A CPE-Based HTS Assay for Antiviral Drug Screening Against Dengue Virus

描述（由申请人提供）：登革热病毒（DENV），包括四种血清型，DENV-1, -2， -3和-4，是黄病毒科的成员，被列为NIAID a类优先病原体之一。由于缺乏疫苗和抗病毒药物，登革热现在是世界各地的一个主要公共卫生负担。到目前为止，还没有开发出高通量筛选（HTS）适用、稳健和可靠的方法来筛选NIH分子文库小分子库（MLSMR）对DENV的抗性，这阻碍了抗病毒药物的发现和开发。在此，我们开发、优化并验证了一种基于cpe的、适用于HTS的DENV-2检测方法，该方法在96孔板上的Z′值为0.71，变异系数（CV）为6.3%，信本比（S/B）为6.88。该实验也被缩小为384孔板格式，具有相当的分析稳健性。我们还使用药理学活性化合物文库（LOPAC1280, Sigma-Aldrich, St. Louis， MO）验证了该分析。该试验现在准备转移到指定的分子文库探针生产中心网络（MLPCN）来筛选NIH MLSMR。所有来自初级HTS的“命中”将使用现有的测定方法进一步确认、验证和优先排序，包括剂量-反应验证法、基于复制的二级测定法和添加时间测定法。我们的长期目标是开发针对人类登革热的新型抗病毒药物。本申请的目标将通过执行以下两个特定目标来实现：特定目标1：将针对DENV-2的HTS适用的、基于cpe的检测转移到指定的HTS中心，以筛选NIH MLSMR。具体目标2：使用包括剂量-反应试验、基于复制的试验和添加时间试验在内的分析来确认、验证和优先考虑来自初级HTS的命中。

DESCRIPTION (provided by applicant): Dengue virus (DENV), including the four serotypes, DENV-1, -2, -3, and -4, is a member in the family of Flaviviridae and listed as one of the NIAID Category A priority pathogens. Due to the lack of vaccine and antiviral drugs, dengue disease is now a major public health burden around the world. Up-to-date, there was no high throughput screening (HTS) amenable, robust and reliable assay(s) developed to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR) against DENV, which hindered the discovery and development of antiviral drugs. Here, we showed the development, optimization and validation of a HTS amenable, CPE-based assay against DENV-2, with Z'-value of 0.71, coefficient of variation (CV) of 6.3% and signal-to-background (S/B) ratio of 6.88 in 96-well plate. This assay was also miniaturized into 384- well plate format with comparable assay robustness. We have also validated the assay using the Library of Pharmacologically Active Compounds (LOPAC1280, Sigma-Aldrich, St. Louis, MO). This assay is now ready to be transferred to the designated Molecular Libraries Probe Production Centers Network (MLPCN) to screen the NIH MLSMR. All "hits" from the primary HTS will be further confirmed, validated and prioritized using assays available including the dose-response confirmatory assay, replicon-based secondary assay, and time-of-addition assay. Our long term goal is to develop novel antivirals against dengue disease in humans. The objectives of this application will be achieved by carrying out the following two Specific Aims: Specific Aim 1: To transfer the HTS amenable, CPE-based assay against DENV-2 to the designated HTS center to screen the NIH MLSMR. Specific Aim 2: To confirm, validate and prioritized hits from the primary HTS using assays including the dose-response assay, the replicon-based assay and time-of-addition assay. PUBLIC HEALTH RELEVANCE: Dengue virus (DENV) is a member in the family of Flaviviridae and one of the NIAID category A priority pathogens. With more than one-third of the world's population living in areas at risk for transmission, dengue infection is a leading cause of illness and death in the tropics and subtropics. As many as 100 million people are infected yearly, with an estimated 500,000 cases of severe dengue requiring hospitalization, of which a very large proportion is in children, with case fatality up to 5%. In the Americas as of December 14, 2009, there were 893,453 reported cases, including 23,980 Dengue hemorrhagic fever (DHF) cases and 370 deaths. Currently, there is no efficient vaccine and effective anti-viral drugs against Dengue diseases, which makes Dengue disease a huge public health burden.