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)是病毒性肺炎的病原体 2019年爆发冠状病毒病(新冠肺炎)，造成广泛的发病率和死亡率。目前， 目前还没有治疗这种疾病的方法。SARS冠状病毒编码的主要蛋白酶(MPRO) 2处理病毒多蛋白，促进病毒复制。一旦被激活，这种酶就可以逃脱 宿主通过裂解宿主蛋白来进行先天免疫反应。因此，SARSCoV的宿主蛋白底物- 2 MPRO特征不佳。直接检测宿主蛋白的主要挑战 靶标的低丰度和技术局限性与目前方法的特异性和敏感性有关。 因此，这项提议旨在开发一种更具体和更敏感的方法，称为N末端组学，以 发现由MPRO在SARS CoV-2感染期间产生的独特蛋白质片段。具体的 这项建议的目的是：1)开发和验证用于鉴定蛋白酶的新型NHS酯探针 SARS-CoV-2 MprO在细胞裂解物中产生的片段和2)鉴定和定量内源性 SARS-CoV-2 MPRO在感染哺乳动物细胞中的底物。建议进行这项研究的理由是 它的成功将有助于更好地了解SARS CoV-2 Mpro蛋白酶是如何促进 新冠肺炎的严重性。此外，它还将帮助开发新冠肺炎的治疗方案。这个 这项研究的预期结果是，我们的方法将更敏感，更容易适应 宿主底物分析任何致病的蛋白水解酶，它将有助于绘制细胞路径图 被病原体劫持以入侵宿主系统。成功地执行了建议的研究 预计这将对减少由以下原因引起的发病率和死亡率产生显著的积极影响 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.