Small molecule approaches to studying T.gondii invasion

研究弓形虫入侵的小分子方法

• 批准号: 6857131
• 负责人: GARY E WARD
• 金额: $ 56.07万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6857131
• 关键词: MDCK cell; Plasmodium knowlesi; SDS polyacrylamide gel electrophoresis; Toxoplasma gondii; adenosinetriphosphatase; animal tissue; cyclic GMP; enzyme activity; enzyme inhibitors; fluorescence microscopy; high throughput technology; host organism interaction; intracellular parasitism; liquid chromatography mass spectrometry; mass spectrometry; molecular pathology; myosins; nanotechnology; pathologic process; phosphorylation; protein structure function; tissue /cell culture; western blottings

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.

描述（由申请人提供）：弓形虫Gondii是一种广泛的Apicomplexan寄生虫，在免疫强化者和先天感染的胎儿中导致毁灭性疾病。弓形虫病的病理是由于t. gondii的主动分裂形式，thezoite的宿主细胞侵袭和裂解的反复循环所致。尽管入侵对寄生虫的生命周期和弓形虫病的病理非常重要，但对于介导侵袭的次氮岩蛋白知之甚少。因为弓形虫是一种单倍体，因此具有强大的细胞内寄生虫，前进和反向研究入侵的方法是有问题的。小分子将被用作规避这一难度并确定在入侵中起重要作用的基因产物的一种手段。在高通量入侵测定中已经筛选了超过14000个结构上多样的小分子的集合，并且已经鉴定出了28个浸润抑制剂。出乎意料的是，还发现了7个入侵增强剂。在次要测定中，显示出几个生物活性小分子会影响寄生虫和/或从其顶端细胞器中分泌的运动。该提案的目标是：（i）测试对其他几个相关和无关的寄生虫的入侵抑制剂和增强子，以确定它们是否针对Apicomplexan入侵机制的保守成分； （ii）测试任何运动抑制剂/增强剂是否通过其所属的多蛋白质复合物通过寄生虫肌球蛋白A或多蛋白质发挥作用； （iii）测试筛网中鉴定出的CGMP磷酸二酯酶的抑制剂是否会影响侵袭过程中的寄生虫CGMP水平； （iv）筛选> 500个已知生物学功能的> 500个小分子的全面集合，以对侵袭产生影响； （v）使用合成，生化和遗传学方法确定最高优先侵入抑制剂/增强子的3-5个体内靶标，而优先级基于I-IV的结果。最近发布的弓形虫基因组序列将极大地促进目标识别。该提案的优势是将生物学实验和合成化学的整合以解决项目的目标的水平。这项工作有可能提供对T. gondii和相关寄生虫涉及宿主细胞侵袭的蛋白质和途径的重要新见解，并且它可以同时鉴定出铅化合物，以开发新的，急需的抗寄生虫药物。