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

描述(申请人提供)：登革病毒(DENV)，包括DENV-1、-2、-3和-4四个血清型，是黄病毒科家族的成员，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，登革热现在是全球主要的公共卫生负担。到目前为止，还没有针对DENV的高通量筛选(HTS)方法(S)，这阻碍了抗病毒药物的发现和发展。在96孔板上，我们建立、优化和验证了一种基于高温气相色谱的抗乙型新城疫病毒的方法，其Z‘值为0.71，变异系数(CV)为6.3%，信号背景比(S/B)为6.88。该分析也被小型化为384孔板格式，具有类似的分析稳定性。我们还使用药理活性化合物库(LOPAC1280，Sigma-Aldrich，St.Louis，MO)验证了该方法。该分析现已准备好转移到指定的分子文库探针生产中心网络(MLPCN)，以筛选NIH MLSMR。来自一次HTS的所有“命中”将使用可用的分析进一步确认、验证和优先排序，包括剂量-反应确证试验、基于复制子的二次试验和添加时间试验。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。这项申请的目标将通过执行以下两个具体目标来实现：具体目标1：将HTS服从的、基于CPE的针对DENV-2的检测转移到指定的HTS中心，以筛选NIH MLSMR。具体目标2：使用包括剂量反应分析、基于复制子的分析和添加时间分析的方法来确认、验证和优先排序来自初级HTS的HITS。 公共卫生相关性：登革病毒(DENV)是黄病毒科家族的成员，也是NIAID A类优先病原体之一。由于世界上三分之一以上的人口生活在有传播风险的地区，登革热感染是热带和亚热带地区疾病和死亡的主要原因。每年有多达1亿人感染，估计有50万严重登革热病例需要住院治疗，其中很大一部分是儿童，病死率高达5%。在美洲，截至2009年12月14日，有893,453例报告病例，包括23,980例登革出血热和370例死亡。目前，还没有针对登革热的有效疫苗和有效的抗病毒药物，这使得登革热成为巨大的公共卫生负担。

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.