A high-throughput screen for antivirals targeting filovirus replication

描述(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，丝状病毒已在人类中造成了一些严重出血性疾病的爆发，其特点是死亡率非常高。人们对这些人畜共患病病毒的传播知之甚少，这主要是由于疫情的零星性质及其与赤道非洲地区的地理隔离。目前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，再加上高死亡率和人与人之间的传播，所有含有传染性丝状病毒的材料都在最高级别的生物安全下处理(BSL4)。这对抗病毒药物的发现工作造成了严格的限制，因为高通量筛选技术不能轻易地转移到BSL4环境中；当然不是在工业中通常用于药物筛选计划的规模。由于担心丝状病毒可能被用作生物恐怖因子，识别丝状病毒抗病毒药物的动力进一步增强。因此，埃博拉和马尔堡病毒都被归类为NIAID优先A生物防御病原体，针对这些病原体的诊断、疫苗和疗法的开发被强调为研究优先事项。为了实现这一目标，我们描述了一项计划，即开发一种高通量的埃博拉病毒复制抑制物筛选试验，该试验可以在BSL2上进行，因此可以兼容专业的小分子筛查设施。该检测以扎伊尔埃博拉病毒微基因组检测为基础，因为它是以质粒为基础的，不涉及传染性病毒，可以在BSL2条件下进行。该检测方法测量病毒基因组复制和转录，到目前为止，还没有描述过埃博拉病毒复制复合体的抑制剂(例如聚合酶抑制剂)。除了是潜在的候选药物外，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。在目标1中，将对该分析进行优化和小型化，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中试筛选，以判断检测性能并建立命中选择的标准。利用莱斯顿埃博拉病毒和马尔堡病毒微型基因组系统进行的二次分析将有助于识别那些具有潜在泛丝病毒抗病毒活性的化合物。该项目的成功完成将扩大小分子筛选活动，增加发现具有强大抗丝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.