Broad-spectrum inhibitors targeting the viral replication promoter conserved in dengue and Zika viruses

项目摘要 寨卡病毒(ZIKV)和登革热病毒(DENV)是由蚊子传播的黄病毒，可导致多种疾病 严重程度从轻微症状到致命的出血性登革热。ZIKV在五年前迅速传播 南美洲和中美洲的20多个国家，ZIKV感染达到大流行水平， 威胁要扩张到美国南部。在过去的两年中，DENV的威胁急剧增加 几十年来，它是热带地区最严重的蚊媒病原体之一。应对新城疫暴发疫情 寨卡病毒流行地区，反之亦然，针对蚊媒黄病毒的广谱抑制药物 将提供一种有效的治疗方法，降低免疫相关恶化的风险， 例如，在以前感染或接种疫苗的患者中。 DENV和ZIKV临床分离株序列比对及二级结构预测 使我们发现了一个保守的RNA三向连接，它在 黄病毒。在这里，我们建议通过靶向启动子rna来鉴定抑制病毒复制的化合物。 作为DENV和ZIKV广谱疗法未来发展的主要候选者。 这项提议的具体目标是：1)开发一种基于FRET的结合分析来评估小分子 作为DENV和ZIKV复制启动子RNA的选择性配体，2)建立体外复制 启动实验鉴定启动子RNA结合配体为ZIKV复制的抑制物，以及3)发现小分子 通过靶向作为病毒的RNA 3WJ基序在细胞培养中抑制DENV和ZIKV的分子 复制促进剂。

Project Summary Zika virus (ZIKV) and dengue virus (DENV) are mosquito-borne flaviviruses that cause disease ranging in severity from mild symptoms to deadly hemorrhagic dengue fever. ZIKV was rapidly spreading five years ago to over 20 countries in South and Central America, where infections by ZIKV reached pandemic levels and threatened to expand to the southern US. The threat by DENV has increased dramatically over the last two decades as one of the worst mosquito-borne pathogens in tropical regions. To treat outbreaks of DENV in ZIKV-endemic regions, and vice versa, broad-spectrum inhibitor drugs against mosquito-borne flaviviruses would provide an efficient therapeutic approach that reduces the risk of immuno-associated exacerbation, for example in previously infected or vaccinated patients. Sequence alignment of DENV and ZIKV clinical isolates in combination with secondary structure prediction have led us to identify a conserved RNA three-way junction that serves as the replication promoter in flaviviruses. Here, we propose to identify compounds that inhibit viral replication by targeting the promoter RNA as lead candidates for the future development of DENV and ZIKV broad-spectrum therapeutics. The Specific Aims of this proposal are to 1) develop a FRET-based binding assay to assess small molecules as selective ligands of the DENV and ZIKV replication promoter RNA, 2) establish an in vitro replication initiation assay to identify promoter RNA-binding ligands as inhibitors of ZIKV replication, and 3) discover small molecules that inhibit DENV and ZIKV in cell culture by targeting the RNA 3WJ motif that serves as the viral replication promoter.