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，是一种由蚊子传播的急性发热 对人和动物都有影响的人畜共患病。大裂谷热疫情暴发 在整个非洲，最近在阿拉伯半岛。因为RVFV不再局限于非洲 在其他国家，它引发了人们对这种疾病可能在全球范围内传播的担忧。RVFV是一种选择代理，它 需要高度的生物控制设施。这一限制阻碍了RVFV抗病毒药物和 疫苗。尽管这种疾病对经济和公共卫生造成了重大影响，但没有完全 获得许可的疫苗和抗病毒药物在美国可供人类和动物使用。识别和发展是当务之急。 针对RVFV的有效抑制剂，用于治疗暴露和感染的人和动物。 马博士的团队已经开发出一种基于RVFV MP12疫苗毒株的基于细胞的筛选试验，该疫苗株 以Renilla为读数表达Renilla荧光素酶以鉴定RVFV抑制剂，并建立了STAT1-KO 易受MP12疫苗株感染的小鼠模型，可在BSL-2设施中使用。此外， 他们已经从NIH收集的化合物中筛选出727种化合物，其中有两种候选化合物，包括6-氮卓烷 米托蒽醌可抑制MP12的复制。他们假设，有效的RVFV抑制剂可能是 通过筛选大的化合物集合并进一步优化其结构和活性来鉴定， 并且可以确定已确定的候选者的抑制作用的机制。因此，他们计划使用 建立了高通量试验，以确定针对RVFV的抑制剂，评估它们在体外和小鼠中的有效性， 并通过两个特定的途径了解已确定的候选者的抑制作用的潜在机制 目标在这份R21提案中。在具体目标1中，由堪萨斯大学高吞吐量组装的图书馆 将对含有约26,000种化合物的筛选实验室进行筛查。26,000人 据预测，化合物会穿过血脑屏障。来自图书馆的点击量将有助于克服挑战 使RVFV抗病毒药物通过血脑屏障到达大脑，保护脑炎。主要的热门歌曲将是 经体外和体内检测证实。在特定的目标2，6-氮卓烷和米托蒽醌以及其他 在AIM 1中鉴定的病毒将在体外和在BALB/c小鼠身上使用强毒RVFV进行评估。要了解 其抑制作用的潜在机制，无论是化合物阻止病毒进入，还是抑制病毒 将使用不同的设计方法来研究复制和聚合酶活性。这项研究的结果 可以发现新的有效的RVFV抑制剂，并了解其潜在的机制 抑制作用，并为设计针对这种人畜共患病的新型抗病毒药物提供了新的见解 这对人类和动物的健康都有好处。

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.