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

项目总结 新冠肺炎是目前流行的流行病，已使世界大部分地区陷入瘫痪。该病的病毒病原体 新冠肺炎是SARS-CoV-2，一种冠状病毒，基因组呈阳性。它在人体细胞中的产生 宿主需要其主要蛋白酶(MPRO)的活性。作为SARS-CoV-2、MPRO必不可少的一种独特的酶 对宿主细胞有剧毒，表明它在病毒感染和复制过程中受到严格调控。它的活动 也代表在受感染的宿主体内的器官和组织中活跃的病毒复制。因此， 检测SARS-CoV-2感染细胞和动物的MPRO活性将提供有关MPRO的基本信息 宿主细胞内的活性调节和SARS-CoV-2在宿主体内的传播和分布 用于阐明SARS-CoV-2的复制和发病机制。非侵入性成像试剂将是关键 用于检测MPRO活性，以避免对病毒和宿主的干扰。以终极的 了解SARS-CoV-2在感染细胞中的复制和阐明SARS-CoV-2的传播 感染动物的模式和器官/组织分布，目前的建议旨在开发非侵入性 荧光和化学发光MPRO衬底，具有高度的细胞稳定性和组织穿透性 对MPRO活动进行成像。三个具体目标：1)发展荧光和化学发光 MPRO底物；2)表征荧光和化学发光MPRO底物的选择性、细胞 稳定性和组织渗透性；以及3)对SARS-CoV-2感染进行细胞和体内成像。这个 拟议研究的成功完成将导致1)产生荧光和化学发光的MPRO衬底 具有最佳的细胞稳定性和组织渗透性，可被新冠肺炎研究人员广泛采用 SARS-CoV-2复制和致病机制的研究和2)提供关键信息 SARS-CoV-2感染细胞中MPRO活性调控及SARS-CoV-2传播模式的研究 在转基因hACE2+小鼠体内的分布。

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.