Identification of Small Molecular Inhibitors of Rift Valley Fever Virus Replication

项目摘要 裂谷热由裂谷热病毒（RVFV）引起，是一种急性、蚊媒、发热性疾病， 人畜共患的疾病。大裂谷热爆发已经发生 在整个非洲，最近在阿拉伯半岛。因为RVFV不再局限于非洲 它引起了人们对这种疾病可能在世界范围内蔓延的担忧。RVFV是一种选择代理， 需要高度的生物防护设施这种限制阻碍了RVFV抗病毒药物的开发， 疫苗。尽管这种疾病对经济和公共卫生产生了重大影响，但目前还没有充分的 在美国获得许可的疫苗和抗病毒药物可用于人类和动物。迫切需要识别和开发 治疗暴露和感染的人类和动物。 Ma博士的小组已经开发了一种基于RVFV MP 12疫苗株的细胞筛选试验， 使用海肾作为读数表达海肾荧光素酶以鉴定RVFV抑制剂，并建立了STAT 1-KO 对可用于BSL-2设施的MP 12疫苗株感染易感的小鼠模型。此外，委员会认为， 他们从NIH的收藏中筛选了727种化合物，其中包括6-氮尿苷在内的两种候选化合物， 米托蒽醌抑制MP 12的复制。他们假设，有效的抑制剂对RVFV可以是 通过筛选大的化合物集合并通过进一步优化其结构和活性来鉴定， 并且可以确定所鉴定的候选物的抑制作用的机制。因此，他们计划使用 开发了高通量测定法来鉴定针对RVFV的抑制剂，评估它们在体外和小鼠中的功效， 并通过两个具体的方法来了解所识别候选物的抑制作用的潜在机制。 在R21提案中的目标。在具体的目标1中，由堪萨斯高中的图书馆组成 将对含有大约26，000种化合物的实验室进行筛选。2.6万 预计化合物会穿过血脑屏障。图书馆的点击将有助于克服挑战 RVFV抗病毒药物通过血脑屏障到达大脑以保护脑炎。主要的打击将是 通过体外和体内测定证实。在具体目标中，2，6-氮杂尿苷和米托蒽醌以及其他 将在体外和BALB/c小鼠中使用毒力RVFV评价目标1中鉴定的病毒。了解 其抑制作用的潜在机制，无论化合物阻断病毒进入，抑制病毒进入， 复制和聚合酶活性将使用不同设计的测定法进行研究。本研究结果 可以识别针对RVFV的新型有效抑制剂，并了解其潜在机制。 抑制作用，并提供了新的见解，设计新的抗病毒药物，对这种人畜共患疾病 这将有利于人类和动物的健康。

Project Summary Rift Valley fever caused by Rift Valley fever virus (RVFV) is an acute, mosquito-borne, fever-causing zoonotic disease that affects both humans and animals. Large Rift Valley fever outbreaks have occurred throughout Africa and more recently in the Arabian Peninsula. Because RVFV is no longer restricted to African countries, it has raised concerns that the disease could spread worldwide. The RVFV is a select agent that requires high biocontainment facilities. This limitation has hampered the development of RVFV antivirals and vaccines. Despite the significant impact of the disease to the economy and public health, there are no fully licensed vaccine and antivirals available in the US for human and animal use. It is urgent to identify and develop effective inhibitors against RVFV to treat exposed and infected humans and animals. Dr. Ma’s group has developed a cell-based screening assay based on the RVFV MP12 vaccine strain that expresses Renilla Luciferase using Renilla as readout to identify RVFV inhibitors, and established a STAT1-KO mouse model susceptible to infection with MP12 vaccine strain that can be used in a BSL-2 facility. Furthermore, they have screened 727 compounds from the NIH collections of which two candidates including 6-azauridine and mitoxantrone inhibited replication of MP12. They hypothesize that effective inhibitors against RVFV can be identified by screening large compound collections and by further optimization of their structures and activities, and the mechanisms of inhibitory effects of identified candidates can be determined. Thus, they plan to use the developed high-throughput assay to identify inhibitors against RVFV, evaluate their efficacy in vitro and in mice, and understand the underlying mechanisms of inhibitory effects of identified candidates through two specific aims in this R21 proposal. In specific aim 1, the libraries assembled by the University of Kansas High Throughput Screening Laboratory that contain approximately 26,000 chemical compounds will be screened. The 26,000 compounds are predicted to cross the blood-brain barrier. Hits from the library will help overcome the challenge for RVFV antivirals to reach the brain through the blood-brain barrier to protect encephalitis. Primary hits will be confirmed by in vitro and in vivo assays. In specific aim 2, 6-azauridine and mitoxantrone as well as others identified in Aim 1 will be evaluated in vitro and in the BALB/c mice using virulent RVFV. To understand the underlying mechanisms of their inhibitory effects, whether the compounds block virus entry, inhibit virus replication and polymerase activity will be investigated using different designed assays. The results of this study could identify novel effective inhibitors against RVFV and understand the underlying mechanisms of their inhibitory effects, and offer novel insights toward the design of novel antiviral drugs against this zoonotic disease that will benefit both human and animal health.