Anthelminthic Activity of Assassin Bug Venom against the Blood Fluke Schistosoma mansoni

Helminths such as the blood fluke Schistosoma mansoni represent a major global health challenge due to limited availability of drugs. Most anthelminthic drug candidates are derived from plants, whereas insect-derived compounds have received little attention. This includes venom from assassin bugs, which contains numerous bioactive compounds. Here, we investigated whether venom from the European predatory assassin bug Rhynocoris iracundus has antischistosomal activity. Venom concentrations of 10–50 µg/mL inhibited the motility and pairing of S. mansoni adult worms in vitro and their capacity to produce eggs. We used EdU-proliferation assays to measure the effect of venom against parasite stem cells, which are essential for survival and reproduction. We found that venom depleted proliferating stem cells in different tissues of the male parasite, including neoblasts in the parenchyma and gonadal stem cells. Certain insect venoms are known to lyse eukaryotic cells, thus limiting their therapeutic potential. We therefore carried out hemolytic activity assays using porcine red blood cells, revealing that the venom had no significant effect at a concentration of 43 µg/mL. The observed anthelminthic activity and absence of hemolytic side effects suggest that the components of R. iracundus venom should be investigated in more detail as potential antischistosomal leads

First Assessment for the Presence of Phlebotomine Vectors in Bavaria, Southern Germany, by Combined Distribution Modeling and Field Surveys

Leishmaniasis is caused by protozoa of the genus Leishmania and transmitted by sand flies from mammalian reservoirs to humans. In recent years, a northward spread of L. infantum from highly endemic Mediterranean countries into previously non-endemic Central European areas has been suspected based on presumed sporadic cases of autochthonous leishmaniasis. Here, we investigated whether sand flies are prevalent in Bavaria in Southern Germany, a federal state in which autochthonous cases have previously been reported. Considering the present and future climatic conditions, we determined whether Bavaria is suitable for five sand fly species with assumed spreading tendencies towards Central Europe: Phlebotomus ariasi, P. neglectus, P. perfiliewi and P. perniciosus that are known vectors for Leishmania in Europe, and P. mascittii, a suspected but not proven vector. Within Bavaria we defined sampling regions based on their climatic suitability and their spatial distance to the sites of the autochthonous cases and/or to areas of reported sand fly detection in states adjacent to Bavaria. At 155 locations in 7 sampling regions, CDC light traps were placed during 38 nights in the summers of 2009 and 2010, resulting in 202 trap-nights. All traps were negative for sand flies. The results suggest that Bavaria is not yet endemic for sand flies, but do not exclude the possibility of sporadic cases of autochthonous human or zoonotic Leishmania infections. This study, which combined methodological approaches from different disciplines, serves as reference for future surveys and risk analyses of sand flies and leishmaniasis in so far non-endemic areas of Europe

Current and projected (2011-2040) climatic suitability for five sand fly species in Bavaria (southern Germany).

<p>Currently, the climatic suitability for sand fly species is rather low or moderate in Bavaria. The projections for future climate change refer to the A1B emission scenario of greenhouse gases, expecting an economical growth in a globalized world with a balanced use of fossil and non-fossil energy resources. During the next decades it can be expected that the suitability will increase. This is true for all analyzed sand fly species. A rather high climatic suitability can be expected for the valley of the river Main, especially for Lower Franconia in the most north-western part of Bavaria, along the river Danube and for the alpine foothills.</p

Insects in anthelminthics research: Lady beetle-derived harmonine affects survival, reproduction and stem cell proliferation of Schistosoma mansoni.

Natural products have moved into the spotlight as possible sources for new drugs in the treatment of helminth infections including schistosomiasis. Surprisingly, insect-derived compounds have largely been neglected so far in the search for novel anthelminthics, despite the generally recognized high potential of insect biotechnology for drug discovery. This motivated us to assess the antischistosomal capacity of harmonine, an antimicrobial alkaloid from the harlequin ladybird Harmonia axyridis that raised high interest in insect biotechnology in recent years. We observed remarkably pleiotropic effects of harmonine on physiological, cellular, and molecular processes in adult male and female Schistosoma mansoni at concentrations as low as 5 μM in vitro. This included tegumental damage, gut dilatation, dysplasia of gonads, a complete stop of egg production at 10 μM, and increased production of abnormally shaped eggs at 5 μM. Motility was reduced with an EC50 of 8.8 μM and lethal effects occurred at 10-20 μM within 3 days of culture. Enzyme inhibition assays revealed acetylcholinesterase (AChE) as one potential target of harmonine. To assess possible effects on stem cells, which represent attractive anthelminthic targets, we developed a novel in silico 3D reconstruction of gonads based on confocal laser scanning microscopy of worms after EdU incorporation to allow for quantification of proliferating stem cells per organ. Harmonine significantly reduced the number of proliferating stem cells in testes, ovaries, and also the number of proliferating parenchymal neoblasts. This was further supported by a downregulated expression of the stem cell markers nanos-1 and nanos-2 in harmonine-treated worms revealed by quantitative real-time PCR. Our data demonstrate a multifaceted antischistosomal activity of the lady beetle-derived compound harmonine, and suggest AChE and stem cell genes as possible targets. Harmonine is the first animal-derived alkaloid detected to have antischistosomal capacity. This study highlights the potential of exploiting insects as a source for the discovery of anthelminthics

Drug Repurposing and De Novo Drug Discovery of Protein Kinase Inhibitors as New Drugs against Schistosomiasis

Schistosomiasis is a neglected tropical disease affecting more than 200 million people worldwide. Chemotherapy relies on one single drug, praziquantel, which is safe but ineffective at killing larval stages of this parasite. Furthermore, concerns have been expressed about the rise in resistance against this drug. In the absence of an antischistosomal vaccine, it is, therefore, necessary to develop new drugs against the different species of schistosomes. Protein kinases are important molecules involved in key cellular processes such as signaling, growth, and differentiation. The kinome of schistosomes has been studied and the suitability of schistosomal protein kinases as targets demonstrated by RNA interference studies. Although protein kinase inhibitors are mostly used in cancer therapy, e.g., for the treatment of chronic myeloid leukemia or melanoma, they are now being increasingly explored for the treatment of non-oncological conditions, including schistosomiasis. Here, we discuss the various approaches including screening of natural and synthetic compounds, de novo drug development, and drug repurposing in the context of the search for protein kinase inhibitors against schistosomiasis. We discuss the status quo of the development of kinase inhibitors against schistosomal serine/threonine kinases such as polo-like kinases (PLKs) and mitogen-activated protein kinases (MAP kinases), as well as protein tyrosine kinases (PTKs)

Map of southern Germany with sampling regions of the sand fly survey in the years 2009 and 2010.

<p>The insert shows a map of Germany with the Federal States of Bavaria and Baden-Württemberg highlighted. CDC light traps for sand fly trapping were put up in seven sampling areas in Bavaria (indicated by the numbers 1-7 and shaded in gray) which were allocated to the following administrative districts: 1, Aschaffenburg/Main-Spessart; 2, Würzburg/Kitzingen; 3, Erlangen-Höchstadt/Forchheim; 4, Regensburg/Kelheim; 5, Aichach-Friedberg/Landsberg am Lech/Weilheim-Schongau; 6, Passau; 7, Lindau am Bodensee/Oberallgäu. Sites of reported cases of autochthonous leishmaniasis in Bavaria and Baden-Württemberg and some of the reported sites of sand flies in Baden-Württemberg are marked [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081088#B12" target="_blank">12</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081088#B14" target="_blank">14</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081088#B16" target="_blank">16</a>].</p

First In Silico Screening of Insect Molecules for Identification of Novel Anti-Parasitic Compounds

Schistosomiasis is a neglected tropical disease caused by blood flukes of the genus Schistosoma. In silico screenings of compounds for the identification of novel anti-parasitic drug candidates have received considerable attention in recent years, including the screening of natural compounds. For the first time, we investigated molecules from insects, a rather neglected source in drug discovery, in an in silico screening approach to find novel antischistosomal compounds. Based on the Dictionary of Natural Products (DNP), we created a library of 1327 insect compounds suitable for molecular docking. A structure-based virtual screening against the crystal structure of a known druggable target in Schistosoma mansoni, the thioredoxin glutathione reductase (SmTGR), was performed. The top ten compounds predominantly originated from beetles and were predicted to interact particularly with amino acids in the doorstop pocket of SmTGR. For one compound from a jewel beetle, buprestin H, we tested and confirmed antischistosomal activity against adult and juvenile parasites in vitro. At concentrations with anti-parasitic activity, we could also exclude any unspecific cytotoxic activity against human HepG2 cells. This study highlights the potential of insect molecules for the identification of novel antischistosomal compounds. Our library of insect-derived molecules could serve not only as basis for future in silico screenings against additional target proteins of schistosomes, but also of other parasites

Hepatocyte integrity depends on c-Jun-controlled proliferation in Schistosoma mansoni infected mice

Abstract Schistosomiasis is a parasitic disease affecting more than 250 million people worldwide. The transcription factor c-Jun, which is induced in S. mansoni infection-associated liver disease, can promote hepatocyte survival but can also trigger hepatocellular carcinogenesis. We aimed to analyze the hepatic role of c-Jun following S. mansoni infection. We adopted a hepatocyte-specific c-Jun knockout mouse model (Alb-Cre/c-Jun loxP) and analyzed liver tissue and serum samples by quantitative real-time PCR array, western blotting, immunohistochemistry, hydroxyproline quantification, and functional analyses. Hepatocyte-specific c-Jun knockout (c-JunΔli) was confirmed by immunohistochemistry and western blotting. Infection with S. mansoni induced elevated aminotransferase-serum levels in c-JunΔli mice. Of note, hepatic Cyclin D1 expression was induced in infected c-Junf/f control mice but to a lower extent in c-JunΔli mice. S. mansoni soluble egg antigen-induced proliferation in a human hepatoma cell line was diminished by inhibition of c-Jun signaling. Markers for apoptosis, oxidative stress, ER stress, inflammation, autophagy, DNA-damage, and fibrosis were not altered in S. mansoni infected c-JunΔli mice compared to infected c-Junf/f controls. Enhanced liver damage in c-JunΔli mice suggested a protective role of c-Jun. A reduced Cyclin D1 expression and reduced hepatic regeneration could be the reason. In addition, it seems likely that the trends in pathological changes in c-JunΔli mice cumulatively led to a loss of the protective potential being responsible for the increased hepatocyte damage and loss of regenerative ability