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      Targeting Echinococcus multilocularis Stem Cells by Inhibition of the Polo-Like Kinase EmPlk1

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          Abstract

          Background

          Alveolar echinococcosis (AE) is a life-threatening disease caused by larvae of the fox-tapeworm Echinococcus multilocularis. Crucial to AE pathology is continuous infiltrative growth of the parasite's metacestode stage, which is driven by a population of somatic stem cells, called germinative cells. Current anti-AE chemotherapy using benzimidazoles is ineffective in eliminating the germinative cell population, thus leading to remission of parasite growth upon therapy discontinuation.

          Methodology/Principal findings

          We herein describe the characterization of EmPlk1, encoded by the gene emplk1, which displays significant homologies to members of the Plk1 sub-family of Polo-like kinases that regulate mitosis in eukaryotic cells. We demonstrate germinative cell-specific expression of emplk1 by RT-PCR, transcriptomics, and in situ hybridization. We also show that EmPlk1 can induce germinal vesicle breakdown when heterologously expressed in Xenopus oocytes, indicating that it is an active kinase. This activity was significantly suppressed in presence of BI 2536, a Plk1 inhibitor that has been tested in clinical trials against cancer. Addition of BI 2536 at concentrations as low as 20 nM significantly blocked the formation of metacestode vesicles from cultivated Echinococcus germinative cells. Furthermore, low concentrations of BI 2536 eliminated the germinative cell population from mature metacestode vesicles in vitro, yielding parasite tissue that was no longer capable of proliferation.

          Conclusions/Significance

          We conclude that BI 2536 effectively inactivates E. multilocularis germinative cells in parasite larvae in vitro by direct inhibition of EmPlk1, thus inducing mitotic arrest and germinative cell killing. Since germinative cells are decisive for parasite proliferation and metastasis formation within the host, BI 2536 and related compounds are very promising compounds to complement benzimidazoles in AE chemotherapy.

          Author Summary

          The lethal disease AE is characterized by continuous and infiltrative growth of the metacestode larva of the tapeworm E. multilocularis within host organs. This cancer-like progression is exclusively driven by a population of parasite stem cells (germinative cells) that have to be eliminated for an effective cure of the disease. Current treatment options, using benzimidazoles, are parasitostatic only, and thus obviously not effective in germinative cell killing. We herein describe a novel, druggable parasite enzyme, EmPlk1, that specifically regulates germinative cell proliferation. We show that a compound, BI 2536, originally designed to inhibit the human ortholog of EmPlk1, can also inhibit the parasite protein at low doses. Furthermore, low doses of BI 2536 eliminated germinative cells from Echinococcus larvae in vitro and prevented parasite growth and development. We propose that BI 2536 and related compounds are promising drugs to complement current benzimidazole treatment for achieving parasite killing.

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          Most cited references46

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          Biological, epidemiological, and clinical aspects of echinococcosis, a zoonosis of increasing concern.

          Echinococcosis in humans is a zoonotic infection caused by larval stages (metacestodes) of cestode species of the genus Echinococcus. Cystic echinococcosis (CE) is caused by Echinococcus granulosus, alveolar echinococcosis (AE) is caused by E. multilocularis, and polycystic forms are caused by either E. vogeli or E. oligarthrus. In untreated cases, AE has a high mortality rate. Although control is essentially feasible, CE remains a considerable health problem in many regions of the northern and southern hemispheres. AE is restricted to the northern hemisphere regions of North America and Eurasia. Recent studies have shown that E. multilocularis, the causative agent of AE, is more widely distributed than previously thought. There are also some hints of an increasing significance of polycystic forms of the disease, which are restricted to Central and South America. Various aspects of human echinococcosis are discussed in this review, including data on the infectivity of genetic variants of E. granulosus to humans, the increasing invasion of cities in Europe and Japan by red foxes, the main definitive hosts of E. multilocularis, and the first demonstration of urban cycles of the parasite. Examples of emergence or reemergence of CE are presented, and the question of potential spreading of E. multilocularis is critically assessed. Furthermore, information is presented on new and improved tools for diagnosing the infection in final hosts (dogs, foxes, and cats) by coproantigen or DNA detection and the application of molecular techniques to epidemiological studies. In the clinical field, the available methods for diagnosing human CE and AE are described and the treatment options are summarized. The development of new chemotherapeutic options for all forms of human echinococcosis remains an urgent requirement. A new option for the control of E. granulosus in the intermediate host population (mainly sheep and cattle) is vaccination. Attempts are made to reduce the prevalence of E. multilocualaris in fox populations by regular baiting with an anthelmintic (praziquantel). Recent data have shown that this control option may be used in restricted areas, for example in cities, with the aim of reducing the infection risk for humans.
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            BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo.

            Fine-mapping of the cell-division cycle, notably the identification of mitotic kinase signaling pathways, provides novel opportunities for cancer-drug discovery. As a key regulator of multiple steps during mitotic progression across eukaryotic species, the serine/threonine-specific Polo-like kinase 1 (Plk1) is highly expressed in malignant cells and serves as a negative prognostic marker in specific human cancer types . Here, we report the discovery of a potent small-molecule inhibitor of mammalian Plk1, BI 2536, which inhibits Plk1 enzyme activity at low nanomolar concentrations. The compound potently causes a mitotic arrest and induces apoptosis in human cancer cell lines of diverse tissue origin and oncogenome signature. BI 2536 inhibits growth of human tumor xenografts in nude mice and induces regression of large tumors with well-tolerated intravenous dose regimens. In treated tumors, cells arrest in prometaphase, accumulate phosphohistone H3, and contain aberrant mitotic spindles. This mitotic arrest is followed by a surge in apoptosis, detectable by immunohistochemistry and noninvasive optical and magnetic resonance imaging. For addressing the therapeutic potential of Plk1 inhibition, BI 2536 has progressed into clinical studies in patients with locally advanced or metastatic cancers.
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              Multifaceted polo-like kinases: drug targets and antitargets for cancer therapy.

              The polo-like kinase 1 (PLK1) acts in concert with cyclin-dependent kinase 1-cyclin B1 and Aurora kinases to orchestrate a wide range of critical cell cycle events. Because PLK1 has been preclinically validated as a cancer target, small-molecule inhibitors of PLK1 have become attractive candidates for anticancer drug development. Although the roles of the closely related PLK2, PLK3 and PLK4 in cancer are less well understood, there is evidence showing that PLK2 and PLK3 act as tumour suppressors through their functions in the p53 signalling network, which guards the cell against various stress signals. In this article, recent insights into the biology of PLKs will be reviewed, with an emphasis on their role in malignant transformation, and progress in the development of small-molecule PLK1 inhibitors will be examined.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                June 2014
                5 June 2014
                : 8
                : 6
                : e2870
                Affiliations
                [1 ]University of Würzburg, Institute of Hygiene and Microbiology, Würzburg, Germany
                [2 ]Universidad de la República, Facultad de Ciencias, Sección Bioquímica y Biología Molecular, Montevideo, Uruguay
                [3 ]EA4479, IFR147, Laboratoire de Régulation des Signaux de Division, SN3, University Lille 1, Villeneuve d'Ascq, France
                [4 ]Center for Infection and Immunology of Lille, Inserm U1019, CNRS-UMR 8204, University Lille 2, Institut Pasteur de Lille, Lille, France
                Center for Discovery and Innovation in Parasitic Diseases, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: KB CD. Performed the experiments: AS UK KC MV. Analyzed the data: AS UK KC MV CD KB. Contributed reagents/materials/analysis tools: AS UK MV. Wrote the paper: KB CD.

                Article
                PNTD-D-14-00184
                10.1371/journal.pntd.0002870
                4046951
                24901228
                e3883412-e2aa-43ce-91ef-9e9f8b8ba338
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 3 February 2014
                : 1 April 2014
                Page count
                Pages: 13
                Funding
                This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG; BR 2045/4-1) and the Wellhöfer Foundation (all to KB), and by the Institut de la Sante et de la Recherche Medicale and Université Lille Nord de France. AS and UK were supported by grants of the German Excellence Initiative to the Graduate School of Life Sciences, University of Würzburg. MV was supported by the Ministere de l'Education Nationale et de la Recherche, France. This publication was funded by the German Research Foundation (DFG) and the University of Würzburg in the funding programme Open Access Publishing. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Cell Cycle and Cell Division
                Molecular Cell Biology
                Developmental Biology
                Parasitology
                Veterinary Science
                Veterinary Diseases
                Zoonoses
                Zoology
                Helminthology
                Medicine and Health Sciences
                Clinical Medicine
                Infectious Diseases
                Parasitic Diseases
                Pharmacology
                Drug Research and Development
                Tropical Diseases
                Neglected Tropical Diseases

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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