TARGETING ESSENTIAL ROP KINASES IN TOXOPLASMA

描述（由申请人提供）：刚地弓形虫是一种广泛存在于野生、家养和伴侣动物中的原生动物寄生虫，通常也会感染人类。严重感染通常只在免疫功能低下的患者中发现，包括HIV感染、癌症化疗、器官移植或子宫内感染的婴儿。此外，新出现的研究表明，严重的眼部弓形虫病也可能发生在健康成年人身上，慢性感染是某些精神疾病的潜在危险因素。分离弓形虫在动物模型和人类感染中的毒力差别很大。最近的研究结果表明，这种寄生虫的主要毒力决定因素是在入侵时注射到宿主细胞中的来自腺状体（ROPs）的分泌蛋白。许多rop含有一个保守的丝氨酸/苏氨酸激酶结构域，并被预测为激酶，可能通过磷酸化特定靶标改变宿主信号通路。在初步研究中，我们已经开发了一种系统的基因破坏策略，以基因组中所有已知的活性ROP激酶为目标。在小鼠模型中，ROP激酶的破坏将用于确定它们在体外和体内生长过程中的作用。另外，我们设计了一个单步插入策略来生成必需ROP激酶的条件表达等位基因。单个rop的敲除将用于探索由这些激酶控制的功能途径。我们还将采用生化分析和质谱分析来确定ROP激酶的底物特异性，从而为开发抑制剂检测提供特定的底物。重组蛋白的表达将用于开发体外活性测定和筛选重点激酶抑制剂文库，以确定有效的抑制剂。这些研究的目的是提供弓形虫必需激酶的遗传验证，并表征抑制ROP激酶的化学支架，为未来的发展提供参考。这些研究将有助于确定一类重要的寄生虫毒力因子的作用，并可能导致改进针对弓形虫病的治疗干预。

DESCRIPTION (provided by applicant): Toxoplasma gondii is a widespread protozoan parasite of wild, domestic, and companion animals that also commonly infects humans. Severe infections are normally only found in immunocompromised patients, including HIV infection, cancer chemotherapy, organ transplant, or infants infected in utero. Additionally, emerging studies indicate that severe ocular toxoplasmosis can also occur in healthy adults, and that chronic infection is an underlying risk factor for some forms of psychiatric disease. Toxoplasma isolates differ dramatically in their virulence in animal models and also in human infections. Recent findings reveal that the major virulence determinants of this parasite are secretory proteins derived from the rhoptries (ROPs), which are injected to the host cell at the time of invasion. Many ROPs contain a conserved serine / threonine kinase domain and are predicted to function as kinases, potentially altering host signaling pathways by phosphorylation of specific targets. In preliminary studies, we have developed a systematic gene disruption strategy to target all of the known active ROP kinases in the genome. Disruption of ROP kinases will be used to define their roles during growth in vitro vs. in vivo in the mouse model. Separately, we have designed a single-step insertional strategy to generate conditional expression alleles of essential ROP kinases. Knockdown of individual ROPs will be used to explore the functional pathways controlled by these kinases. We will also employ biochemical assays and mass spectrometry to identify the substrate specificity of ROP kinases, there by providing specific substrates for development of inhibitor assays. Expression of recombinant proteins will be used to develop in vitro activity assays and to screen focused kinase inhibitor libraries to indentify potent inhibitors. The goal of these studies to provide genetic validation of essential kinases in T. gondii and to characterize chemical scaffolds that inhibitor ROP kinases for future development. These studies will help define the roles of an important class of parasite virulence factors and may lead to improved therapeutic intervention against toxoplasmosis.