Project 1 - Coronavirus

项目摘要 人畜共患冠状病毒（CoV），如SARS-CoV和MERS-CoV是大流行的威胁。MERS-CoV 继续造成新的人畜共患和人类传播疾病，死亡率约为35%。目前有 没有FDA批准的治疗冠状病毒的疗法新的人畜共患病CoV可能会从异质病毒中出现 因此，需要针对完全保守和脆弱的 目标的CoV快速选择对多种类型的抑制剂的抗性，表明需要 防止出现耐药性的方法。SARS和MERS感染都表现为严重的 免疫病理学损伤，可能限制直接作用抗病毒药物（DAA）的治疗窗口。 在没有抗病毒药物的情况下，免疫调节已被证明是无益的，甚至会加剧 SARS和MERS疾病因此，DAA和靶向免疫调节剂的组合可能是必要的， 有效治疗已建立的感染。我们项目的总体目标是开发抗冠状病毒药物 广泛抑制已知和未来潜在大流行的人畜共患病CoV的策略， 并通过靶向宿主免疫病理反应来延长治疗窗口。的 拟议的研究将推进CoV抑制性核苷类似物EIDD的临床前开发， 1931/2801和其他核苷类似物在管道和测试两个小分子命中确定为高度 有效对抗SARS-CoV，用于治疗和预防流行性和突发前的CoV。具体目标1、 将定义化合物的抗病毒活性谱和治疗功效。抗病毒功效， 将在原代人肺细胞培养物中测定每种化合物的代谢和细胞毒性 被SARS-CoV和MERS-CoV攻击。先导化合物的预防和治疗功效将是 在SARS和MERS发病机制的年轻、老年和免疫抑制小鼠模型中进行评估。在 具体目标2，将确定先导化合物的作用机制和耐药性动力学。 将评估化合物对病毒复制、保真度和先天免疫诱导的抗病毒作用。 MERS-CoV和SARS-CoV基因组中的耐药突变在存在增加的情况下传代 药物浓度将通过深度测序来确定。耐药性对SARS-CoV和MERS的影响- 将确定CoV毒力、对其他药物的敏感性和先导化合物的治疗效果。 具体目标3将侧重于开发治疗新冠病毒的联合方案。的 DAA对野生型和耐药SARS-CoV感染的联合治疗效果， MERS-CoV将使用培养的细胞和小鼠进行定义。联合DAA治疗的疗效 将在SARS和MERS的小鼠模型中评估免疫调节剂的作用。这些研究将产生 IND申请和人体临床试验发起所需的机制和疗效数据。

PROJECT SUMMARY Zoonotic coronaviruses (CoVs) such as SARS-CoV and MERS-CoV are pandemic threats. MERS-CoV continues to cause new zoonotic and human transmission and illness with ~35% mortality. Currently, there are no FDA-approved therapies to treat any CoV. New zoonotic CoVs likely will emerge from heterogeneous virus pools in animal reservoirs, thus requiring antiviral strategies aimed at completely conserved and vulnerable targets. CoVs rapidly select for resistance to multiple classes of inhibitors, demonstrating the need for approaches to prevent resistance emergence. Both SARS and MERS infections manifest as severe immunopathologic damage, potentially limiting the therapeutic window for direct-acting antivirals (DAAs). Immunomodulation in the absence of antivirals has been shown to not be beneficial and to even exacerbate SARS and MERS disease. Thus, combinations of DAAs and targeted immunomodulators may be necessary for effective treatment of established infection. The overall goal of our program is to develop CoV antiviral strategies that broadly inhibit known and future potential pandemic zoonotic CoVs, prevent emergence of resistance, and extend the therapeutic window by targeting host immunopathologic responses. The proposed research will advance preclinical development of the CoV-inhibitory nucleoside analogue EIDD- 1931/2801 and other nucleoside analogues in the pipeline and test two small-molecule hits identified as highly active against SARS-CoV for treatment and prevention of epidemic and pre-emergent CoVs. In Specific Aim 1, the spectrum of antiviral activity and therapeutic efficacy of compounds will be defined. The antiviral efficacy, metabolism, and cytotoxicity of each compound will be determined in cultures of primary human lung cells targeted by SARS-CoV and MERS-CoV. The prophylactic and therapeutic efficacy of lead compounds will be evaluated in young, aged, and immunosuppressed murine models of SARS and MERS pathogenesis. In Specific Aim 2, the mechanism of action of lead compounds and kinetics of drug resistance will be determined. The antiviral effect of compounds on virus replication, fidelity, and induction of innate immunity will be assessed. Resistance mutations in genomes of MERS-CoV and SARS-CoV passaged in the presence of increasing concentrations of drug will be determined by deep sequencing. The impact of resistance on SARS-CoV and MERS- CoV virulence, sensitivity to other drugs, and therapeutic efficacy of lead compounds will be determined. Specific Aim 3 will focus on the development of combination regimens for the treatment of emerging CoVs. The combined therapeutic efficacy of DAAs against infections with both wild-type and drug-resistant SARS-CoV and MERS-CoV will be defined using cultured cells and mice. The therapeutic effect of treatment combining a DAA with an immunomodulator will be assessed in mouse models of SARS and MERS. These studies will generate mechanistic and efficacy data necessary for IND filing and origination of human clinical trials.