Small molecule approaches to studying T.gondii invasion

描述(由申请人提供)：弓形虫是一种广泛传播的细粒复合体寄生虫，会在免疫功能受损的人和先天性感染的胎儿中引起毁灭性的疾病。弓形虫病的病理是由于弓形虫的活跃分裂形式--速殖子--反复的宿主细胞入侵和裂解。尽管入侵对寄生虫的生命周期和弓形虫病的病理很重要，但人们对介导入侵的速殖子蛋白知之甚少。由于弓形虫是一种单倍体的专性细胞内寄生虫，正向和反向遗传方法研究入侵是有问题的。小分子将被用作绕过这一困难的手段，并识别在入侵中发挥重要作用的基因产品。在高通量侵袭试验中筛选了14000多种结构不同的小分子，并鉴定了28种侵袭抑制剂。出乎意料的是，还发现了7种侵袭促进剂。在二次检测中，几个生物活性小分子被证明影响寄生虫的活动和/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其他几种相关和不相关的寄生虫的侵袭作用，以确定它们是否针对Apicomplexan入侵机制的保守组件；(Ii)测试是否有任何运动抑制剂/增强剂通过寄生虫的肌球蛋白A或其所属的多蛋白复合体发挥作用；(Iii)测试屏幕上识别的cGMP磷酸二酯酶抑制剂是否在入侵期间影响寄生虫的cGMP水平；(Iv)筛选已知生物学功能的&GT；500小分子的综合集合，以影响入侵；以及(V)使用合成、生化和遗传方法来确定3-5种最优先的侵袭抑制/增强剂的体内靶点，其中优先顺序基于I-IV的结果。最近公布的弓形虫基因组序列将极大地促进靶标识别。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。这项工作有可能为了解弓形虫和相关寄生虫入侵宿主细胞所涉及的蛋白质和途径提供重要的新见解，并可能同时识别先导化合物，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.