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

背景：土壤传播的蠕虫寄生虫感染威胁着四分之一的人类，导致严重的发病率，特别是在儿童中。寄生蠕虫Trichuris trichiura(鞭虫)也不例外，但由于目前的驱虫治疗对这种蠕虫效果不佳，因此提出了另一个挑战。目前还不存在疫苗：迫切需要新的创新药物。这一建议建立在我们最近发现的一类新型药物(IMPS)的基础上，这些药物在体内和体外都能杀死毛滴虫。然而，它们的作用机制以及它们是如何进入寄生虫组织的尚不清楚。事实上，寄生虫一般是如何摄取化合物的还没有确定，甚至连蠕虫的摄食机制和营养来源也仍然存在争议。NanoSIMS成像通过确定新药物化合物在寄生虫和宿主组织中的亚细胞定位，有可能彻底改变我们对新药物化合物的药理潜力的理解。此外，NanoSIMS还提供了探索寄生虫基本生物学的机会，并阐明了鞭虫生物学最神秘的特征之一--摄食机制。AIMS：1.开发和应用NanoSIMS成像到寄生虫组织，以便在亚细胞水平上定位新的药物化合物。2.通过确定鞭虫的摄食机制和营养来源，围绕鞭虫寄生虫的基本生物学产生新的知识。方法：用新的化合物和标记的营养物质治疗蠕虫：首先，我们将研究鞭虫T.Muris的成虫阶段。随后，我们可能会在这些分析中包括寄生虫的早期幼虫阶段和卵子阶段，对这些阶段的成像将需要额外的技术开发。成虫阶段的寄生虫将在体内生长，在尸检时取出，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，以加入奇异元素或同位素标签(例如13C)。用于生物组织的NanoSIMS的开发和应用：NanoSIMS分析需要良好的样品准备，才能获得有意义的结果，因为该技术在超高真空下运行。样品制备将通过比较化学固定和冷冻固定来优化，以确定哪种固定保留了药物的体内位置和细胞结构。然后，样品将被树脂嵌入并进行显微切割。用新的化合物或标记的食物来源治疗小鼠：我们开发了NanoSIMS方法来在体外检测寄生虫T.Muris的单个细胞内的药物化合物和标记的营养物质，我们将扩展工作以实现一定程度的敏感性，以便我们可以在喂食标记食物或体内剂量药物的小鼠身上检测到药物化合物/标记的营养物质。这一点很重要，因为它将使我们(I)了解体内从宿主那里实际摄取的食物以及体内药物的可获得性和在蠕虫中的定位，蠕虫生活在宿主组织中。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是重要的。

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