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.

PROJECT NARRATIVE The proposed research is relevant to public health because it aims to develop noninvasive imaging reagents to study SARS-CoV-2, the pathogen of COVID-19 in infected host cells and organisms. The project is relevant to NIH’s mission in promoting health and combating diseases because it will assist revealing the SARS-CoV-2 regulation in infected cells and the spreading pattern of SARS-CoV-2 in an animal model to facilitate the identification of therapeutics for both treatment and prevention of COVID-19.