Identification and Characterization of Novel Flavivirus Antivirals

RNA病毒会引起各种新出现的、与生物防御相关的、对公共卫生具有重要意义的疾病。这个 病毒来自多个家庭，引起的疾病也是多种多样的。其中包括西尼罗河 由黄病毒、委内瑞拉马脑炎和基孔肯雅热引起的脑炎和登革热 由甲型病毒引起的甲型病毒和由本亚病毒引起的裂谷热。目前治疗这些疾病的方法 抗病毒药物是有限的，确实需要开发有效的小分子抗病毒药物。中环 这一五年计划的目标是通过发现和开发新的抗病毒药物来满足这一需求 对医学上重要的RNA病毒的活性，特别强调广泛活跃的候选者。 在目标1中，我们将从不同的化学线索中开发出对黄病毒具有活性的化合物，这些化学线索由 使用西尼罗河病毒复制子颗粒(VRP)分析的高通量筛查(HTS)，可以 在低生物遏制条件下使用。这些化合物将经历一个迭代程序，其中包括 几轮化学修饰，以及对黄病毒的体外毒性和抗病毒活性测试，将 结果鉴定了改进的抗病毒药物。在目标2中，我们将把一种甲型病毒VRP试验发展成一种 HTS-适合的格式，并使用此测试来筛选先导化合物，然后将使用这些化合物来启动 使用目标1中描述的范例的甲型病毒抗病毒药物的发现和开发计划。在目标3中，我们 将开发一种HTS检测方法，利用一种表达报告基因的裂谷热减毒病毒 启动一个本亚病毒抗病毒计划。在每个目标中，活动将使用多个病毒进行评估 科，以鉴定具有广泛活性的化合物。这一进程将通过评估Active继续进行 化合物对其他感兴趣的病毒家族成员的作用。在这样做的过程中，我们认识到早期 其中许多病毒引起的临床症状很难区分；因此，广谱抗病毒药物 可以在感染早期使用，当它们有最大机会产生临床影响时是临床上的 优先考虑。伴随药物发现研究，在目标4中，我们将探索其作用机制。 化合物，产生抗药性突变以定义它们发生的频率，并确定 抗性机制。此外，我们还将探索活性化合物组合使用的效果， 试图确定具有潜在未来临床实用价值的协同组合。最后，在目标5中，我们将 在黄病毒病小鼠模型中评估两种有希望的化合物。

RNA viruses cause a variety of emerging and biodefense-related diseases of public health importance. The viruses come from multiple families, and the diseases they cause are diverse. They include West Nile encephalitis and dengue fever both caused by flaviviruses, Venezuelan equine encephalitis and chikungunya caused by alphaviruses and Rift Valley fever caused by a bunyavirus. Current therapies for these diseases are limited, and there is a real need to develop effective small molecule antiviral agents. The central objectives of this five year project are to meet this need by identifying and developing new antivirals with activity against medically important RNA viruses giving particular emphasis to broadly active candidates. In aim 1 we will develop compounds active against flaviviruses from diverse chemical leads identified by high throughput screening (HTS) using a West Nile virus virus-replicon particle (VRP) assay that can be used under low biocontainment conditions. The compounds will undergo an iterative program with multiple rounds of chemical modifications, and in vitro toxicity and antiviral activity testing against flaviviruses that will result in the identification of improved antivirals. In aim 2 we will develop an alphavirus VRP assay into a HTS-suitable format and use this assay to screen for lead compounds that will then be used to initiate an alphavirus antiviral discovery and development program using the paradigm described in aim 1. In aim 3 we will develop an HTS assay that utilizes an attenuated Rift Valley fever virus that expresses a reporter gene to initiate a bunyavirus antiviral program. In each aim activity will be evaluated using multiple viruses within the family, to identify compounds with broad activity. This process will be continued by evaluating active compounds against members of the other virus families of interest. In doing this we recognize that the early clinical signs caused by many of these viruses are difficult to distinguish; thus, broad spectrum antivirals that could be used early in infection when they would have the best chance of clinical impact are a clinical priority. Concomitant with the drug discovery studies, in aim 4 we will explore the mechanism of action of compounds, generate resistant mutations to define the frequency with which they occur, and determine the mechanism of resistance. In addition, we will explore the effects of using active compounds in combination, in attempts to identify synergistic combinations with potential future clinical utility. Finally in aim 5 we will evaluate two promising compounds in vivo in mouse models of flavivirus diseases.