Project 6 - Development of Antivirals against Alphaviruses

甲病毒是有包膜的正义RNA病毒，包括许多重要的人类病原体，如 如致关节炎甲病毒基孔肯雅病毒（CHIKV）、马亚罗病毒和罗斯河病毒，和 脑炎甲病毒，如东方和委内瑞拉马脑炎病毒。这些病毒具有 作为世界范围的公共卫生和/或生物防御威胁出现，但迄今为止没有许可的疫苗， 抗病毒治疗。AC/DC的项目6旨在开发口服直接作用的抗病毒药物， 甲病毒感染我们专注于靶向RNA复制复合体，它是由协调的 四种甲病毒非结构蛋白（nsP 1 -4）的活性。我们现有的初步数据 化学资产证明通过核苷和CHIKV RNA复制的抑制剂抑制CHIKV RNA复制。 nsP 2的必需蛋白酶活性此外，用我们的核糖核苷EIDD-2749预防性治疗小鼠， 预防CHIKV病毒血症和疾病。最后，我们将在这些发现的基础上通过以下目标再接再厉 与AC/DC核心的合作： 1.优化和表征先前鉴定的核苷抑制剂EIDD-2749、EIDD-2749的抑制作用。 2997，和从AC/DC SAR研究开发的另外的前药/类似物。我们将定义他们的机制， 使用我们的可用蛋白质，细胞为基础的，和病毒感染检测面板的行动。我们将决定 并确定耐药性特征和对细胞中病毒复制的影响 文化 2.使用基于细胞的复制子进行CHIKV RNA复制抑制剂的高通量筛选 报告人制度将与Cores B和C合作，将其进展到临床前开发， 目标1中定义的机制。 3.优化和表征nsP 2蛋白酶的抑制剂。我们将优化和进一步发展我们最初的 nsP 2蛋白酶抑制剂，并使用基于细胞的筛选来鉴定另外的nsP 2蛋白酶抑制剂。我们将 通过使用无细胞nsP 2酶测定和病毒感染，以及通过应用 目标1和2所述的战略。 4.表征体内功效。我们将使用已建立的小鼠模型来确定 EIDD-2749和来自针对急性和慢性CHIKV感染和疾病的目的1-3的早期线索。我们还将 开发一种具有整合的甲病毒微型基因组模板的新型报告小鼠系， 甲病毒介导的RNA复制具有高特异性和灵敏度。这一战略将用于遵循 通过未修饰的甲病毒感染，以在CHIKV的急性和慢性阶段期间鉴定靶细胞 感染，并评估本提案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.