Identifying inhibitors of West Nile and dengue viruses

描述（由申请人提供）：西尼罗河（WN）和登革热（DEN）病毒是新出现的黄病毒和潜在的生物防御病原体，可引起重大人类疾病。目前没有疫苗或有效的化疗可用于人类WN和DEN感染。本项目的目的是鉴定WN和DEN病毒的新型抑制剂。我们将实现以下两个临时目标。(1)开发新的高通量检测方法来筛选WN和DEN复制抑制剂。利用表达报告基因的亚基因组WN和DEN复制子，我们将开发报告细胞系，可用于高通量筛选针对所有涉及病毒复制的靶标的抑制剂。作为一个原理证明，我们已经建立了这样一个高通量筛选WN抑制剂的报告细胞系。我们将开发一种类似的高通量检测方法来发现抗den药物。(2)确定新的WN和DEN抑制剂类别，并分析其作用模式。通过与ViroPharma公司的合作，我们将通过筛选大量化合物文库，使用高通量分析来鉴定新的WN和DEN抑制剂类别。我们将通过(i)检查化合物是否可以抑制黄病毒的其他成员，以及（ii）通过生化（酶测定）和遗传方法（选择化合物抗性病毒）分析化合物的作用模式来研究潜在的抑制剂。在本应用中提出的高通量测定方法已被证明在抗病毒药物发现（如丙型肝炎病毒）方面是富有成效的。在这些研究完成后，我们期望能够鉴定出WN和DEN的新型抑制剂，并确定这些抑制剂的作用模式。这些结果将具有重要意义，因为它们将为开发治疗WN和DEN感染的有效化疗奠定基础。本项目获得的报告细胞系也将在黄病毒基因组包装和病毒颗粒形成等方面的研究中发挥重要作用。

DESCRIPTION (provided by applicant): West Nile (WN) and dengue (DEN) viruses are emerging flaviviruses and potential biodefense pathogens that cause significant human diseases. Neither vaccine nor effective chemotherapy is currently available for WN and DEN infections in humans. The objective of this project is to identify novel inhibitors of WN and DEN viruses. We will accomplish the following two interim objectives. (1) Develop novel high-throughput assays to screen for inhibitors of WN and DEN replication. Using subgenomic WN and DEN replicons expressing reporter genes, we will develop reporting cell lines that can be used for high-throughput screening of inhibitors against all targets involved in viral replication. As a proof-of-principle, we have already established such a reporting cell line for high-throughput screening of WN inhibitors. We will develop a similar high-throughput assay for anti-DEN drug discovery. (2) Identify new classes of inhibitors of WN and DEN and analyze their modes of action. In collaboration with ViroPharma, Inc., we will use the high-throughput assays to identify new classes of WN and DEN inhibitors through screening a large number of compound libraries. We will study the potential inhibitors by (i) examining whether the compounds can inhibit other members of the flaviviruses, and (ii) analyzing the modes of action of the compounds through both biochemical (enzyme assays) and genetic approaches (selection of compound-resistant viruses). The high-throughput assay-approach proposed in this application has been proven to be fruitful in antiviral drug discovery (e.g. hepatitis C virus). At the completion of these studies, we expect to have identified novel inhibitors of WN and DEN, and to have determined the modes of action of these inhibitors. These results will be significant, because they will form a foundation for development of an efficacious chemotherapy of WN and DEN infections. The reporting cell lines developed in this project will also be useful for many aspects of flavivirus research such as studying genome packaging and viral particle formation.