Discovery of SARS-CoV-2 antivirals using a replicon assay

项目总结 严重急性呼吸综合征冠状病毒2(SARS-CoV-2)导致人类严重疾病(COVID- 19)这对全球公共卫生构成了重大威胁。自2019年12月首次报道以来，新冠肺炎已经 成为一种继续蔓延的流行病，截至目前，已有2.46亿确诊病例和500万死亡病例 2021年11月02日除了人类悲剧之外，由大流行引发的全球经济内爆的规模 经济是巨大的。尽管现在有疫苗可用，但其效力似乎随着 传播病毒株。Remdevir是唯一被批准的针对SARS-CoV-2的抗病毒药物，但对 新冠肺炎死亡率。因此，确定和开发更多的抗病毒药物来对抗病毒感染至关重要。 以下强有力的初步数据使SARS-CoV-2成为靶向：1)构建广泛的 SARS-CoV-2复制子系统集合，支持尖端、快速和经济的高通量 放映。2)稳定表达SARS-CoV-2复制子的细胞系的建立。3)初步筛选 化学图书馆导致发现了一种新的SARS-CoV-2抗病毒药物，该药物已经得到了改进 通过药物化学努力进行一轮优化。4)单基因组或单基因组的多重可视化 亚基因组(+)或(-)SARS-CoV-2RNA以及同时存在于单个感染细胞中的病毒和/或宿主蛋白。 5)尖端快速高通量传染性病毒BSL3检测，使动力学、机械性、药物 耐药性研究。6)10个SARS-CoV-2非结构蛋白的克隆、表达和纯化 (NSPS)。7)用生化和生物物理方法测定几种SARS-CoV和 SARS-CoV-2 NSP蛋白，并测定化合物与NSP的结合。 我们假设我们最近开发的复制子系统可以用于发现抗SARS- CoV-2 Hits，经过点击到领先的优化，可以成为新冠肺炎的候选药物。要解决这个问题 假设，我们提出了以下具体目标： 1.利用SARS-CoV-2复制子载体系统和SARS-CoV-2复制子表达细胞株进行筛选 抗病毒药物的化学库。 2.优先使用抑制剂进行抑制和抗药性研究。 3.点击到领先的优化 这些研究将导致具有强大效力和药代动力学特征的抗病毒药物，为 SARS-CoV-2抗病毒药物和联合治疗的进展。

PROJECT SUMMARY Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) cause severe diseases in humans (COVID- 19) that presents a major threat for global public health. Since it was first reported in 12/2019, COVID-19 has become a pandemic that continues to spread, with >246 million confirmed cases and >5 million deaths as of 11/02/2021. In addition to the human tragedy, the magnitude of the pandemic-driven implosion of global economies is enormous. Although vaccines are now available, their efficacy appears to be reduced with spreading viral strains. Remdesivir is the only approved antiviral targeting SARS-CoV-2, but it has little effect on COVID-19 mortality. Therefore, it is critical to identify and develop additional antivirals to combat viral infection. The following strong preliminary data enable targeting of SARS-CoV-2: 1) Construction of an extensive collection of SARS-CoV-2 replicon systems that enable cutting-edge, rapid, and economical high-throughput screening. 2) Preparation of cell lines that stably express SARS-CoV-2 replicon. 3) Preliminary screening of chemical libraries led to the discovery of a novel SARS-CoV-2 antiviral, which has already been improved with one round of optimization through medicinal chemistry efforts. 4) Multiplex visualization of single-genomic or subgenomic (+) or (-) SARS-CoV-2 RNA and simultaneously viral and/or host proteins in individual infected cells. 5) Cutting-edge rapid high-throughput infectious virus BSL3 assays that enable kinetic, mechanistic, drug resistance studies. 6) In-house cloning expression and purification of 10 SARS-CoV-2 non-structural proteins (nsps). 7) Biochemical and biophysical assays to measure the enzymatic activities of several SARS-CoV and SARS-CoV-2 nsp proteins and to measure compound binding to nsps. We hypothesize that our recently developed replicon systems can be used for the discovery of anti-SARS- CoV-2 hits, which upon hit-to-lead optimization can become COVID-19 drug candidates. To address this hypothesis, we propose the following specific aims: 1. Use of SARS-CoV-2 replicon plasmid systems and SARS-CoV-2 replicon-expressing cell lines to screen chemical libraries for antiviral hits. 2. Inhibition and resistance studies with prioritized inhibitors. 3. Hit-to-lead optimization These studies will lead to antivirals with strong potency and pharmacokinetic profiles, setting the stage for development of SARS-CoV-2 antivirals and combination therapies.