Host Substrate Profiling of SARS-CoV-2 Virus Protease

严重急性呼吸综合征冠状病毒2（SARS-CoV-2）是引起病毒性肺炎的主要病原体 2019冠状病毒病（COVID-19）爆发，导致广泛的发病率和死亡率。目前， 这种疾病没有治疗方法。SARS-CoV编码的主要蛋白酶（Mpro）- 2处理病毒多聚蛋白并促进病毒复制。一旦被激活，这种酶就可以逃避 宿主通过切割宿主蛋白质进行先天免疫反应。因此，SARSCoV的宿主蛋白底物- 2 Mpro没有很好地表征。直接检测宿主蛋白的主要挑战 靶点是它们的低丰度和目前方法的特异性和灵敏度的技术限制。 因此，该提议旨在开发一种称为N-末端组学的更特异和敏感的方法， 发现Mpro在SARS CoV-2感染过程中产生的独特蛋白片段。具体 本研究的目的是：1）开发和验证新型NHS酯探针，以鉴定蛋白酶 SARS-CoV-2 Mpro在细胞裂解物中产生的片段; 2）鉴定和定量内源性 SARS-CoV-2 Mpro在感染的哺乳动物细胞中的底物。拟议研究的基本原理是 它的成功将有助于更好地了解SARS CoV-2 Mpro蛋白酶如何有助于 COVID-19的严重性。此外，它将有助于开发COVID-19的治疗方案。的 这项研究的预期结果是，我们的方法将更敏感，更容易适应 宿主底物分析任何病原性蛋白酶，它将有助于绘制细胞途径， 被病原体劫持入侵宿主系统成功执行研究建议 预期在降低由以下原因引起的发病率和死亡率方面具有显著的积极影响： 新冠肺炎。

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.