Host Substrate Profiling of SARS-CoV-2 Virus Protease

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the viral pneumonia outbreak of coronavirus disease 2019 (COVID-19) causing widespread morbidity and mortality. Currently, there are no treatment available for this disease. Main protease (Mpro) encoded by SARS-CoV- 2 processes the viral polyproteins and facilitate viral replication. Once activated, this enzyme could evade the host innate immune responses by cleaving host proteins. As such, the host protein substrates of SARSCoV- 2 Mpro are not well characterized. The major challenges with the direct detection of host protein targets is their low abundance and technical limitations with specificity and sensitivity of current methods. Thus, this proposal is designed to develop a more specific and sensitive approach termed N-terminomics to discover unique protein fragments that are generated by Mpro during SARS CoV-2 infection. The Specific Aims of this proposal are: 1) Development and validation of novel NHS ester probes to identify protease fragments generated by SARS-CoV-2 Mpro in cell lysate and 2) Identify and quantify the endogenous substrates of SARS-CoV-2 Mpro in infected mammalian cells. The rationale for the proposed research is that its success would facilitate a greater understanding of how SARS CoV-2 Mpro protease contributes to the severity of COVID-19. Moreover, it will aid in developing treatment regimens for COVID-19. The expected outcome of this research is that our approach will be more sensitive and readily adaptable for the host substrate profiling of any pathogenic proteases and it will aid in mapping cellular pathways which are hijacked by pathogens to invade the host system. The successful execution of the research proposed herein is expected to have a significant positive impact in reducing morbidity and mortality caused by COVID-19.