Screening for anti-RSV compounds with indicator cells

描述(由申请人提供)：该项目的目标是使用一种不依赖感染的细胞检测来鉴定抑制呼吸道合胞病毒(RSV)的化合物。这种生物测定的基础是在转基因细胞的细胞质中表达存在于人工病毒基因组中的报告基因。在识别包括RSV在内的许多致病负链RNA病毒复制过程中涉及的顺式作用元件和反式作用因子方面已经取得了重大进展。负链RNA病毒包括一些最臭名昭著的人类病原体，由于严重的呼吸道疾病和出血热综合征，在世界范围内造成重大的发病率和死亡率。不幸的是，很少有治疗剂可用于治疗这些病毒的感染。寻找新的抗病毒药物的努力将需要符合HTS的方法。在这项申请中，研究人员描述了他们计划使用新的基于细胞的分析来鉴定具有抗RSV活性的化合物。为了确定抑制RSV复制的“HITS”，申请者计划筛选一个独特的天然化合物文库和几个新的合成化合物文库。阿帕斯的合作伙伴开发了一种专有系统，用于从植物材料中制作化合物库。去除缺乏类药物性质的高丰度化合物，其余化合物被层析分离成不同的峰，这些峰作为条形码96孔板中的阵列提供给APath，LLC。合成化学库已经从商业来源获得，这些来源使用新的方法从构思良好的支架中制造不同的化合物。所有HITS的抗病毒活性将在二次检测中确认，以直接测量对RSV的抑制。研究人员将确定具有最有希望的化学和生物学特征的化合物作为潜在的抗RSV药物。申请者还计划通过生化和遗传方法确定抑制化合物的目标。

DESCRIPTION (provided by applicant): The goal of this project is to use an infection-independent cell-based assay to identify compounds that inhibit respiratory syncytial virus (RSV). The basis of this bioassay is expression of a reporter gene from an artificial viral genome present within the cytoplasm of transfected cells. Significant advances have been made in identifying the cis-acting elements and trans-acting factors involved in replication of many pathogenic negative-strand RNA viruses including RSV. Negative-strand RNA viruses include some of the most notorious human pathogens and cause significant worldwide morbidity and mortality due to severe respiratory disease and hemorrhagic fever syndromes. Unfortunately very few therapeutic agents are available to treat infection with these viruses. Efforts to find new antiviral agents will require methods that are amenable to HTS. In this application the investigators describe their plans to use novel cell-based assays to identify compounds with anti-RSV activity. To identify 'hits' that inhibit replication of RSV the applicants plan to screen a unique library of natural compounds and several novel libraries of synthetic compounds. Apath's partner has developed a proprietary system for making compound libraries from plant-based material. High abundance compounds that lack drug like properties are removed, and the remaining compounds are chromatographically fractionated into distinct peaks, which are provided to Apath, LLC as arrays in bar-coded 96-well plates. Synthetic chemical libraries have been obtained from commercial sources that use novel approaches to make diverse compounds from well-conceived scaffolds. Antiviral activity of all 'hits' will be confirmed in secondary assays to measure inhibition of RSV directly. The investigators will identify compounds with the most promising chemical and biological profiles to pursue as potential anti-RSV drugs. The applicants also plan to identify the targets of the inhibitory compounds through biochemical and genetic approaches.