A high-throughput screen for antivirals targeting filovirus replication

描述（由申请方提供）：自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，已在人类中引起多次严重出血性疾病暴发，其特征为死亡率非常高。人们对这些人畜共患病毒的传播知之甚少，主要是由于疫情的零星性质及其在赤道非洲地区的地理隔离。目前没有疫苗或药物获批用于预防和治疗丝状病毒感染，由于缺乏治疗，加上高死亡率和人际传播，所有含有传染性丝状病毒的材料都在最高生物安全水平（BSL 4）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能轻易转移到BSL 4环境中;当然不是在工业中通常用于药物筛选计划的规模上。由于担心这些病毒可能被用作生物恐怖剂，进一步加强了鉴定丝状病毒抗病毒药物的动力。因此，埃博拉病毒和马尔堡病毒都被列为NIAID生物防御优先A级病原体，并强调开发这些病原体的诊断、疫苗和治疗方法是研究重点。为了实现这一目标，我们描述了一个计划，以开发一种高通量的筛选试验，可以在BSL 2进行埃博拉病毒复制的抑制剂，因此，可兼容用于专业的小分子筛选设施。该试验基于扎伊尔埃博拉病毒微型基因组试验，由于其基于质粒且不涉及感染性病毒，因此可在BSL 2条件下进行。该测定测量病毒基因组复制和转录，并且迄今为止，尚未描述埃博拉病毒复制复合物的抑制剂（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，以进一步了解这种病毒复合体的功能。在目标1中，将对测定进行优化和小型化，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，以判断测定性能并建立命中选择的标准。使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级检测将有助于鉴定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，增加发现具有强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.