Project 6 - Development of Antivirals against Alphaviruses

甲型病毒是包膜正义RNA病毒，包括许多重要的人类病原体，如 作为致关节炎的甲型病毒基孔肯雅病毒(CHIKV)、马亚罗病毒和罗斯河病毒，以及 东部和委内瑞拉马脑炎病毒等脑型甲型病毒。这些病毒有 出现了世界性的公共卫生和/或生物防御威胁，但到目前为止还没有获得许可的疫苗或 抗病毒疗法。AC/DC项目6寻求开发口服可用直接作用抗病毒药物 甲型病毒感染。我们专注于靶向RNA复制复合体，它是由协调的 四种甲型病毒非结构蛋白(nsP1-4)的活性。有希望的初步数据与我们现有的 化学资产显示核苷和CHIKV核糖核酸抑制剂对CHIKV RNA复制有抑制作用 NsP2必需的蛋白酶活性。此外，我们的核糖核苷EIDD-2749对小鼠的预防性治疗 预防了CHIKV病毒血症和疾病。我们将通过以下目标在这些发现的基础上再接再厉 与AC/DC核心协作： 1.优化和表征已鉴定的核苷抑制剂EIDD-2749、EIDD-1的抑制作用。 2997，以及从AC/DC SAR研究中开发的其他前体药物/类似物。我们将定义它们的机制 使用我们可用的蛋白质、基于细胞的和病毒感染分析小组采取行动。我们将确定广度 对其他甲型病毒的抑制作用，并确定耐药谱和对细胞内病毒复制的影响 文化。 2.使用基于细胞的复制子高通量筛选CHIKV RNA复制抑制物 记者制度。HITS将与B和C核心合作进行临床前开发，以及 目标1中定义的机制。 3.对nsP2蛋白水解酶抑制剂进行优化和表征。我们将优化和进一步发展我们最初的 NsP2蛋白酶抑制剂，并使用基于细胞的筛选来识别其他nsP2蛋白酶抑制剂。我们会 通过使用无细胞的nsP2酶分析和病毒感染以及通过应用 目标1和目标2所述的战略。 4.体内药效的表征。我们将使用已建立的小鼠模型来确定其在体内的疗效 EIDD-2749和AIMS 1-3的早期领先药物，用于治疗急性和慢性CHIKV感染和疾病。我们还将 开发一种新型报告鼠系，其整合的甲病毒微基因组模板可以检测到 甲型病毒介导的RNA复制具有高度的特异性和敏感性。这一战略将被用来遵循 在CHIKV急性期和慢性期通过未经修饰的甲型病毒感染来识别靶细胞 感染，并评估在这项建议中开发的抗病毒疗法。

Alphaviruses are enveloped plus-sense RNA viruses that include a number of important human pathogens such as the arthritogenic alphaviruses chikungunya virus (CHIKV), Mayaro virus, and Ross River virus, and encephalitic alphaviruses such as Eastern and Venezuelan equine encephalitis viruses. These viruses have emerged as world-wide public health and/or biodefense threats, but to date there are no licensed vaccines or antiviral therapies. Project 6 in the AC/DC seeks to develop orally available direct-acting antivirals against alphavirus infection. We focus on targeting the RNA replication complex, which is formed by the coordinated activities of the four alphavirus non-structural proteins (nsP1-4). Promising preliminary data with our existing chemical assets demonstrate inhibition of CHIKV RNA replication by nucleosides and by inhibitors of the essential protease activity of nsP2. Moreover, prophylactic treatment of mice with our ribonucleoside EIDD-2749 prevented CHIKV viremia and disease. We will build on these findings through the following Aims, in close collaboration with the AC/DC Cores: 1. Optimize and characterize inhibition by the previously identified nucleoside inhibitors EIDD-2749, EIDD- 2997, and additional prodrugs/analogs developed from AC/DC SAR studies. We will define their mechanism of action using our panel of available protein, cell-based, and virus infection assays. We will determine the breadth of inhibition across other alphaviruses and determine resistance profiles and effects on virus replication in cell culture. 2. Perform high throughput screening for inhibitors of CHIKV RNA replication using a cell-based replicon reporter system. Hits will be progressed to preclinical development in collaboration with Cores B and C, and mechanisms defined as in Aim 1. 3. Optimize and characterize inhibitors of the nsP2 protease. We will optimize and further develop our initial inhibitors of nsP2 protease, and use a cell-based screen to identify additional nsP2 protease inhibitors. We will define their mechanisms by using cell-free nsP2 enzymatic assays and virus infection, as well as by applying the strategies described in Aims 1 and 2. 4. Characterize in vivo efficacy. We will use established mouse models to determine the in vivo efficacy of EIDD-2749 and early leads from Aims 1-3 against acute and chronic CHIKV infection and disease. We will also develop a novel reporter mouse line with an integrated alphavirus minigenome template that can detect alphavirus-mediated RNA replication with high specificity and sensitivity. This strategy will be used to follow infection by unmodified alphaviruses, to identify target cells during the acute and chronic phases of CHIKV infection, and to evaluate antiviral therapies developed in this proposal.