 of 4.4 µM. The cellular active inhibitors showed binding to Plk1 PBD and compete with PBD substrate in microscale thermophoresis analysis.

Usp16 regulates kinetochore localization of Plk1 to promote proper chromosome alignment in mitosis.

During the G2 to M phase transition, a portion of mitotic regulator Plk1 localizes to the kinetochores and regulates the initiation of kinetochore-microtubule attachments for proper chromosome alignment. Once kinetochore-microtubule attachment is achieved, this portion of Plk1 is removed from the kinetochores as a result of ubiquitination. However, the crucial molecular mechanism that promotes the localization and the maintenance of Plk1 on the kinetochores until metaphase is still unclear. We report that ubiquitin-specific peptidase 16 (Usp16) plays a key role during this process. Usp16 deubiquitinates Plk1, resulting in an enhanced interaction with kinetochore-localized proteins such as BubR1, and thereby retains Plk1 on the kinetochores to promote proper chromosome alignment in early mitosis. Down-regulation of Usp16 causes increased ubiquitination and decreased kinetochore localization of Plk1. Thus, our data unveil a unique mechanism by which Usp16 promotes the localization and maintenance of Plk1 on the kinetochores for proper chromosome alignment.

Modeling the mitotic regulatory network identifies highly efficient anti-cancer drug combinations.

Targeting mitotic regulation is recognized as an important strategy for cancer therapy. Aurora A/B kinase and polo-like kinase 1 (PLK1) are the key mitotic regulators, and many inhibitors have been developed. Combinations of these inhibitors are anticipated to be more effective therapeutics compared with single-inhibitor treatments; however, a systematic analysis of the combined effects is lacking. Here, we constructed the first mammalian cell mitotic regulation network model, which spans from mitotic entry to anaphase initiation, and contains all key mitotic kinase targets. The combined effects of different kinase inhibitors and microtubule inhibitors were systematically explored. Simultaneous inhibition of Aurora B and PLK1 strongly induces polyploidy. Microtubule inhibitor dosage can be significantly reduced when combined with a PLK1 inhibitor. The efficacy of these inhibitor combinations was validated by our experimental results. The mitotic regulatory network model provides a platform to study the complex interactions during mitosis, enables identification of mitotic regulators, and determines targets for drug discovery research. The suggested use of combining microtubule inhibitors with PLK1 inhibitors is anticipated to enhance microtubule-inhibitor tolerance in a wide range of patients.

PCM1 recruits Plk1 to the pericentriolar matrix to promote primary cilia disassembly before mitotic entry.

Primary cilia, which emanate from the cell surface, exhibit assembly and disassembly dynamics along the progression of the cell cycle. However, the mechanism that links ciliary dynamics and cell cycle regulation remains elusive. In the present study, we report that Polo-like kinase 1 (Plk1), one of the key cell cycle regulators, which regulate centrosome maturation, bipolar spindle assembly and cytokinesis, acts as a pivotal player that connects ciliary dynamics and cell cycle regulation. We found that the kinase activity of centrosome enriched Plk1 is required for primary cilia disassembly before mitotic entry, wherein Plk1 interacts with and activates histone deacetylase 6 (HDAC6) to promote ciliary deacetylation and resorption. Furthermore, we showed that pericentriolar material 1 (PCM1) acts upstream of Plk1 and recruits the kinase to pericentriolar matrix (PCM) in a dynein-dynactin complex-dependent manner. This process coincides with the primary cilia disassembly dynamics at the onset of mitosis, as depletion of PCM1 by shRNA dramatically disrupted the pericentriolar accumulation of Plk1. Notably, the interaction between PCM1 and Plk1 is phosphorylation dependent, and CDK1 functions as the priming kinase to facilitate the interaction. Our data suggest a mechanism whereby the recruitment of Plk1 to pericentriolar matrix by PCM1 plays a pivotal role in the regulation of primary cilia disassembly before mitotic entry. Thus, the regulation of ciliary dynamics and cell proliferation share some common regulators.

Pericentrin contains five NESs and an NLS essential for its nucleocytoplasmic trafficking during the cell cycle.

Pericentrin, a conserved centrosomal component, provides the structural scaffold to anchor numerous centrosomal proteins, and thus plays an essential role in the organization and function of the centrosome and the mitotic spindle. Although pericentrin was shown to localize in the cytoplasm and reported to be sensitive to leptomycin B (LMB), a specific inhibitor of Crm1, the regions within pericentrin that serve as signals for transporting in and out of the nucleus have not yet been identified. In this study, we identified five novel nuclear export signals (NESs) in pericentrin with diverse export activities. All of the five NESs could bind to Crm1 in a LMB-sensitive way when mediating the nuclear export of pericentrin. We also demonstrated that the region of amino acids 8-42 in pericentrin contains a tripartite nuclear localization signal (NLS) consisting of three clusters of basic amino acids. The NLS of pericentrin binds to importin beta directly or via the adaptor importin alpha to form the import complex, which could be disrupted by RanQ69L, a dominant-negative Ran GTPase possessing high affinity for importin beta. Furthermore, we found that mutation of the NESs in full-length pericentrin results in both nuclear and cytoplasmic localization, and mutation of the NLS abolishes the nuclear import of pericentrin. On the basis of these results, we suggest that the NESs and NLS of pericentrin are essential for its subcellular localization and nucleocytoplasmic trafficking during the cell cycle.