Development of fluorogenic and chemiluminogenic main protease substrates for cellular and in vivo imaging of SARS-CoV-2

开发用于 SARS-CoV-2 细胞和体内成像的荧光和化学发光主要蛋白酶底物

• 批准号: 10432895
• 负责人: Wenshe Ray Liu
• 金额: $ 22.46万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10432895
• 关键词: 2019-nCoV; Acute; Adopted; Aldehydes; Animal Model; Animals; Apoptosis; Back; COVID-19; COVID-19 pandemic; COVID-19 prevention; COVID-19 therapeutics; COVID-19 vaccine; Caco-2 Cells; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Chemicals; Communicable Diseases; Coronavirus; Country; Data; Detection; Development; Disease; Drug or chemical Tissue Distribution; Emergency Situation; Enzymes; Genome; Goals; Health Promotion; Human; Hydrolysis; Image; In Vitro; Investigation; Measures; Mission; Mus; Mutate; Nonstructural Protein; Open Reading Frames; Organ; Organism; Paralysed; Pathogenesis; Pattern; Peptide Hydrolases; Peptides; Permeability; Pharmaceutical Preparations; Production; Protocols documentation; Public Health; RNA; RNA Viruses; Reagent; Regulation; Reporting; Reproduction; Research; Research Personnel; Resistance; SARS-CoV-2 infection; Ships; Structure; Testing; Therapeutic; Tissue imaging; Tissues; Transgenic Organisms; Translating; United States National Institutes of Health; Viral; Viral Pathogenesis; Virus; Virus Diseases; Virus Replication; World Health Organization; analog; base; cellular imaging; fluorophore; improved; in vivo imaging; inhibitor; monolayer; non-invasive imaging; pandemic disease; pathogen; pathogenic virus; polypeptide; resorufin; stable cell line; unnatural amino acids

PROJECT SUMMARY COVID-19 is the currently prevailing pandemic that has paralyzed much of the world. The viral pathogen of COVID-19 is SARS-CoV-2, a coronavirus with a positive sense RNA genome. Its production in the human cell host requires the activity of its main protease (MPro). As a unique enzyme that is essential to SARS-CoV-2, MPro is acutely toxic to the host cell indicating that it is tightly regulated during viral infection and replication. Its activity also represents active viral reproduction in organs and tissues in an infected host organism. Therefore, the detection of MPro activity in SARS-CoV-2-infected cells and animals will provide essential information about MPro activity regulation in the host cell and SARS-CoV-2 spreading and distribution in a host organism respectively for the elucidation of SARS-CoV-2 replication and pathogenesis. Noninvasive imaging reagents will be critical for the detection of MPro activity in order to avoid interference with both the virus and the host. With an ultimate goal of understanding the SARS-CoV-2 replication in infected cells and elucidating SARS-CoV-2 spreading patterns and organ/tissue distribution in infected animals, the current proposal aims to develop noninvasive fluorogenic and chemiluminogenic MPro substrates that have highly cellular stability and tissue penetrability for imaging MPro activity. Three specific aims will be pursued by 1) developing fluorogenic and chemiluminogenic MPro substrates; 2) characterizing fluorogenic and chemiluminogenic MPro substrates on their selectivity, cellular stability and tissue penetrability; and 3) conducting cellular and in vivo imaging of SARS-CoV-2 infection. The successful completion of the proposed study will 1) result in fluorogenic and chemiluminogenic MPro substrates with optimal cellular stability and tissue penetratability that can be broadly adopted by COVID-19 researchers for the investigation of SARS-CoV-2 replication and pathogenesis and 2) provide critical information for the elucidation of Mpro activity regulation in SARS-CoV-2-infected cells and SARS-CoV-2 spreading patterns and distribution in transgenic hACE2+ mice.

项目摘要 COVID-19是目前流行的大流行病，已使世界大部分地区陷入瘫痪。病毒病原体 COVID-19是SARS-CoV-2，一种具有正义RNA基因组的冠状病毒。它在人体细胞中的产生 宿主需要其主要蛋白酶（MPro）的活性。MPro作为SARS-CoV-2所必需的一种独特酶， 对宿主细胞具有急性毒性，表明其在病毒感染和复制期间受到严格调控。其活性 也表示在受感染的宿主生物体的器官和组织中活跃的病毒繁殖。因此 在SARS-CoV-2感染的细胞和动物中检测MPro活性将提供关于MPro的基本信息 宿主细胞中的活性调节和SARS-CoV-2在宿主生物体中的传播和分布 用于阐明SARS-CoV-2的复制和发病机制。非侵入性成像试剂将至关重要 用于检测MPro活性，以避免干扰病毒和宿主。优异的最大 目的是了解SARS-CoV-2在感染细胞中的复制并阐明SARS-CoV-2的传播 模式和器官/组织分布在受感染的动物，目前的建议旨在开发非侵入性 具有高度细胞稳定性和组织穿透性的荧光和荧光MPro底物， 成像MPro活动。三个具体目标将追求1）发展荧光和荧光 MPro底物; 2）表征荧光性和荧光性MPro底物的选择性，细胞 稳定性和组织穿透性;和3）进行SARS-CoV-2感染的细胞和体内成像。的 成功完成拟定研究将：1）产生荧光和荧光MPro底物 具有最佳的细胞稳定性和组织穿透性，可被COVID-19研究人员广泛采用， SARS-CoV-2复制和致病机制的研究; 2）为研究 阐明SARS-CoV-2感染细胞中Mpro活性调节和SARS-CoV-2传播模式， 在转基因hACE 2+小鼠中的分布。