Small Molecule Approaches to Studying T. gondi Invasion

项目摘要 弓形虫是一种分布广泛的尖端复合体寄生虫，可在免疫功能低下的人群中引起严重疾病。 人和先天感染的胎儿。宿主细胞入侵和裂解的重复循环 弓形虫的活跃分裂形式，即速殖子，是急性期内组织损伤的重要原因。 感染。尽管入侵对寄生虫的生命周期和 弓形虫病，人们对介导这一过程的速殖子蛋白知之甚少。在一个小分子中- 基于对这一问题的探讨，24种新的小分子抑制剂和6种侵袭促进剂 都被确认了。这些化合物的一个子集影响所有被测试的顶复合体寄生虫的入侵， 包括疟疾寄生虫，这表明它们针对顶端复合体入侵的保守成分 机械设备。化合物的生物学效应的表征和广泛的合成化学 导致了关于它们的作用机制的具体假说的发展；这些假说中有三个 将在AIMS 1-3进行测试。这项工作也证明了确定目标的一般方法的必要性。 对寄生虫有活性的小分子，即允许识别 许多小分子/靶分子对。这种方法，再加上高通量筛查，将代表 一种在寄生虫中识别新的可用药靶点的有效方法。AIM 4将解决这个问题，使用 酵母-3-杂交法(Y3H)。这项建议的具体目的是： (1)验证化合物115556通过修饰以下物质来抑制寄生虫活动和入侵的假设 肌球蛋白轻链(TgMLC1)； (2)检验化合物112762引起的侵袭增强是通过 对寄生虫蛋白精氨酸甲基转移酶活性的影响； (3)检验化合物144146通过作用于以下物质而抑制锥形延伸和侵袭的假设 寄生虫蛋白Peroxiredoxin2；以及 (4)建立和使用酵母-3-杂交法(Y3H)鉴定弓形虫侵袭抑制剂的靶点 这项工作有可能对宿主参与的蛋白质和途径提供重要的新见解 弓形虫的细胞入侵。结果也很可能被转移到其他更难实验获得的地方 人类寄生虫，如疟原虫和隐孢子虫。这项提议的一个主要优点是 为了解决这个项目，将整合哪些生物实验和合成化学？S的目标是。

Project Summary Toxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in immunocompromised persons and the congenitally-infected fetus. Repeated cycles of host cell invasion and lysis by the actively dividing form of T. gondii, the tachyzoite, are a significant cause of tissue damage during acute infection. Despite the importance of invasion to the life cycle of the parasite and the pathology of toxoplasmosis, relatively little is known about the tachyzoite proteins that mediate the process. In a smallmolecule- based approach to this question, 24 novel small molecule inhibitors and 6 enhancers of invasion were identified. A subset of these compounds affects the invasion of all apicomplexan parasites tested, including malaria parasites, suggesting that they target conserved components of the apicomplexan invasion machinery. Characterization of the biological effects of the compounds and extensive synthetic chemistry has led to the development of specific hypotheses regarding their mechanism of action; three of these hypotheses will be tested in Aims 1-3. The work has also demonstrated the need for general methods to identify the targets of small molecules that are active against the parasite, i.e., methods that would allow for the identification of many small-molecule/target pairs. Such an approach, coupled with high-throughput screening, would represent a powerful way to identify new druggable targets within the parasite. Aim 4 will address this issue, using Yeast-3-Hybrid (Y3H) methodology. The Specific Aims of the proposal are to: (1) Test the hypothesis that compound 115556 inhibits parasite motility and invasion through modification of myosin light chain (TgMLC1); (2) Test the hypothesis that the enhancement of invasion caused by compound 112762 occurs through an effect on parasite protein arginine methyltransferase activity; (3) Test the hypothesis that compound 144146 inhibits conoid extension and invasion through an effect on the parasite protein peroxiredoxin2; and (4) Develop and use Yeast-3-Hybrid (Y3H) methods to identify targets of T. gondii invasion inhibitors This work has the potential to provide important new insights into the proteins and pathways involved in host cell invasion by T. gondii. The results are also likely to be transferable to other, less experimentally accessible human parasites, such as Plasmodium and Cryptosporidium. A major strength of the proposal is the extent to which biological experiments and synthetic chemistry will be integrated to address the project?s goals.