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，例如 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，以及 脑炎甲病毒，例如东部和委内瑞拉马脑炎病毒。这些病毒有 已成为世界范围内的公共卫生和/或生物防御威胁，但迄今为止还没有获得许可的疫苗或疫苗 抗病毒疗法。 AC/DC 中的项目 6 旨在开发口服直接作用抗病毒药物 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 (nsP1-4) 的活性。我们现有的初步数据有希望 化学资产证明核苷和抑制剂可抑制 CHIKV RNA 复制 nsP2 的必需蛋白酶活性。此外，用我们的核糖核苷 EIDD-2749 对小鼠进行预防性治疗 预防 CHIKV 病毒血症和疾病。我们将通过以下目标，以这些发现为基础，密切关注 与 AC/DC 核心的协作： 1. 优化并表征先前鉴定的核苷抑制剂EIDD-2749、EIDD-的抑制作用 2997，以及根据 AC/DC SAR 研究开发的其他前药/类似物。我们将定义他们的机制 使用我们可用的蛋白质、基于细胞和病毒感染检测的面板来采取行动。我们将确定宽度 对其他甲病毒的抑制并确定耐药性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对 CHIKV RNA 复制抑制剂进行高通量筛选 记者系统。命中将与核心 B 和 C 合作进入临床前开发，并且 机制如目标 1 中定义。 3. nsP2 蛋白酶抑制剂的优化和表征。我们将优化并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鉴定其他 nsP2 蛋白酶抑制剂。我们会 通过使用无细胞 nsP2 酶测定和病毒感染以及应用 目标 1 和 2 中描述的策略。 4.表征体内功效。我们将使用已建立的小鼠模型来确定其体内功效 EIDD-2749 和针对急性和慢性 CHIKV 感染和疾病的目标 1-3 的早期线索。我们还将 开发一种新型报告小鼠系，具有集成的甲病毒小基因组模板，可以检测 甲病毒介导的 RNA 复制具有高特异性和敏感性。该策略将用于遵循 未经修饰的甲病毒感染，以识别 CHIKV 急性期和慢性期的靶细胞 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.