Small molecule approaches to studying T.gondii invasion

描述（由申请方提供）：弓形虫是一种广泛分布的顶复门寄生虫，可导致免疫功能低下者和先天性感染胎儿发生毁灭性疾病。弓形虫病的病理是由于T细胞的活跃分裂形式导致宿主细胞侵入和溶解的重复循环。弓形虫速殖子尽管入侵的重要性，寄生虫的生活周期和弓形虫病的病理，很少有人知道的速殖子蛋白介导的入侵。因为T.弓形虫是一种单倍体、专性细胞内寄生虫，研究入侵的正向和反向遗传方法是有问题的。小分子将被用作一种手段来规避这一困难，并确定在入侵中发挥重要作用的基因产物。在高通量侵袭试验中筛选了超过14000种结构多样的小分子，并鉴定了28种侵袭抑制剂。出乎意料的是，还发现了7种入侵增强剂。在二次试验中，几种生物活性小分子显示出影响寄生虫的运动性和/或其顶端细胞器的分泌。该提案的目标是：（I）测试针对其他几种相关和不相关寄生虫的入侵抑制剂和增强剂，以确定它们是否靶向顶复门入侵机制的保守组分;（II）测试任何运动抑制剂/增强剂是否通过寄生虫肌球蛋白A或其所属的多蛋白复合物发挥作用;（III）测试在筛选中鉴定的cGMP磷酸二酯酶抑制剂是否影响寄生虫入侵期间的cGMP水平;和（V）使用合成、生物化学和遗传方法来确定3-5种最高优先级的入侵抑制剂/增强剂的体内靶标，其中优先级是基于I-IV的结果。最近公布的弓形虫基因组序列将极大地促进目标识别。该提案的一个优点是生物实验和合成化学将被整合到一个水平，以实现该项目的目标。这项工作有可能为T.它还可以同时鉴定先导化合物，用于开发新的和迫切需要的抗寄生虫药物。

DESCRIPTION (provide by the applicant): Toxoplasma gondii is a widespread Apicomplexan parasite that causes devastating disease in immunocompromised persons and the congenitally infected fetus. The pathology of toxoplasmosis is due to repeated cycles of host cell invasion and lysis by the actively dividing form of T. gondii, the tachyzoite. Despite the importance of invasion to the life cycle of the parasite and the pathology of toxoplasmosis, little is known about the tachyzoite proteins that mediate invasion. Because T. gondii is a haploid, obligate intracellular parasite, forward and reverse genetic approaches to studying invasion are problematic. Small molecules will be used as a means to circumvent this difficulty and identify gene products that play an important role in invasion. A collection of over 14000 structurally diverse small molecules has been screened in a high-throughput invasion assay, and 28 invasion inhibitors have been identified. Unexpectedly, 7 invasion enhancers were also discovered. In secondary assays, several of the bioactive small molecules were shown to affect motility of the parasite and/or secretion from its apical organelles. The goals of the proposal are to: (I) Test the invasion inhibitors and enhancers against several other related and unrelated parasites, to determine whether they target conserved components of the Apicomplexan invasion machinery; (II) Test whether any of the motility inhibitors/enhancers exert their effects through parasite myosin A or the multi-protein complex to which it belongs; (III) Test whether an inhibitor of cGMP phosphodiesterase identified in the screen affects parasite cGMP levels during invasion; (IV) Screen a comprehensive collection of >500 small molecules of known biological function for an effect on invasion; and (V) Use synthetic, biochemical and genetic methods to determine the in vivo targets of 3-5 of the highest priority invasion inhibitors/enhancers, where prioritization is based on the results of I-IV. Target identification will be greatly facilitated by the recently released sequence of the Toxoplasma genome. A strength of the proposal is the level to which biological experiments and synthetic chemistry will be integrated to address the project's goals. This work has the potential to provide important new insights into the proteins and pathways involved in host cell invasion by T. gondii and related parasites, and it may simultaneously identify lead compounds for the development of new and urgently needed anti-parasitic drugs.