Plk1 Polo-box Domain Research Articles

Structural basis for recognition of Emi2 by Polo-like kinase 1 and development of peptidomimetics blocking oocyte maturation and fertilization.

In a mammalian oocyte, completion of meiosis is suspended until fertilization by a sperm, and the cell cycle is arrested by a biochemical activity called cytostatic factor (CSF). Emi2 is one of the CSFs, and it maintains the protein level of maturation promoting factor (MPF) by inhibiting ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Degradation of Emi2 via ubiquitin-mediated proteolysis after fertilization requires phosphorylation by Polo-like kinase 1 (Plk1). Therefore, recognition and phosphorylation of Emi2 by Plk1 are crucial steps for cell cycle resumption, but the binding mode of Emi2 and Plk1 is poorly understood. Using biochemical assays and X-ray crystallography, we found that two phosphorylated threonines (Thr152 and Thr176) in Emi2 are each responsible for the recruitment of one Plk1 molecule by binding to its C-terminal polo box domain (PBD). We also found that meiotic maturation and meiosis resumption via parthenogenetic activation were impaired when Emi2 interaction with Plk1-PBD was blocked by a peptidomimetic called 103-8. Because of the inherent promiscuity of kinase inhibitors, our results suggest that targeting PBD of Plk1 may be an effective strategy for the development of novel and specific contraceptive agents that block oocyte maturation and/or fertilization.

Optimized Plk1 PBD Inhibitors Based on Poloxin Induce Mitotic Arrest and Apoptosis in Tumor Cells.

Polo-like kinase 1 (Plk1) is a central regulator of mitosis and has been validated as a target for antitumor therapy. The polo-box domain (PBD) of Plk1 regulates its kinase activity and mediates the subcellular localization of Plk1 and its interactions with a subset of its substrates. Functional inhibition of the Plk1 PBD by low-molecular weight inhibitors has been shown to represent a viable strategy by which to inhibit the enzyme, while avoiding selectivity issues caused by the conserved nature of the ATP binding site. Here, we report structure-activity relationships and mechanistic analysis for the first reported Plk1 PBD inhibitor, Poloxin. We present the identification of the optimized analog Poloxin-2, displaying significantly improved potency and selectivity over Poloxin. Poloxin-2 induces mitotic arrest and apoptosis in cultured human tumor cells at low micromolar concentrations, highlighting it as a valuable tool compound for exploring the function of the Plk1 PBD in living cells.

JIP4 is a PLK1 binding protein that regulates p38MAPK activity in G2 phase

Cell cycle progression from G2 phase into mitosis is regulated by a complex network of mechanisms, all of which finally control the timing of Cyclin B/CDK1 activation. PLK1 regulates a network of events that contribute to regulating G2/M phase progression. Here we have used a proteomics approach to identify proteins that specifically bind to the Polobox domain of PLK1. This identified a panel of proteins that were either associated with PLK1 in G2 phase and/or mitosis, the strongest interaction being with the MAPK scaffold protein JIP4. PLK1 binding to JIP4 was found in G2 phase and mitosis, and PLK1 binding was self-primed by PLK1 phosphorylation of JIP4. PLK1 binding is required for JIP4-dependent p38MAPK activation in G2 phase during normal cell cycle progression, but not in either G2 phase or mitotic stress response. Finally, JIP4 is a target for caspase-dependent cleavage in mitotically arrested cells. The role for the PLK1–JIP4 regulated p38MAPK activation in G2 phase is unclear, but it does not affect either progression into or through mitosis.

Abstract 3773: Plk1-mediated stabilization of 53BP1 suppresses centrosome abnormal amplification

Abstract Mammalian Plk1 has been studied as an essential factor in regulating mitotic events through the phosphorylation of several Plk1-specific substrates. Here we show that in mitosis 53BP1 binds to the C-terminal polo-box domain of Plk1 and is a substrate for Plk1 in vitro. 53BP1 is hyper-phosphorylated and its level is up-regulated in cells expressing wild type Plk1 (Plk1-WT), but not in cells expressing a kinase-defective mutant (Plk1-KM). Depletion of Plk1 reduces the level of 53BP1 which is restored in cells treated with MG132. In cells expressing Plk1-KM but not in Plk1-WT cells, 53BP1 interacts with Mdm2 suggesting that 53BP1 turnover is regulated by Plk1-mediated phosphorylation. In mitosis, 53BP1 colocalizes with Plk1 to the centrosome and the spindle pole. Down-regulation of 53BP1 by shRNA induces multiple centrioles, multiple spindle poles, and mis-orientation of poles in HeLa cells. The data suggest that the phosphorylation of 53BP1 by Plk1 regulates its stability, and that this biochemical event plays a crucial role in modulating chromosomal amplification which ensures bipolarity in mitosis. Citation Format: Hyungshin Yim, Sang-Uk Woo, Sol-Bi Shin, Raymond L. Erikson. Plk1-mediated stabilization of 53BP1 suppresses centrosome abnormal amplification. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3773. doi:10.1158/1538-7445.AM2015-3773

Structural analysis of the polo-box domain of human Polo-like kinase 2.

Polo-like kinases (Plks) are the key regulators of cell cycle progression, the members of which share a kinase domain and a polo-box domain (PBD) that serves as a protein-binding module. While Plk1 is a promising target for antitumor therapy, Plk2 is regarded as a tumor suppressor even though the two Plks commonly recognize the S-pS/T-P motif through their PBD. Herein, we report the crystal structure of the PBD of Plk2 at 2.7 Å. Despite the overall structural similarity with that of Plk1 reflecting their high sequence homology, the crystal structure also contains its own features including the highly ordered loop connecting two subdomains and the absence of 310 -helices in the N-terminal region unlike the PBD of Plk1. Based on the three-dimensional structure, we furthermore could model its interaction with two types of phosphopeptides, one of which was previously screened as the optimal peptide for the PBD of Plk2.

Crystal structure of the polo-box domain of polo-like kinase 2

Polo-like kinase 2 (PLK2) is a crucial regulator in cell cycle progression, DNA damage response, and neuronal activity. PLK2 is characterized by the conserved N-terminal kinase domain and the unique C-terminal polo-box domain (PBD). The PBD mediates diverse functions of PLK2 by binding phosphorylated Ser-pSer/pThr motifs of its substrates. Here, we report the first crystal structure of the PBD of PLK2. The overall structure of the PLK2 PBD is similar to that of the PLK1 PBD, which is composed by two polo boxes each contain β6α structures that form a 12-stranded β sandwich domain. The edge of the interface between the two polo boxes forms the phosphorylated Ser-pSer/pThr motifs binding cleft. On the hand, the peripheral regions around the core binding cleft of the PLK2 PBD is distinct from that of the PLK1 PBD, which might confer the substrate specificity of the PBDs of the polo-like kinase family.

The molecular docking of 1,4-naphthoquinone derivatives as inhibitors of Polo-like kinase 1 using Molegro Virtual Docker

Polo-like kinase 1 (Plk1) is overexpressed in many types of human cancers, and has been implicated as an adverse prognostic marker for cancer patients. Plk1 is localized to its intracellular anchoring sites via its polo-box domain (PBD). The PBD of Plk1 has a crucial role in proper subcellular localization and mitotic functions of Plk1. Plk1 is the preferential target for inhibition of the mitotic processing therefore it can be chosen as drug target for the treatment of cancer. The aim of the study is to find plk1 inhibitor potential from naphthoquinone derivatives through binding free energy analysis into plk1 using molecular docking. We conducted docking simulation to naphthoquinone derivatives as ligands into plk1 as receptor. The 3D structure of plk1 was downloaded from PDB (Code ID:3THB). The structure of ligands and protein were prepared using ChemBioDrawUltra 12.0. Docking process, the interaction and binding of ligands – protein were done and visualized using software Molegro Virtual Docking.(MVD). The results showed no hydrogen bonding and electrostatic interaction between compound NO11(modified naphthoquinone) with Plk1, but this compound have more steric interaction with Phe 133, Asp 194, Glu 101, Lys 82, Cys 133 and Glu 140 of Plk1. Moldock scores of compound NO11, is -134.73 kcal/mol. It is predicted that compound NO11 has potency as lead compound to find a new anticancer candidates for possible therapeutic agents.

A new class of peptidomimetics targeting the polo-box domain of Polo-like kinase 1.

Recent progress in the development of peptide-derived Polo-like kinase (Plk1) polo-box domain (PBD) inhibitors has led to the synthesis of multiple peptide ligands with high binding affinity and selectivity. However, few systematic analyses have been conducted to identify key Plk1 residues and characterize their interactions with potent Plk1 peptide inhibitors. We performed systematic deletion analysis using the most potent 4j peptide and studied N-terminal capping of the minimal peptide with diverse organic moieties, leading to the identification of the peptidomimetic 8 (AB-103) series with high binding affinity and selectivity. To evaluate the bioavailability of short peptidomimetic ligands, PEGylated 8 series were synthesized and incubated with HeLa cells to test for cellular uptake, antiproliferative activity, and Plk1 kinase inhibition. Finally, crystallographic studies of the Plk1 PBD in complex with peptidomimetics 8 and 22 (AB-103-5) revealed the presence of two hydrogen bond interactions responsible for their high binding affinity and selectivity.

Abstract 4574: Polo box-targeted PLK1 inhibitors: structure activity relationships and activity in cells resistant to ATP-based inhibitors

Abstract Polo-like Kinase 1 (PLK1) performs critical roles in the coordination of mitosis. PLK1 is also an oncogene over-expressed in many cancer types and is being targeted therapeutically. ATP-binding site inhibitors of PLK1 have progressed to clinical trials, and one was recently given FDA breakthrough status to treat acute myeloid leukemia. However, recent data suggests that there are major disadvantages of the conventional approach to blocking the kinase activity of PLK1. First, both general kinome and PLK family specificity is an issue with ATP competitive compounds because they commonly inhibit all paralogs in the PLK family, including PLK3, a known tumor suppressor. Second, a single point mutant in the active site of PLK1 (Cys67Val) results in dramatic resistance to structurally distinct ATP competitive inhibitors in clinical trials (Burkard, Santamaria, and Jallepalli, ACS Chem. Biol. 2012, 7, 978−981), suggesting that the emergence of resistance in the clinic against these agents is a near certainty. We have validated the innovative REPLACE strategy by discovering non-ATP competitive and PLK1 selective inhibitors as a potential therapeutic strategy. The Polo-box domain (PBD) of each PLK is a phospho-peptide binding motif that determines substrate recognition and subcellular localization. Our approach is to target the PBD of PLK1 to achieve desired PLK1 selectivity and improve efficacy. Our first generation of fragment-ligated inhibitory peptides (FLIPs) (Mol. Cancer Ther., 2012;11:1683-1692) included a compound that contained a butyl-benzamide group and possessed a binding affinity to the PLK1 PBD of 8.6 μM, and insignificant levels of PLK3 binding (IC50 > 600 μM). Subsequent modeling and design produced a FLIP with an octyl-benzamide group, which possessed IC50 of 0.3 μM for the PLK1 PBD, and an IC50 of 93.5 μM for PLK3, a >300-fold selectivity (unpublished results). We also report the investigation of our PBD inhibitors in cells expressing the mutant C67V PLK1 that is resistant to ATP-based inhibitors. Indeed, we find that our compounds are equally active in cells expressing wild-type or C67V PLK1, whereas cells expressing C67V PLK1 are dramatically resistant to a model ATP-based inhibitor, BI 2536. We also further explore the structure-activity relationship and structural basis of PLK1 versus PLK3 selectivity with selected modifications to PBD binding peptides and FLIPs. In summary, these exciting developments demonstrate the validity of our approach to produce drug-like lead PBD inhibitors that are PLK1 selective and will be active against tumors resistant to ATP inhibitors. Citation Format: Merissa Baxer, Sandra Craig, Campbell McInnes, Michael Wyatt. Polo box-targeted PLK1 inhibitors: structure activity relationships and activity in cells resistant to ATP-based inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4574. doi:10.1158/1538-7445.AM2014-4574

Design and Synthesis of a Cell-Permeable, Drug-Like Small Molecule Inhibitor Targeting the Polo-Box Domain of Polo-Like Kinase 1

BackgroundPolo-like kinase-1 (Plk1) plays a crucial role in cell proliferation and the inhibition of Plk1 has been considered as a potential target for specific inhibitory drugs in anti-cancer therapy. Several research groups have identified peptide-based inhibitors that target the polo-box domain (PBD) of Plk1 and bind to the protein with high affinity in in vitro assays. However, inadequate proteolytic resistance and cell permeability of the peptides hinder the development of these peptide-based inhibitors into novel therapeutic compounds.Methodology/Principal FindingsIn order to overcome the shortcomings of peptide-based inhibitors, we designed and synthesized small molecule inhibitors. Among these molecules, bg-34 exhibited a high binding affinity for Plk1-PBD and it could cross the cell membrane in its unmodified form. Furthermore, bg-34-dependent inhibition of Plk1-PBD was sufficient for inducing apoptosis in HeLa cells. Moreover, modeling studies performed on Plk1-PBD in complex with bg-34 revealed that bg-34 can interact effectively with Plk1-PBD.Conclusion/SignificanceWe demonstrated that the molecule bg-34 is a potential drug candidate that exhibits anti-Plk1-PBD activity and possesses the favorable characteristics of high cell permeability and stability. We also determined that bg-34 induced apoptotic cell death by inhibiting Plk1-PBD in HeLa cells at the same concentration as PEGylated 4j peptide, which can inhibit Plk1-PBD activity 1000 times more effectively than bg-34 can in in vitro assays. This study may help to design and develop drug-like small molecule as Plk1-PBD inhibitor for better therapeutic activity.

Plk1-targeted therapies in TP53- or RAS-mutated cancer

Despite advances in treatment, prognosis for many types of carcinoma remains poor. Polo-like kinase 1 (Plk1) has been explored as a target for the development of anticancer drugs. As a mitotic master Ser/Thr kinase, Plk1 is involved in centrosomal maturation, microtubule nucleation, chromosomal segregation, and cytokinesis. Additional functions in interphase and in response to DNA damage have been revealed. The multiple locations of Plk1 correspond to distinct functions, mediated by phosphorylation of multiple substrates. Since it is highly expressed in several carcinomas, and expression of Plk1 is inversely correlated with the survival rate of patients in non-small cell lung, head and neck, and esophageal cancer, Plk1 is recognized as a valid prognostic marker. Connections between Plk1 and p53 or KRAS in carcinoma provide a rationale and several possible routes to the development of therapies. Tumors with both p53-deficiency and high Plk1 expression may be particularly sensitive to Plk1 inhibitors, although some controversial data exist. In KRAS-mutant cancers, on the other hand, Plk1 may be essential for tumor cell survival, but detailed studies as to whether Plk1 inhibitors are more effective in KRAS-mutant cancers must be performed in order to determine whether this is the case. Here, we present evidence for Plk1 as a prognostic marker and potentially effective target for the treatment of patients with carcinoma, to demonstrate the value of Plk1 as a target for the development of cancer treatment, especially for patients with solid tumors. In addition, the effects of Plk1 inhibition in p53- or KRAS-mutated cancer are discussed with respect to clinical implications. Structural specifics of Plk1 are presented, as well as current strategies for discovering new Plk1 inhibitors by targeting the conserved ATP binding site or polo-box domain of Plk1, in order to develop Plk1-specific anticancer drugs.

Design of Druglike Small Molecules with LYN-Specific Binding

Glioblastoma multiforme (GBM), a very aggressive brain tumor, has a median survival of only 14 months. LYN, an important kinase involved in regulation of hematopoietic cells, is significantly overexpressed in GBM. It is believed that LYN promotes migration of cancer cells, thus advancing the malignancy. This research addresses computational design of small druglike molecules that could potentially inhibit LYN and thus stave off the cancer advancement. LYN has a very similar binding site to the polo-box domain (PBD) in Polo-like kinase 1 (Plk1). Plk1 is a main regulator of mitosis. Considering the key cellular roles of both LYN and Plk1, it is important to design inhibitors that will specifically bind to LYN. In this work, physical and chemical properties of the binding sites of LYN and Plk1 were investigated and compared. Pertinent atomic distances within the LYN binding site were found to be smaller than those within the PBD of Plk1. The two sites also differed in their flexibilities. By utilizing the differences, novel molecules were designed that could potentially bind LYN with higher affinities than they could Plk1. Previously designed molecules that bonded both LYN and Plk1 were used as initial templates to design more specific inhibitors. Potential toxicities and drug-likeness of the molecules were evaluated. Molecules with no implied toxicities and optimal druglike properties were used for docking studies. Molecules that made the most stable docking configurations with LYN and with no other kinases were identified as LYN-specific. Binding energies of the stable complexes that these molecules formed with LYN were calculated. Possible utilization of the designed molecules against tumors with overexpressed LYN is discussed.

CIP2A Modulates Cell-Cycle Progression in Human Cancer Cells by Regulating the Stability and Activity of Plk1

Abnormal cell-cycle control can lead to aberrant cell proliferation and cancer. The oncoprotein cancerous inhibitor of protein phosphatase 2A (CIP2A) is an inhibitor of protein phosphatase 2A (PP2A) that stabilizes c-Myc. However, the precise role of CIP2A in cell division is not understood. Herein, we show that CIP2A is required for mitotic progression by regulating the polo-like kinase (Plk1). With mitotic entry, CIP2A translocated from the cytoplasm to the nucleus, where it was enriched at spindle poles. CIP2A depletion delayed mitotic progression, resulting in mitotic abnormalities independent of PP2A activity. Unexpectedly, CIP2A interacted directly with the polo-box domain of Plk1 during mitosis. This interaction was required to maintain Plk1 stability by blocking APC/C-Cdh1-dependent proteolysis, thereby enhancing the kinase activity of Plk1 during mitosis. We observed strong correlation and in vivo interactions between these two proteins in multiple human cancer specimens. Overall, our results established a novel function for CIP2A in facilitating the stability and activity of the pivotal mitotic kinase Plk1 in cell-cycle progression and tumor development.

Exploring the Binding Nature of Pyrrolidine Pocket-Dependent Interactions in the Polo-Box Domain of Polo-Like Kinase 1

BackgroundOver the years, a great deal of effort has been focused on the design and synthesis of potent, linear peptide inhibitors targeting the polo-like kinase 1 (Plk1), which is critically involved in multiple mitotic processes and has been established as an adverse prognostic marker for tumor patients. Plk1 localizes to its intracellular anchoring sites via its polo-box domain, and inhibiting the Plk1 polo-box domain has been considered as an approach to circumvent the specificity problems associated with inhibiting the conserved adenosine triphosphate-binding pocket. The polo-box domain consists of two different binding regions, such as the unique, broader pyrrolidine-binding pocket and the conserved, narrow, Tyr-rich hydrophobic channel, among the three Plk polo-box domains (Plks 1–3), respectively. Therefore, the studies that provide insights into the binding nature of the unique, broader pyrrolidine-binding pocket might lead to the development of selective Plk1-inhibitory compounds.Methodology/Principal FindingsIn an attempt to retain the monospecificity by targeting the unique, broader pyrrolidine-binding pocket, here, for the first time, a systematic approach was undertaken to examine the structure-activity relationship of N-terminal-truncated PLHSpTM derivatives, to apply a site-directed ligand approach using bulky aromatic and non-aromatic systems, and to characterize the binding nature of these analogues using X-ray crystallographic studies. We have identified a new mode of binding interactions, having improved binding affinity and retaining the Plk1 polo-box domain specificity, at the pyrrolidine-binding pocket. Furthermore, our data revealed that the pyrrolidine-binding pocket was very specific to recognize a short and bulky hydrophobic ligand like adamantane, whereas the Tyr-rich hydrophobic channel was specific with lengthy and small hydrophobic groups.Conclusion/SignificanceThe progress made using our site-directed ligands validated this approach to specifically direct the ligand into the unique pyrrolidine-binding region, and it extends the applicability of the strategy for discovering selective protein-protein interaction inhibitors.

RETRACTED: Peptide-Based Inhibitors of Plk1 Polo-box Domain Containing Mono-anionic Phosphothreonine Esters and Their Pivaloyloxymethyl Prodrugs

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides may be used to disrupt PBD function, the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising in part from the di-anionic nature of the phosphoryl group. We report five-mer peptides containing mono-anionic pThr phosphoryl esters that exhibit single-digit nanomolar PBD binding affinities in extracellular assays and improved antimitotic efficacies in whole cell assays. The cellular efficacies of these peptides have been further enhanced by the application of bio-reversible pivaloyloxymethyl (POM) phosphoryl protection to a pThr-containing polypeptide. Our findings may redefine structural parameters for the development of PBD-binding peptides and peptide mimetics.

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.

Abstract 2156: Targeting polo-like kinase 1 with novel inhibitors of the polo-box domain.

Abstract Polo-like Kinase 1 (PLK1) is a central player in cell cycle regulation and mitotic progression. Observations that PLK1 is over-expressed in many cancer types have led to efforts to target this protein therapeutically. Several ATP-binding site inhibitors of PLK1 have advanced to clinical trials, but these compounds inhibit at least three of the four known mammalian PLKs to varying degrees. This is problematic because, for example, PLK3 acts as a tumor suppressor and has opposing functions to PLK1. Moreover, a recent study identified a single point mutation near the ATP binding site that renders PLK1 resistant to several structurally unrelated ATP-based inhibitors, while retaining kinase activity. The Polo-box domain (PBD) of each PLK is a phospho-peptide binding motif that determines substrate recognition and subcellular localization. Our approach is to target the PBD of PLK1 to achieve desired PLK1 selectivity and improve efficacy. We recently reported a structure-activity relationship for a series of fragment-ligated inhibitory peptides (FLIPs) designed to be selective for the PBD of PLK1 (Mol. Cancer Ther., 2012, 11, 1683-1692). Our most potent compound in that class possessed a binding affinity to the PLK1 PBD (IC50 value of 8.6 μM), and insignificant levels of PLK3 binding up to 600 μM, which represents at least a 100-fold selectivity. The compound was able to induce cellular phenotypes consistent with a PLK1 knockdown, including aberrant mitoses and induction of apoptosis. Here, we report the development of new FLIP analogues with improved potency in binding to the PLK1 PBD. Specifically, we have designed, synthesized, and tested new benzoic acid capping groups to exploit a hydrophobic cleft in the PBD binding site. This newer generation of compounds possess binding affinities to the PLK1 PBD with IC50 values below 1 μM. Studies are in progress to determine PLK1 selectivity and activity in cells. Future studies will explore the ability of PBD-targeted compounds to circumvent resistance to ATP-based compounds. Citation Format: Merissa Baxter, Sandra Craig, Campbell McInnes, Michael D. Wyatt. Targeting polo-like kinase 1 with novel inhibitors of the polo-box domain. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2156. doi:10.1158/1538-7445.AM2013-2156

Development of cyclic peptomer inhibitors targeting the polo-box domain of polo-like kinase 1

The polo-box domain (PBD) of polo-like kinase 1 (Plk1) is essentially required for the function of Plk1 in cell proliferation. The availability of the phosphopeptide-binding pocket on PBD provides a unique opportunity to develop novel protein–protein interaction inhibitors. Recent identification of a minimal 5-residue-long phosphopeptide, PLHSpT, as a Plk1 PBD-specific ligand has led to the development of several peptide-based inhibitors, but none of them is cyclic peptide. Through the combination of single-peptoid mimics and thio-ether bridged cyclization, we successfully demonstrated for the first time two cyclic peptomers, PL-116 and PL-120, dramatically improved the binding affinity without losing mono-specificity against Plk1 PBD in comparison with the linear parental peptide, PLHSpT. These cyclic peptomers could serve as promising templates for future drug designs to inhibit Plk1 PBD.

Small Druglike Molecules with Specific Binding to Plk1 Or LYN Kinases

The research addresses computational design of small druglike molecules for possible anticancer applications. Polo-like kinase 1 (Plk1) is a main regulator of mitosis. Its inhibition can lead to apoptosis of cancer cells. Most kinases interact with inhibitors via their highly conserved ATP sites. This makes the design of Plk1-specific inhibitors challenging. However, Plk1 also has the polo-box domain (PBD) that is absent from other kinases. Previously, we designed several molecules that interacted with the PBD of Plk1 and with only one other kinase, LYN. The LYN kinase regulates hematopoietic cells from which cellular blood components are derived. Yet, LYN's greatly elevated activity in glioblastoma tumors suggests that it promotes the malignancy in these cells. In light of their respective cellular roles, it is important to design inhibitors that are either Plk1- or LYN-specific. This is not trivial because of their very similar binding sites. Physicochemical differences between the sites were analyzed in the DeepView program. Relevant atomic distances within the PBD of Plk1 were found greater than those within LYN. Thus, a larger molecule could potentially fit into the PBD and would be excluded from the LYN's smaller and less flexible site. Previously designed molecules that bonded both Plk1 and LYN were used as templates to design more specific inhibitors. Designed molecules with no indicated toxicities and optimal values of other drug-related properties were used for docking in ArgusLab. Molecules that made stable complexes specifically with either Plk1 or LYN, but with no other kinases, were identified. Drugs based on these molecules could be useful against tumors with overexpressed Plk1 or LYN. Such drugs could selectively bind one of the kinases and lead to fewer side effects than a less selective drug.

High Mitotic Activity of Polo-like Kinase 1 Is Required for Chromosome Segregation and Genomic Integrity in Human Epithelial Cells

Protein kinases play key roles in regulating human cell biology, but manifold substrates and functions make it difficult to understand mechanism. We tested whether we could dissect functions of a pleiotropic mitotic kinase, Polo-like kinase 1 (Plk1), via distinct thresholds of kinase activity. We accomplished this by titrating Plk1 activity in RPE1 human epithelial cells using chemical genetics and verifying results in additional lines. We found that distinct activity thresholds are required for known functions of Plk1 including (from low to high activity) bipolar spindle formation, timely mitotic entry, and formation of a cytokinesis cleavage furrow. Subtle losses in Plk1 activity impaired chromosome congression and produced severe anaphase dysfunction characterized by poor separation of chromosome masses. These two phenotypes were separable, suggesting that they stem from distinct phosphorylation events. Impaired chromosome segregation in anaphase was the most sensitive to modest loss in Plk1 activity. Mechanistically, it was associated with unpaired sister chromatids with stretched kinetochores, suggestive of merotelic attachments. The C-terminal Polo box domain of Plk1 was required for its anaphase function, although it was dispensable for forming a bipolar spindle. The ultimate effect of partial inhibition of Plk1 was the formation of micronuclei, an increase in tetraploid progeny, and senescence. These results demonstrate that different thresholds of Plk1 activity can elicit distinct phenotypes, illustrating a general method for separating pleiotropic functions of a protein kinase even when these are executed close in time.