New treatments for the neglected human pathogen whipworm: exploring mechanism of action at a subcellular level.

土壤传播的蠕虫寄生虫感染威胁着四分之一的人类，造成严重的发病率，特别是儿童。寄生蠕虫毛首鞭虫（鞭虫）也不例外，但由于目前的驱虫治疗对这种蠕虫物种的疗效不佳，因此提出了额外的挑战。没有疫苗存在：迫切需要新的创新药物。这个建议建立在我们最近发现的一类新的药物（IMP），杀死T。体内和离体小鼠。然而，它们的作用机制以及它们如何被吸收到寄生虫组织中尚不清楚。事实上，寄生虫一般如何吸收化合物尚不清楚，甚至蠕虫的进食机制和营养来源也仍有争议。NanoSIMS成像有可能通过确定其在寄生虫和宿主组织内的亚细胞定位来彻底改变我们对新型药物化合物的药理学潜力的理解。此外，NanoSIMS还提供了探索寄生虫的基本生物学的机会，并揭示了鞭虫生物学最神秘的特征之一，即进食机制。开发和应用NanoSIMS成像寄生虫组织，以便在亚细胞水平上定位新型药物化合物。2.通过定义鞭虫的摄食机制和营养源，围绕鞭虫寄生虫的基本生物学产生新的知识。方法：用新化合物和标记的营养物治疗蠕虫：首先，我们将研究鞭虫T的小鼠种的成虫阶段。穆里斯。随后，我们可能在这些分析中包括寄生虫的早期幼虫阶段和卵阶段，其成像将需要额外的技术开发。成虫期寄生虫将在体内生长，尸检时取出，并在加/减新药物化合物或加/减同位素标记的葡萄糖或氨基酸的培养基中培养24小时。此外，我们将与牛津大学的长期合作伙伴合作，设计和合成我们新型驱虫化合物系列的衍生物，以掺入外来元素或同位素标记（例如13 C）。NanoSIMS在生物组织中的开发和应用：NanoSIMS分析需要良好的样品制备，以获得有意义的结果，因为该技术在超高真空下运行。将通过比较化学固定和冷冻固定来优化样品制备，以确定哪种方法保留了药物的体内位置和细胞的结构。然后对样本进行树脂包埋和切片。用新型化合物或标记的食物来源治疗小鼠：开发了NanoSIMS方法来检测寄生虫T. muris离体，我们将扩展工作以实现一定水平的灵敏度，使得我们可以检测从喂食标记食物或体内给药药物的小鼠中回收的蠕虫中的药物化合物/标记的营养物。这很重要，因为它将使我们（i）了解体内宿主的实际食物摄取以及体内蠕虫内的药物可及性和定位，其中蠕虫部分嵌入宿主组织中。我们还将能够评估药物进入宿主肠道组织的水平，这对于告知健康和安全筛选以及药代动力学参数非常重要。

BACKGOUND: Infection with soil-transmitted helminth parasites threatens a quarter of humanity causing significant morbidity, particularly in children. The parasitic helminth Trichuris trichiura (whipworm) is no exception to this, but presents an additional challenge as current anthelmintic treatments show poor efficacy against this helminth species. No vaccine exists: new innovative drugs are urgently needed. This proposal builds on our recent identification of a novel class of drugs (the IMPs) which kill T. muris in vivo and ex vivo. However, their mechanism of action and how they are taken up into parasite tissues is unknown. Indeed, how compounds are taken up by the parasite in general is undefined, with even the feeding mechanisms and nutrient sources of the worm still debated. NanoSIMS imaging has the potential to revolutionise our understanding of the pharmacological potential of novel drug compounds by determining their subcellular localisation within the parasite and host tissue. Further, NanoSIMS also offers the opportunity to explore the basic biology of the parasite and shed light on one of the most enigmatic features of whipworm biology, the feeding mechanism.AIMS: 1. To develop and apply NanoSIMS imaging to parasite tissues in order to localise novel drug compounds at a subcellular level. 2. To generate new knowledge surrounding the basic biology of whipworm parasites by defining the mechanism of whipworm feeding and nutrient sourcesMETHODS: TREATMENT OF WORMS WITH NOVEL COMPOUNDS AND LABELLED NUTRIENTS: initially we will work with the adult stage of the mouse species of whipworm T. muris. Subsequently we may include the early larval stages of the parasite and the egg stage in these analyses, the imaging of which will require additional technological development. Adult stage parasites will be grown in vivo, removed at necropsy and cultured in medium plus/minus the novel drug compound or plus/minus isotopically labelled glucose or amino acids for 24 hours. In addition, we will work with our long standing collaborators at the University of Oxford to design and synthesise derivatives of our novel anthelmintic compound series to incorporate exotic elements, or isotopic labels (e.g. 13C). DEVELOPMENT AND APPLICATION OF NANOSIMS FOR BIOLOGICAL TISSUES: Excellent sample preparation is required for NanoSIMS analysis to achieve meaningful results as the technique operates under ultra-high vacuum. Sample preparation will be optimised by comparing chemical- and cryo-fixation to determine which preserves the in-vivo location of the drugs and structure of the cells. Samples will then be resin embedded and microtomed. TREATMENT OF MICE WITH NOVEL COMPOUNDS OR LABELLED FOOD SOURCES: having developed the NanoSIMS methodology to detect drug compounds and labelled nutrients within individual cells of the parasite T. muris ex vivo, we will extend the work to enable a level of sensitivity such that we can detect drug compounds/labelled nutrients in worms recovered from mice fed labelled chow or dosed with the drugs in vivo. This is important as it will allow us (i) to understand actual food uptake from the host in vivo and drug accessibility and localisation within the worm in vivo, where it lives partially embedded within the host tissue. We will also be able to assess the level of drug penetrance into the host intestinal tissue which is important to inform health and safety screens and pharmacokinetic parameters