Protein Synthesis Inhibitors as anti-T. Cruzi agents

项目摘要 克氏锥虫是恰加斯病的病原体。尽管慢性T。 克鲁兹感染对人类健康的影响，目前还没有令人满意的治疗方案。这项提案的目标是 是开发有效、负担得起和安全的治疗方法来对抗慢性弓形虫感染。这种疗法必须 以克氏锥虫生存所必需的生物过程为目标，而不破坏人类的这种过程。这一概念具有 成功地用于开发抗生素，其中许多以细菌蛋白质合成为目标 对人类的毒性最小。克鲁兹毛滴虫蛋白质的合成与人类在以下水平上有很大的不同 信使核糖核酸结构，翻译起始和延伸因子，核糖体RNA和蛋白质及核糖体 结构。这些差异使得克氏锥虫蛋白质合成的每个阶段都有可能适合 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 建议对本实验室建立的克鲁兹毛滴虫体外翻译试验进行优化，使之适用于 吞吐量筛选(特定目标1)并实施HTS活动，以确定特定的代理 抑制克氏毛滴虫的蛋白质合成并选择性地杀死这些寄生虫(特定目标2)。 PI开发并实施了几种高通量筛选(HTS)试验，获得了三项第一 分类化合物，并通过评估聚焦文库和结构-活性-关系来优化这些化合物 学习。这些探测器已被科学界广泛采用，并促进了 结果，派、他的合作者和其他人发表了100多篇同行评议的出版物。其中两个是领先的 该系列已被批准用于进一步开发用于癌症治疗的药物。 在这一应用中，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，并 实施以鉴定特异性/选择性的T.Cruzi蛋白合成抑制剂。除了主要之外 筛选试验我们将采用绿色荧光蛋白报道的T.ruzi体外翻译试验来消除假阳性。 和一种反双荧光素酶哺乳动物体外翻译试验，以消除非特异性的命中化合物 克氏支原体，也就是那些也抑制哺乳动物蛋白质合成的那些。我们将测试克氏锥虫的特定抑制剂 抗寄生虫活性和对寄生虫与宿主细胞的选择性毒性的蛋白质合成。而我们所做的 建议这里是一个试点屏幕，我们拥有进行大规模所需的所有专业知识和设施 针对我们将识别的点击量进行屏幕和/或点击到主打的优化。努力实现从点击到领先的优化 将要求我们的一个或多个特定的/选择性的T.ruzi蛋白质合成抑制剂化合物 对克鲁兹毛滴虫有毒性，但对哺乳动物细胞没有毒性，并将在化学上可用于设计 聚焦图书馆和结构-活性-关系研究。命中化合物直接靶标的鉴定 未来研究的另一个领域将阐明翻译机制之间的生物学差异 人类和T·克鲁兹，都在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.