Coronavirus antiviral nucleoside analogs: inhibition and reduced susceptibility

冠状病毒抗病毒核苷类似物：抑制和降低敏感性

• 批准号: 9763429
• 负责人: Maria Agostini
• 金额: $ 0.49万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2019-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9763429
• 关键词: 5' -exoribonuclease; Adenosine; Affect; Antiviral Agents; Antiviral Therapy; Blood Circulation; Collaborations; Common Cold; Complex; Coronavirus; Coronavirus Infections; Defective Viruses; Development; Disease; Excision; Exons; Exoribonucleases; Family; Fingers; Future; Genome; Goals; HIV; Health; Hepatitis B Virus; Hepatitis C virus; Herpesviridae; Human; Individual; Infection; Institutes; Interruption; Life Cycle Stages; Lung diseases; Mediating; Mediator of activation protein; Middle East Respiratory Syndrome Coronavirus; Modeling; Murine hepatitis virus; Mutagenesis; Mutation; Nonstructural Protein; Northern Blotting; Nucleic Acids; Nucleosides; Nucleotides; Polymerase; Population; Predisposition; RNA Viruses; RNA chemical synthesis; RNA replication; RNA-Directed RNA Polymerase; Regulation; Reporting; Research; Ribavirin; Role; Science; Severe Acute Respiratory Syndrome; Syndrome; Testing; Therapeutic; Time; Vaccine Therapy; Vaccines; Viral; Virus; Virus Replication; Zoonoses; antiviral nucleoside analog; clinical development; clinically relevant; combat; cytotoxicity; deep sequencing; drug development; drug discovery; experimental study; fitness; improved; inhibitor/antagonist; insight; mortality; nucleoside analog; pandemic disease; resistance mutation; respiratory; tool; viral RNA; viral fitness

PROJECT SUMMARY Coronaviruses (CoVs) are a family of positive-sense RNA viruses that cause respiratory illnesses in humans ranging from the common cold to severe and lethal disease. The emergence of SARS-coronavirus (CoV) in 2002 and the continued circulation of MERS-CoV emphasize the capacity of CoVs to cause new zoonotic infections with pandemic potential. Despite the high mortality rates of these infections, no therapeutics or vaccines against any CoVs are currently available. Broadly active antiviral nucleoside analogs such as Ribavirin (RBV) are ineffective against CoVs. This limitation is attributed to a unique proofreading exoribonuclease (ExoN) in nonstructural protein 14 (nsp14-ExoN) that aids the RNA-dependent RNA polymerase (RdRp) encoded in nonstructural protein 12 (nsp12-RdRp) in high fidelity replication of these large positive-strand RNA viruses. We have identified two antiviral nucleoside analogs, GS-5734 and EIDD-1931, in collaboration with Gilead Sciences and the Emory Institute for Drug Development, respectively, which are broadly active against multiple CoVs with minimal cytotoxicity. In addition, we have identified two mutations within the predicted fingers domain of the nsp12-RdRp that reduce susceptibility to GS-5734, a C-adenosine nucleoside analog. The goals of this proposal are to define mechanisms through which these antiviral compounds inhibit CoV replication and determine the impact of resistance mutations on viral fitness, replication fidelity, and nucleotide selectivity. In Specific Aim 1, the basis of GS-5734 and EIDD-1931-mediated inhibition of CoV replication will be defined using deep sequencing, RT-qPCR, and Northern blot analysis to distinguish between the two most common mechanisms of antiviral action displayed by nucleoside analogs: chain termination and lethal mutagenesis. Experiments proposed in Specific Aim 2 will determine the impact of mutations that reduce susceptibility to GS-5734 and EIDD-1931 on coronavirus replication fidelity, viral fitness, and susceptibility to other nucleoside analogs. Together, these studies will probe mechanisms of GS-5734 and EIDD-1931 inhibition of CoV replication and explore the potential for these antiviral nucleoside analogs to individually and cooperatively serve as potent therapies against existing and emerging CoVs. This research also will inform the development of broadly active and complementary antiviral approaches to combat CoV infections. Finally, these studies will utilize GS-5734 and EIDD-1931 as tools to better understand mechanisms and viral mediators of CoV replication efficiency and fidelity.

项目摘要 冠状病毒（Coronaviruses，CoV）是一种引起人类呼吸道疾病的正义RNA病毒家族 从普通感冒到严重致命的疾病。SARS冠状病毒（CoV）的出现 2002年和中东呼吸综合征冠状病毒的持续传播强调了冠状病毒引起新的人畜共患病的能力， 可能导致大流行的感染。尽管这些感染的死亡率很高，但没有治疗方法或 针对任何CoV的疫苗目前都是可用的。广泛活性的抗病毒核苷类似物，例如 利巴韦林（RBV）对CoV无效。这种局限性是由于一种独特的校对 非结构蛋白14（nsp 14-ExoN）中的核糖核酸外切酶（ExoN），有助于RNA依赖性RNA 非结构蛋白12（nsp 12-RdRp）编码的聚合酶（RdRp）在这些大的 正链RNA病毒。我们已经鉴定了两种抗病毒核苷类似物，GS-5734和EIDD-1931， 分别与吉利德科学公司和埃默里药物开发研究所合作， 对多种冠状病毒具有广泛的活性，细胞毒性最小。此外，我们还发现了两种突变， 在nsp 12-RdRp的预测指状结构域内，降低对GS-5734的敏感性，C-腺苷 核苷类似物该提案的目标是确定这些抗病毒药物 化合物抑制CoV复制并确定抗性突变对病毒适应性、复制 保真度和核苷酸选择性。在特异性目的1中，GS-5734和EIDD-1931介导的抑制的基础 将使用深度测序、RT-qPCR和北方印迹分析来确定CoV复制的百分比，以区分 在核苷类似物显示的两种最常见的抗病毒作用机制之间： 终止和致死诱变。具体目标2中提出的实验将确定 降低GS-5734和EIDD-1931对冠状病毒复制保真度，病毒适应性， 和对其他核苷类似物的敏感性。总之，这些研究将探索GS-5734和 EIDD-1931抑制CoV复制，并探索这些抗病毒核苷类似物的潜力， 单独和合作作为有效的疗法对现有的和新出现的冠状病毒。本研究 还将为开发广泛有效和互补的抗病毒方法以对抗CoV提供信息 感染.最后，这些研究将利用GS-5734和EIDD-1931作为工具，以更好地了解机制 以及CoV复制效率和保真度的病毒介质。