Protein Synthesis Inhibitors as anti-T. Cruzi agents

项目摘要 克氏锥虫是南美锥虫病的病原体。尽管慢性T. 克氏病对人类健康的影响，目前还没有令人满意的治疗方案。这项提案的目的是 是开发有效的，负担得起的，安全的治疗慢性T。克鲁兹感染。这些疗法必须 以T所必需生物过程为目标。而不会破坏人体内的克氏生存能力。这一概念 已成功地用于开发抗生素，其中许多靶向细菌蛋白质合成， 对人体毒性极小。t. cruzi蛋白质合成在以下水平上与人类的显著不同： mRNA结构、翻译起始和延伸因子、核糖体RNA和蛋白质以及核糖体 结构这些差异使得T. Cruzi蛋白质合成可能适用于 用于治疗慢性感染的T，Cruzi特异性蛋白质合成抑制剂的开发。因此我们 建议优化和调整T.本实验室开发的Cruzi体外翻译试验， 通量筛选（具体目标1），并实施HTS活动，以确定具体 抑制T. cruzi蛋白质的合成，并选择性地杀死这些寄生虫（具体目标2）。 PI开发并实施了几种高通量筛选（HTS）试验，获得了三个首次在 分类化合物，并通过评估重点库和构效关系优化这些化合物 问题研究这些探测器已被科学界广泛采用，并促进了 PI、他的合作者和其他人发表了100多篇同行评议的出版物。其中两个导致 该系列已被授权给工业界进一步开发成癌症治疗药物。 在本申请中，我们建议利用我们在翻译生物学和HTS检测开发方面的专业知识， 实施以鉴定特异性/选择性抑制剂T. Cruzi蛋白质合成。除了主要的 筛选试验我们将使用GFP报告子T。消除假阳性的Cruzi体外翻译测定 和计数器双荧光素酶哺乳动物体外翻译测定，以消除非特异性的命中化合物， t. cruzi，即也抑制哺乳动物蛋白质合成的那些。我们将测试特定的T. cruzi 抗寄生虫活性和对寄生虫相对于宿主细胞的选择性毒性的蛋白质合成。虽然我们 这里提出的是一个试点屏幕，我们拥有进行大规模测试所需的所有专业知识和设施。 筛选和/或针对我们将识别的点击进行点击到销售线索优化。努力实现点击到领先的优化 需要一种或多种我们的特异性/选择性克氏锥虫蛋白合成抑制剂化合物 显示对克氏锥虫的毒性，但对哺乳动物细胞没有毒性， 重点文库和构效关系研究。确定命中化合物的直接靶标是 未来研究的另一个领域，将阐明翻译机器之间的生物差异， human和T. cruzi，和所有在PI的能力范围内。

Project Abstract Trypanosoma cruzi is the causative agent of the Chagas’ disease. Despite the devastating effects of chronic T. cruzi infections on human health, there are no satisfactory options for their treatment. The goal of this proposal is to develop effective, affordable, and safe therapies against chronic T. cruzi infections. Such therapies must target a biological process essential for T. cruzi viability without disrupting it in humans. This concept has successfully been used for the development of antibiotics, many of which target bacterial protein synthesis with minimal toxicity to humans. T. cruzi protein synthesis differs substantially from that of humans at the levels of mRNA structure, translation initiation and elongation factors, ribosomal RNA and proteins and ribosome structure. These differences render every stage of T. Cruzi protein synthesis potentially suitable for the development of T,Cruzi specific protein synthesis inhibitors for the treatment of chronic infections. We therefore propose to optimize and adapt a T. cruzi in vitro translation assay developed in our laboratory to high throughput screening (Specific Aim 1) and implement an HTS campaign to identify agents that specifically inhibit T. cruzi protein synthesis and selectively kill these parasites (Specific Aim 2). PI has developed and implemented several high throughput screening (HTS) assays, obtained three first-in- class compounds and optimized these by evaluating focused libraries and structure-activity-relationships studies. These probes have been widely adapted by the scientific community and fostered research that resulted in more than 100 peer-reviewed publications by PI, his collaborators and others. Two of these lead series have been licensed to industry for further development into drugs for cancer therapy. In this application we propose to utilize our expertise in biology of translation and HTS assay development and implementation to identify the specific/selective inhibitors T.Cruzi protein synthesis. In addition to primary screening assay we will employ a GFP reporter T. cruzi in vitro translation assay to eliminate false positives and a counter dual luciferase mammalian in vitro translation assay to eliminate hit compounds not specific for T. cruzi, i.e. those that also inhibit mammalian protein synthesis. We will test specific inhibitors of T. cruzi protein synthesis for anti-parasitic activity and selective toxicity to parasite vs. host cells. While what we propose here is a pilot screen, we possess all the expertise and facilities needed to conduct a large scale screen and/or hit-to-lead optimization for the hits we will identify. Effort towards the hit-to-lead optimization would require that one or more of our specific/selective T.cruzi protein synthesis inhibitor compounds will display toxicity towards T.cruzi but not the mammalian cells and will be chemically accessible for design of focused libraries and structure-activity-relationship studies. Identification of the direct target of hit compounds is another area of future research that will elucidate biological differences between translation machineries of human and T. cruzi, and all well within the PI’s capabilities.