Small molecule approaches to studying T.gondii invasion

描述（由申请人提供）：刚地弓形虫是一种广泛存在的顶端复合体寄生虫，可在免疫功能低下的人和先天感染的胎儿中引起毁灭性疾病。弓形虫病的病理是由于宿主细胞的侵袭和裂解的积极分裂形式的弓形虫，速殖子的重复周期。尽管入侵对寄生虫的生命周期和弓形虫病的病理很重要，但对介导入侵的速殖子蛋白知之甚少。由于弓形虫是单倍体，专性细胞内寄生虫，正向和反向遗传方法研究入侵是有问题的。小分子将被用作规避这一困难的手段，并识别在入侵中起重要作用的基因产物。在高通量入侵试验中筛选了超过14000种结构多样的小分子，并确定了28种入侵抑制剂。出乎意料的是，还发现了7种入侵增强剂。在二次分析中，一些生物活性小分子被证明可以影响寄生虫的运动和/或其顶端细胞器的分泌。该提案的目标是：(I)测试入侵抑制剂和增强剂对几种其他相关和不相关寄生虫的攻击，以确定它们是否针对顶复合体入侵机制的保守成分；（II）检测运动抑制剂/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；（三）检测筛选到的cGMP磷酸二酯酶抑制剂是否影响寄生虫入侵时的cGMP水平；（四）全面筛选已知生物学功能的bb50 500个小分子，研究其对入侵的影响；(V)使用合成、生化和遗传方法确定3-5种最优先的入侵抑制剂/增强剂的体内靶点，其中优先级基于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。该提案的一个优势是将生物实验和合成化学结合起来以实现项目的目标。这项工作有可能为弓形虫和相关寄生虫入侵宿主细胞的蛋白和途径提供重要的新见解，并可能同时为开发新的和迫切需要的抗寄生虫药物提供先导化合物。

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.