A high-throughput screen for antivirals targeting filovirus replication

描述（由申请人提供）：自从30-40年前首次发现丝状病毒（埃博拉病毒和马尔堡病毒）以来，它们已经在人类中引起了许多严重出血性疾病的暴发，其特点是死亡率非常高。人们对这些人畜共患病毒的传播知之甚少，这主要是由于疫情的散发性质以及它们在赤道非洲地区的地理隔离。目前还没有批准用于预防和治疗丝状病毒感染的疫苗或药物，由于缺乏治疗，再加上死亡率高和人际传播，所有含有传染性丝状病毒的材料都在最高生物安全级别（BSL4）下处理。这严重限制了抗病毒药物的发现工作，因为高通量筛选技术不容易转移到BSL4环境中；当然不是通常用于工业药物筛选项目的规模。由于担心这些病毒可能被用作生物恐怖制剂，鉴定丝状病毒抗病毒药物的动力进一步增强。因此，埃博拉病毒和马尔堡病毒都被列为NIAID生物防御的优先A类病原体，并强调为这些病原体开发诊断、疫苗和治疗方法是研究重点。为了实现这一目标，我们描述了一项计划，即开发一种高通量筛选方法，用于埃博拉病毒复制抑制剂，这种方法可以在BSL2上进行，因此可以兼容于专门的小分子筛选设施。该测定是基于扎伊尔埃博拉病毒微小基因组测定，因为它是基于质粒的，不涉及传染性病毒，可以在BSL2条件下进行。该检测测量病毒基因组复制和转录，迄今为止，尚未描述埃博拉病毒复制复合体的抑制剂（例如聚合酶抑制剂）。除了作为潜在的候选药物外，这些分子还将作为有用的探针，进一步了解这种病毒复合体的功能。在Aim 1中，该分析将被优化和小型化，以满足用于小分子高通量筛选的标准。在目标2中，我们将进行生物活性分子的试点筛选，以判断分析性能并建立命中选择标准。利用雷斯顿埃博拉病毒和马尔堡病毒小基因组系统进行的二次分析将有助于确定那些具有潜在泛丝状病毒抗病毒活性的化合物。该项目的成功完成将允许扩大小分子筛选活动，增加发现具有有效抗丝状病毒活性的化合物的可能性。

DESCRIPTION (provided by applicant): Since they were first identified 30-40 years ago, the filoviruses (Ebola viruses and Marburg viruses) have caused a number of outbreaks of severe hemorrhagic disease in humans that are characterized by a very high mortality rate. Little is understood regarding the transmission of these zoonotic viruses, mostly due to the sporadic nature of the outbreaks and their geographic isolation to regions of equatorial Africa. There are currently no vaccines or drugs approved for the prevention and treatment of filovirus infections and as a result of this lack of therapy, combined with the high mortality and human-to-human transmission, all material containing infectious filovirus is handled under the highest level of biosafety (BSL4). This places a severe restriction on antiviral drug discovery efforts, as technology for high-throughput screening cannot easily be transferred to a BSL4 setting; certainly not on the scale normally used for drug screening programs in industry. The impetus for identifying filovirus antivirals has been further heightened by concerns that these viruses may be used as bioterror agents. Consequently both Ebola and Marburg viruses are classified as NIAID priority A pathogens for biodefense, and development of diagnostics, vaccines and therapeutics for these agents is emphasized as a research priority. Towards this goal we describe a plan to develop a high-throughput screening assay for inhibitors of Ebola virus replication that can be performed at BSL2, and therefore be compatible for use in specialist small molecule screening facilities. The assay is based on the Zaire Ebola virus minigenome assay, which because it is plasmid-based and does not involve infectious virus, can be performed under BSL2 conditions. The assay measures viral genome replication and transcription and to date, no inhibitors of the Ebola virus replication complex (e.g. polymerase inhibitors) have been described. In addition to being potential drug candidates, such molecules will serve as useful probes to further our understanding of how this viral complex functions. In Aim 1 the assay will be optimized and miniaturized to meet the criteria for use in a small molecule high-throughput screen. In Aim 2 we will conduct a pilot screen of biologically active molecules to judge assay performance and establish the criteria for hit selection. Secondary assays performed with the Reston Ebola and Marburg virus minigenome systems will assist in identifying those compounds that have potential pan-filovirus antiviral activity. Successful completion of this project will allow for expanded small molecule screening campaigns with increased possibility of discovering compounds with potent anti-filovirus activity. PUBLIC HEALTH RELEVANCE: Ebola viruses and Marburg viruses cause highly fatal hemorrhagic infections in humans. There are currently no approved drugs to prevent or treat these infections, and the development of novel screening approaches will facilitate identification of candidate antiviral drugs.