Kinase Domain Of Plk1 Research Articles

An investigation of Plk1 PBD inhibitor KBJK557 as a tumor growth suppressor in non-small cell lung cancer

Lung cancer is the second most commonly reported type of cancer worldwide. Approximately 80–85% of lung cancer occurrences are accounted by non-small cell lung cancer (NSCLC). Polo-like kinase-1 (Plk1) plays multiple roles in cell cycle progression and its overexpression is observed in majority of malignancies, including NSCLC. A combination of frontline drugs and inhibitors targeting the Plk kinase domain (KD) has been used to overcome drug resistance in NSCLC. Plk1 KD inhibitors are highly prone to cross-reactivity with similar kinases, eventually leading to undesirable side effects. Moreover, there have been no reports of Plk1 PBD inhibitors showing antitumorigenic effects on NSCLC cells or animal models so far. To address this issue herein, for the first time, our recently reported Plk1 PBD inhibitor KBJK557 was evaluated for the anticancer potential against NSCLC cells. KBJK557 displayed notable cytotoxic effects in A549, PC9, and H1975 cells. Mechanistic investigations revealed that KBJK557-treated cells underwent G2/M cell cycle arrest, triggering subsequent apoptosis. In vivo antitumorigenic activity in xenograft mice model demonstrates that KBJK557-treated mice showed a considerable decrease in tumor size, proving the significances of Plk1 in lung cancer. Collectively, this study demonstrates that KBJK557 can serve as a promising drug candidate for treating the lung cancer through Plk1 PBD inhibition.

QSAR-guided pharmacophoric modeling reveals important structural requirements for Polo kinase 1 (Plk1) inhibitors

Targeting Polo-like kinase 1 (Plk1) by molecular inhibitors is being a promising approach for tumor therapy. Nevertheless, insufficient methodical analyses have been done to characterize the interactions inside the Plk1 binding pocket. In this study, an extensive combined ligand and structure-based drug design workflow was conducted to data-mine the structural requirements for Plk1 inhibition. Consequently, the binding modes of 368 previously known Plk1 inhibitors were investigated by pharmacophore generation technique. The resulted pharmacophores were engaged in the context of Genetic function algorithm (GFA) and Multiple linear regression (MLR) analyses to search for a prognostic QSAR model.The most successful QSAR model was with statistical criteria of (r2277 = 0.76, r2adj = 0.76, r2pred = 0.75, Q2 = 0.73). Our QSAR-selected pharmacophores were validated by Receiver Operating Characteristic (ROC) curve analysis. Later on, the best QSAR model and its associated pharmacophoric hypotheses (HypoB-T4-5, HypoI-T2-7, HypoD-T4-3, and HypoC-T3-3) were used to identify new Plk1 inhibitory hits retrieved from the National Cancer Institute (NCI) database. The most potent hits exhibited experimental anti-Plk1 IC50 of 1.49, 3.79. 5.26 and 6.35 μM. Noticeably, our hits, were found to interact with the Plk1 kinase domain through some important amino acid residues namely, Cys67, Lys82, Cys133, Phe183, and Asp194.

Abstract 4712: Selective polo-like kinase 1 PBD inhibitor

Abstract Polo Like Kinase 1 (PLK1) is only expressed in dividing cells and plays a critical role in several stages of mitosis. PLK1 is highly expressed in tumors of various origins while its expression is largely absent in normal tissues. PLK1 inhibition selectively kills cancer cells because cancer cells utterly depend on the mitotic functions of PLK1 overexpression. PLK1 consists of a highly conserved N-terminal catalytic kinase domain and a unique, functionally essential C-terminal Polo Box Domain (PBD). The PBD of each PLK is a phospho-peptide binding motif that determines substrate recognition and sub-cellular localization. PLK1 catalytic inhibitorshave advanced to clinical trials but they inhibit other kinases including family members PLK2 and PLK3. This is problematic because PLK3 is a tumor suppressor and partial inhibition of PLK3 could lead to a long-term malignant progression. Moreover, a single point mutation (C67V) in the PLK1 kinase domain confers substantial cancer cell resistance against PLK1 catalytic inhibitors. An alternative approach to developing potent and selective PLK1 inhibitors is to allosterically target the PBD. We generated fragment ligated inhibitory peptides (FLIPs) through a strategy called REPLACE. This is a computational and synthetic approach that uses structure activity relationships of peptide inhibitors to generate pharmaceutically acceptable lead molecules. Fragments are docked into the crystal structure of a truncated peptide/receptor complex, then prioritized for synthesis with the peptide and subsequently tested in an in vitro binding assay. PBD-interacting protein (PBIP) and Cdc25c are natural substrates of PLK1. We replaced individual amino acid residues in the PBIP (PLHSpTAI) and Cdc25C PBD substrate peptides (LLCSpTPNGL) with benzoic acid fragments. Here we show PLK1 specificity for PBD inhibitors in vitro. A fluorescent polarization (FP) competitive binding assay to the PBD of PLK1 was used to determine binding specificity. A PLK3 counter screen was used to determine the binding selectivity of our PBD inhibitors. To measure the PLK1 inhibitory activity of the FLIPs in cells, phosphorylation of mitotic proteins was measured. Candidate FLIPs induce mitotic arrest, block PLK1 mediated phosphorylation, and inhibit growth of human cancer cells. To make more drug-like molecules, we modified FLIPs to Small Molecule Inhibitors (SMIs). Further, we are optimizing SMIs to improve biochemical selectivity. SMIs bind to the PLK1 PBD as determined by FP assay. Also, SMIs inhibit cancer cell growth as determined by viability measurements and live cell imaging. Overall these results highlight that targeting the PBD of PLK1 has promise as an antitumor strategy. Citation Format: Danda P. Chapagai, Merissa Baxter, Gurusankar Ramamoorthy, Campbell McInnes, Michael Wyatt. Selective polo-like kinase 1 PBD inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4712.

An oncoinformatics study to predict the inhibitory potential of recent FDA-approved anti-cancer drugs against human Polo-like kinase 1 enzyme: a step towards dual-target cancer medication.

Cancer prevalence has increased at an alarming rate worldwide. Complexity, resistance mechanism and multiple compensatory survival pathways of cancer cells have abated the response of currently available cancer medications. Therefore, multi-target agents rather than single target might provide a better solution to these cancer therapy issues. In the present study, anti-PLK1 (Polo-like kinase 1) potential of the eight FDA-approved (2017) anti-cancer drugs have been explored using molecular docking approach. Out of all the tested drugs, brigatinib, niraparib and ribociclib showed better binding affinity towards the 'kinase domain' of PLK1. The Gibbs free binding energy (ΔG) and inhibition constant (K i) values for brigatinib, niraparib and ribociclib interaction with the kinase domain of PLK1 were '- 8.05kcal/mol and 1.26µM', '- 8.35kcal/mol and 0.729µM' and '- 7.29kcal/mol and 4.52µM', respectively. Interestingly, the docking results of these three drugs were better than the known PLK1 inhibitors (BI-2536 and rigosertib). The ΔG and K i values for BI-2536 and rigosertib interaction with the kinase domain of PLK1 were '- 6.8kcal/mol and 10.38µM' and '- 6.6kcal/mol and 14.51µM', respectively. Brigatinib, niraparib and ribociclib have been approved by FDA for the treatment of non-small cell lung cancer, ovarian/fallopian tube cancer and breast cancer, respectively. PLK1 is regarded as a potential cancer target, and it is specifically over-expressed in different types of cancer cells, including aforementioned cancers. Actually, the target enzymes for anti-cancer action of brigatinib, niraparib and ribociclib are tyrosine kinase, poly(ADP-ribose) polymerase and cyclin-dependent kinase 4/6, respectively. However, based on our outcomes, we could safely state that PLK1 might plausibly emerge as an add-on target for each of these three anti-cancer drugs. We strongly believe that this study would assist in the development of better dual-targeting cancer therapeutic agent in the near future.

Impact of Polo-like kinase 1 inhibitors on human adipose tissue-derived mesenchymal stem cells.

Polo-like kinase 1 (Plk1) has been established as one of the most promising targets for molecular anticancer intervention. In fact, various Plk1 inhibitors have been identified and characterized. While the data derived from the bench are prospective, the clinical outcomes are less encouraging by showing modest efficacy. One of the explanations for this discrepancy could be unintendedly targeting of non-malignant cells by Plk1 inhibitors. In this work, we have addressed the effect of Plk1 inhibition in adipose tissue-derived mesenchymal stem cells (ASCs). We show that both visceral and subcutaneous ASCs display monopolar spindles, reduced viability and strong apoptosis induction upon treatment with BI 2536 and BI 6727, the Plk1 kinase domain inhibitors, and with Poloxin, the regulatory Polo-box domain inhibitor. While Poloxin triggers quickly apoptosis, BI 2536 and BI 6727 result in mitotic arrest in ASCs. Importantly, survived ASCs exhibit DNA damage and a pronounced senescent phenotype. In addition, Plk1 inhibition impairs ASCs' motility and homing ability. These results show that Plk1 inhibitors target slowly proliferating ASCs, an important population of anti-inflammation and immune modulation. The toxic effects on primary cells like ASCs could be partially responsible for the reported moderate antitumor activity in patients treated with Plk1 inhibitors.

Discovery of Non-ATP-Competitive Inhibitors of Polo-like Kinase 1.

Polo-like kinase 1 (Plk1) is an evolutionarily conserved serine/threonine kinase, and its N-terminal kinase domain (KD) controls cell signaling through phosphorylation. Inhibitors of Plk1 are potential anticancer drugs. Most known Plk1 KD inhibitors are ATP-competitive compounds, which may suffer from low selectivity. In this study we discovered novel non-ATP-competitive Plk1 KD inhibitors by virtual screening and experimental studies. Potential binding sites in Plk1 KD were identified by using the protein binding site detection program Cavity. The identified site was subjected to molecular-docking-based virtual screening. The activities of top-ranking compounds were evaluated by in vitro enzyme assay with full-length Plk1 and direct binding assay with Plk1 KD. Several compounds showed inhibitory activity, and the most potent was found to be 3-((2-oxo-2-(thiophen-2-yl)ethyl)thio)-6-(pyridin-3-ylmethyl)-1,2,4-triazin-5(4H)-one (compound 4) with an IC50 value of 13.1 ± 1.7 μm. Our work provides new insight into the design of kinase inhibitors that target non-ATP binding sites.

Mechanisms underlying Plk1 polo-box domain-mediated biological processes and their physiological significance.

Mammalian polo-like kinase 1 (Plk1) has been studied intensively as a key regulator of various cell cycle events that are critical for proper M-phase progression. The polo-box domain (PBD) present in Plk1’s C-terminal non-catalytic region has been shown to play a central role in targeting the N-terminal kinase domain of Plk1 to specific subcellular locations. Subsequent studies reveal that PBD binds to a phosphorylated motif generated by one of the two mechanisms - self-priming by Plk1 itself or non-self-priming by a Pro-directed kinase, such as Cdc2. Here, we comparatively review the differences in the biochemical steps of these mechanisms and discuss their physiological significance. Considering the diverse functions of Plk1 during the cell cycle, a better understanding of how the catalytic activity of Plk1 functions in concert with its cis-acting PBD and how this coordinated process is intricately regulated to promote Plk1 functions will be important for providing new insights into different mechanisms underlying various Plk1-mediated biological events that occur at the multiple stages of the cell cycle.

Identification of Potential Plk1 Targets in a Cell-Cycle Specific Proteome through Structural Dynamics of Kinase and Polo Box-Mediated Interactions

Polo like kinase 1 (Plk1) is a key player in orchestrating the wide variety of cell-cycle events ranging from centrosome maturation, mitotic entry, checkpoint recovery, transcriptional control, spindle assembly, mitotic progression, cytokinesis and DNA damage checkpoints recovery. Due to its versatile nature, Plk1 is considered an imperative regulator to tightly control the diverse aspects of the cell cycle network. Interactions among Plk1 polo box domain (PBD) and its putative binding proteins are crucial for the activation of Plk1 kinase domain (KD). To date, only a few substrate candidates have been characterized through the inclusion of both polo box and kinase domain-mediated interactions. Thus it became compelling to explore precise and specific Plk1 substrates through reassessment and extension of the structure-function paradigm. To narrow this apparently wide gap in knowledge, here we employed a thorough sequence search of Plk1 phosphorylation signature containing proteins and explored their structure-based features like conceptual PBD-binding capabilities and subsequent recruitment of KD directed phosphorylation to dissect novel targets of Plk1. Collectively, we identified 4,521 phosphodependent proteins sharing similarity to the consensus phosphorylation and PBD recognition motifs. Subsequent application of filters including similarity index, Gene Ontology enrichment and protein localization resulted in stringent pre-filtering of irrelevant candidates and isolated unique targets with well-defined roles in cell-cycle machinery and carcinogenesis. These candidates were further refined structurally using molecular docking and dynamic simulation assays. Overall, our screening approach enables the identification of several undefined cell-cycle associated functions of Plk1 by uncovering novel phosphorylation targets.

Polo-Box Domain Inhibitor Poloxin Activates the Spindle Assembly Checkpoint and Inhibits Tumor Growth in Vivo

Polo-like kinase 1 (Plk1) is widely established as one of the most promising targets in oncology. Although the protein kinase domain of Plk1 is highly conserved, the polo-box domain (PBD) of Plk1 provides a much more compelling site to specifically inhibit the localization and target binding of Plk1. We recently identified, via fluorescence polarization assay, the natural product derivative, Poloxin, as the first small-molecule inhibitor specifically targeting the function of the Plk1 PBD. In this study, we characterized its mitotic phenotype and its function in vitro and in vivo. Poloxin induces centrosome fragmentation and abnormal spindle and chromosome misalignment, which activate the spindle assembly checkpoint and prolong mitosis. Notably, centrosomal fragmentation induced by Poloxin is partially attributable to dysfunctional Kizuna, a key substrate of Plk1 at centrosomes. Moreover, Poloxin strongly inhibits proliferation of a panel of cancer cells by inducing mitotic arrest, followed by a surge of apoptosis. More important, we report, for the first time to our knowledge, that the PBD inhibitor, Poloxin, significantly suppresses tumor growth of cancer cell lines in xenograft mouse models by lowering the proliferation rate and triggering apoptosis in treated tumor tissues. The data highlight that targeting the PBD by Poloxin is a powerful approach for selectively inhibiting Plk1 function in vitro and in vivo.

PLK1 Phosphorylates Mitotic Centromere-associated Kinesin and Promotes Its Depolymerase Activity

During cell division, interaction between kinetochores and dynamic spindle microtubules governs chromosome movements. The microtubule depolymerase mitotic centromere-associated kinesin (MCAK) is a key regulator of mitotic spindle assembly and dynamics. However, the regulatory mechanisms underlying its depolymerase activity during the cell cycle remain elusive. Here, we showed that PLK1 is a novel regulator of MCAK in mammalian cells. MCAK interacts with PLK1 in vitro and in vivo. The neck and motor domain of MCAK associates with the kinase domain of PLK1. MCAK is a novel substrate of PLK1, and the phosphorylation stimulates its microtubule depolymerization activity of MCAK in vivo. Overexpression of a polo-like kinase 1 phosphomimetic mutant MCAK causes a dramatic increase in misaligned chromosomes and in multipolar spindles in mitotic cells, whereas overexpression of a nonphosphorylatable MCAK mutant results in aberrant anaphase with sister chromatid bridges, suggesting that precise regulation of the MCAK activity by PLK1 phosphorylation is critical for proper microtubule dynamics and essential for the faithful chromosome segregation. We reasoned that dynamic regulation of MCAK phosphorylation by PLK1 is required to orchestrate faithful cell division, whereas the high levels of PLK1 and MCAK activities seen in cancer cells may account for a mechanism underlying the pathogenesis of genomic instability.

Polo-box domain: a versatile mediator of polo-like kinase function.

Members of the polo subfamily of protein kinases have emerged as important regulators in diverse aspects of the cell cycle and cell proliferation. A large body of evidence suggests that a highly conserved polo-box domain (PBD) present in the C-terminal non-catalytic region of polo kinases plays a pivotal role in the function of these enzymes. Recent advances in our comprehension of the mechanisms underlying mammalian polo-like kinase 1 (Plk1)-dependent protein-protein interactions revealed that the PBD serves as an essential molecular mediator that brings the kinase domain of Plk1 into proximity with its substrates, mainly through phospho-dependent interactions with its target proteins. In this review, current understanding of the structure and functions of PBD, mode of PBD-dependent interactions and substrate phosphorylation, and other phospho-independent functions of PBD are discussed.

Plk1 regulates liver tumor cell death by phosphorylation of TAp63

We previously found that Plk1 inhibited the p53/p73 activity through its direct phosphorylation. In this study, we investigated the functional role of Plk1 in modulating the p53 family member TAp63, resulting in the control of apoptotic cell death in liver tumor cells. Immunoprecipitation and in vitro pull-down assay showed that p63 binds to the kinase domain of Plk1 through its DNA-binding region. in vitro kinase assay indicated that p63 is phosphorylated by Plk1 at Ser-52 of the transactivating (TA) domain. Plk1 decreased the protein stability of TAp63 by its phosphorylation and suppressed TAp63-induced cell death. Furthermore, Plk1 knockdown in p53-mutated liver tumor cells transactivated p53 family downstream effectors, PUMA, p21(Cip1/WAF1) and 14-3-3sigma, and induced apoptotic cell death. Double knockdown of Plk1/p63 attenuated Plk1 knockdown-induced apoptotic cell death and transactivation. Intriguingly, both Plk1 and p63 are highly expressed in the side population (SP) fraction of liver tumor cells compared to non-SP fraction cells, suggesting the significance of Plk1/TAp63 in the control of cell death in tumor-initiating SP fraction cells. Thus, Plk1 controls TAp63 by its phosphorylation and regulates apoptotic cell death in liver tumor cells. Plk1/TAp63 may be a suitable candidate as a molecular target of liver tumor treatments.

Discovery and Exploitation of Inhibitor-resistant Aurora and Polo Kinase Mutants for the Analysis of Mitotic Networks

The Aurora and Polo-like kinases are central components of mitotic signaling pathways, and recent evidence suggests that substantial cross-talk exists between Aurora A and Plk1. In addition to their validation as novel anticancer agents, small molecule kinase inhibitors are increasingly important tools to help dissect clinically relevant protein phosphorylation networks. However, one major problem associated with kinase inhibitors is their promiscuity toward "off-target" members of the kinome, which makes interpretation of data obtained from complex cellular systems challenging. Additionally, the emergence of inhibitor resistance in patients makes it clear that an understanding of resistance mechanisms is essential to inform drug design. In this study, we exploited structural knowledge of the binding modes of VX-680, an Aurora kinase inhibitor, and BI 2536, a Polo-like kinase inhibitor, to design and evaluate drug-resistant kinase mutants. Using inducible stable human cell lines, we authenticated mitotic targets for both compounds and demonstrated that Aurora A mutants exhibit differential cellular sensitivity toward the inhibitors VX-680 and MLN8054. In addition, we validated Aurora B as an important anti-proliferative target for VX-680 in model human cancer cells. Finally, this chemical genetic approach allowed us to prove that Aurora A activation loop phosphorylation is controlled by a Plk1-mediated pathway in human cells.

Chaperone-Assisted Crystallography with DARPins

The structure of proteins that are difficult to crystallize can often be solved by forming a noncovalent complex with a helper protein--a crystallization "chaperone." Although several such applications have been described to date, their handling usually is still very laborious. A valuable addition to the present repertoire of binding proteins is the recently developed designed ankyrin repeat protein (DARPin) technology. DARPins are built based on the natural ankyrin repeat protein fold with randomized surface residue positions allowing specific binding to virtually any target protein. The broad potential of these binding proteins for X-ray crystallography is illustrated by five cocrystal structures that have been determined recently comprising target proteins from distinct families, namely a sugar binding protein, two kinases, a caspase, and a membrane protein. This article reviews the opportunities of this technology for structural biology and the structural aspects of the DARPin-protein complexes.

433 POSTER Crystal structures of Plk1 kinase domain in complex with ATP-competitive inhibitors

433 POSTER Crystal structures of Plk1 kinase domain in complex with ATP-competitive inhibitors

Structure of wild type Plk1 kinase domain in complex with a selective DARPin

Structure of wild type Plk1 kinase domain in complex with a selective DARPin

Structure of wild-type Plk-1 kinase domain in complex with a selective DARPin

As a key regulator of mitosis, the Ser/Thr protein polo-like kinase-1 (Plk-1) is a well validated drug target in cancer therapy. In order to enable structure-guided drug design, determination of the crystal structure of the kinase domain of Plk-1 was attempted. Using a multi-parallel cloning and expression approach, a set of length variants were identified which could be expressed in large amounts from insect cells and which could be purified to high purity. However, all attempts to crystallize these constructs failed. Crystals were ultimately obtained by generating designed ankyrin-repeat proteins (DARPins) selective for Plk-1 and using them for cocrystallization. Here, the first crystal structure of the kinase domain of wild-type apo Plk-1, in complex with DARPin 3H10, is presented, underlining the power of selective DARPins as crystallization tools. The structure was refined to 2.3 A resolution and shows the active conformation of Plk-1. It broadens the basis for modelling and cocrystallization studies for drug design. The binding epitope of 3H10 is rich in arginine, glutamine and lysine residues, suggesting that the DARPin enabled crystallization by masking a surface patch which is unfavourable for crystal contact formation. Based on the packing observed in the crystal, a truncated DARPin variant was designed which showed improved binding characteristics.

Inhibitory Role of Plk1 in the Regulation of p73-dependent Apoptosis through Physical Interaction and Phosphorylation

In response to DNA damage, p73 plays a critical role in cell fate determination. In this study, we have found that Plk1 (polo-like kinase 1) associates with p73, phosphorylates p73 at Thr-27, and thereby inhibits its pro-apoptotic activity. During cisplatin-mediated apoptosis in COS7 cells in which the endogenous p53 is inactivated by SV40 large T antigen, p73 was induced to accumulate in association with a significant down-regulation of Plk1. Consistent with these observations, Plk1 reduced the stability of the endogenous p73. Immunoprecipitation and in vitro pulldown assay demonstrated that p73 binds to the kinase domain of Plk1 through its NH(2)-terminal region. Luciferase reporter assay and reverse transcription-PCR analysis revealed that Plk1 is able to block the p73-mediated transcriptional activation. Of note, kinase-deficient Plk1 mutant (Plk1(K82M)) retained an ability to interact with p73; however, it failed to inactivate the p73-mediated transcriptional activation, suggesting that kinase activity of Plk1 is required for the inhibition of p73. Indeed, in vitro kinase assay indicated that p73 is phosphorylated at Thr-27 by Plk1. Furthermore, small interference RNA-mediated knockdown of the endogenous Plk1 in p53-deficient H1299 cells resulted in a significant increase in the number of cells with sub-G(1) DNA content accompanied by the up-regulation of p73 and pro-apoptotic p53(AIP1) as well as the proteolytic cleavage of poly(ADP-ribose) polymerase. Thus, our present results suggest that Plk1-mediated dysfunction of p73 is one of the novel molecular mechanisms to inhibit the p53-independent apoptosis, and the inhibition of Plk1 might provide an attractive therapeutic strategy for cancer treatment.

Purification and Characterization of Active Plk‐1 Produced in E. coli and Baculovirus‐Infected Sf9 Insect Cells

The mammalian protein kinase Polo-Like Kinase-1 (Plk-1) is an important regulator of mitosis. It is involved in the signaling networks that regulate many cell cycle processes such as centrosome maturation and separation, mitotic entry and exit, spindle formation and cytokinesis. Over-expression of Plk-1 was observed in a number of different tumors such as head and neck squamous cell carcinomas, ovarian and breast carcinomas and melanomas1. Cancer patients with over-expressed Plk-1 were predicted to have poor clinical outcome and shorten lifespan compared to those with moderate Plk-1 expression2. Plk-1 knockdown studies using anti-Plk-1 antibodies3 or Plk-1 siRNA4 showed mitotic arrest and apoptosis. This validates Plk-1 as a good candidate for small molecule inhibitor therapy to treat a wide range of cancer diseases. Human Plk-1 consists of a N-terminal Ser/Thr Kinase Domain (KD) and a regulatory C-terminal Polo-Box Domain (PBD). The PBD functions both as a Plk-1 cellular localization domain as well as an inhibitory domain of the kinase. Upon binding to a phosphopeptide, such as Cdc25, PBD relinquishes the inhibitory effect thus allowing for phosphorylation and activation of the kinase domain. The active Plk-1 then carries out the signaling cascade through phosphorylation of various downstream molecules such as Cdc2-CyclinB complex and Anaphase Promoting Complex (APC)5. Presented here is the purification and characterization of Plk-1 Full Length (expressed in Sf9 and E. coli cells) as well as Plk-1 Kinase Domain (expressed in E. coli).

Inhibitors of Polo-like kinase reveal roles in spindle-pole maintenance

Polo-like kinases (Plks) have several functions in mitotic progression and are upregulated in many tumor types. Small-molecule Plk inhibitors would be valuable as tools for studying Plk biology and for developing antitumor agents. Guided by homology modeling of the Plk1 kinase domain, we have discovered a chemical series that shows potent and selective Plk1 inhibition. The effects of one such optimized benzthiazole N-oxide, cyclapolin 1 (1), on purified centrosomes indicate that Plks are required to generate MPM2 epitopes, recruit gamma-tubulin and enable nucleation of microtubules. The compound can also promote loss of centrosome integrity and microtubule nucleating ability apparently through increased accessibility of protein phosphatases. We show that treatment of living S2 cells with cyclapolin 1 leads to collapsed spindles, in contrast to the metaphase-arrested bipolar spindles observed after RNAi. This different response to protein depletion and protein inhibition may have significance in the development of antitumor agents.