TARGETING ESSENTIAL ROP KINASES IN TOXOPLASMA

描述（由申请人提供）：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。严重感染通常只发生在免疫功能低下的患者中，包括HIV感染、癌症化疗、器官移植或宫内感染的婴儿。此外，新的研究表明，严重的眼部弓形虫病也可能发生在健康的成年人中，慢性感染是某些形式的精神疾病的潜在危险因素。弓形虫分离株在动物模型和人类感染中的毒力显著不同。最近的研究结果表明，这种寄生虫的主要毒力决定因素是来源于棒状体（ROP）的分泌蛋白，这些分泌蛋白在入侵时被注入宿主细胞。许多ROP含有保守的丝氨酸/苏氨酸激酶结构域，并且被预测作为激酶起作用，通过特定靶标的磷酸化潜在地改变宿主信号传导途径。在初步研究中，我们已经开发了一种系统的基因破坏策略，以靶向基因组中所有已知的活性ROP激酶。ROP激酶的破坏将用于定义其在小鼠模型中体外与体内生长期间的作用。另外，我们设计了一种单步插入策略来产生必需ROP激酶的条件表达等位基因。敲除单个ROP将用于探索由这些激酶控制的功能途径。我们还将采用生物化学测定和质谱法来鉴定ROP激酶的底物特异性，从而为抑制剂测定的开发提供特异性底物。重组蛋白的表达将用于开发体外活性测定和筛选聚焦激酶抑制剂文库以鉴定有效的抑制剂。这些研究的目的是为T.弓形虫和表征化学支架，抑制ROP激酶的未来发展。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，并可能导致改善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.