Inhibition of coronavirus release and mitigate COVID-19 pathogenesis

抑制冠状病毒释放并减轻 COVID-19 发病机制

基本信息

批准号：
10692222
负责人：
PETER D. SUN
金额：
$ 17.13万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10692222
关键词：
2019-nCoV ACE2 Acute Affect Affinity Binding Binding Proteins Biochemistry Blood Coagulation Factor Blood Pressure Blood Vessels Bradykinin Bronchoalveolar Lavage Fluid CASP1 gene COVID-19 COVID-19 pathogenesis COVID-19 patient COVID-19 severity Cell surface Cells Cessation of life Clinical Coagulation Process Coronavirus Coronavirus spike protein Drug Targeting Epithelial Cells Equilibrium Fiber Fibrin Fibrinogen Fibrosis HIV HIV Envelope Protein gp120 Human Infection Journals Lectin Receptors Liquid substance Lung Manuscripts Mediating Metalloproteases Modeling Neuraminidase Oseltamivir Pathology Peptide Hydrolases Pharmacology Physiological Plasma Polysaccharides Proteins Proteomics Prothrombin Public Health Publications Pulmonary Fibrosis Receptor Inhibition Recombinants SARS-CoV-2 infection SARS-CoV-2 inhibitor SARS-CoV-2 spike protein ST14 gene Serine Protease Sialic Acids Site System Testing Therapeutic Thrombin Trypsin Viral Virus Diseases coronavirus disease glycosylation influenzavirus inhibitor matriptase member nanomolar pandemic disease peptide analog receptor respiratory response

项目摘要

To characterize the SARS-CoV-2 spike protein interaction with lectin receptors, we have expressed recombinant SARS-CoV-2 spike prefusion trimer using 293F freestyle expression system. Given the nanomolar high affinity of the spike protein binding to human ACE2, the entry receptor for SARS-CoV-2, and the physiological importance of ACE2 in balancing blood pressure, we investigated if this interaction would affect the enzymatic activity of ACE2. Surprisingly, SARS-CoV-2 trimeric spike protein increased ACE2 proteolytic activity 3-10 fold when fluorogenic caspase-1 substrate and Bradykinin-analog peptides were used to characterize ACE2 activity. In addition, the enhancement was mediated by ACE2 binding of RBD domain of SARS-CoV-2 spike. These results highlighted the altered activity of ACE2 during SARS-CoV-2 infection and would shed new lights on the pathogenesis of COVID-19 and its complications for better treatments. A manuscript describing this finding is under revision for publication in Journal of Biological Chemistry. COVID-induced lung fibrosis is a major confounding factor associated with the current pandemic related death. Understanding the mechanism of COVID-associated lung fibrosis and developing a therapeutic treatment are an urgent public health need to reduce the current pandemic-associated death. Through proteomic analyses of bronchoalveolar lavage fluid (BALF) from COVID patients, we found dramatically elevated levels of fibrinogen and prothrombin in the acute COVID lung fluid compared to the healthy donors. Using primary human bronchoalveolar epithelial cells as models, we discovered that SARS-CoV-2 infected primary lung cells but not other SARS-CoV-2 susceptible cells, including Vero and 293T cells, induced fibrin to clot in the absence of many plasma coagulation factors. Many fibrin fibers induced by SARS-CoV-2 infection originated from lung cells. These infected lung cell induced fibrin clotting occurred with all strains of SARS-CoV-2 pseudoviruses, including omicron, as well as the circulating replication competent SARS-CoV-2 strains tested. They are indistinguishable from those thrombin clots and can be inhibited by pharmacological direct thrombin inhibitors. Importantly, infected lung cells triggered fibrin clotting in 3 of 4 acute but not recovered COVID nor healthy BALF. We suggest the viral infection activated members of cell surface expressed transmembrane serine proteases to directly activate prothrombin for fibrin clotting, as evidenced from the shedding of one of the transmembrane proteases, ST14, in response to the infection, and both recombinant catalytic matriptase and human airway trypsin-like protease activated prothrombin for fibrin clot formation. Our findings revealed a lung cell-mediated fibrosis triggered by SARS-CoV-2 infection that is independent of plasma coagulation, and suggest the need to focus therapeutic treatments to respiratory airway rather than blood vessels to mitigate COVID-induced lung fibrosis.

为了表征SARS-COV-2尖峰蛋白与凝集素受体的相互作用，我们使用293F自由泳表达系统表达了重组SARS-COV-2尖峰预灌注序列。鉴于峰值蛋白与人ACE2的结合，SARS-COV-2的进入受体以及ACE2在平衡血压中的生理重要性，我们研究了这种相互作用是否会影响ACE2的酶活性，我们研究了ACE2的生理重要性。令人惊讶的是，当使用荧光caspase-1底物和Bradykinin-Analog肽来表征ACE2活性时，SARS-COV-2三聚体峰蛋白增加了ACE2蛋白水解活性3-10倍。另外，增强是由SARS-COV-2 SPIKE的RBD结构域的ACE2结合介导的。这些结果强调了在SARS-COV-2感染期间ACE2的活性改变，并将对Covid-19的发病机理及其并发症的并发症释放出新的灯光。描述这一发现的手稿正在修订生物化学杂志上发表。共vid诱导的肺纤维化是与当前大流行相关死亡相关的主要混杂因素。了解共同相关的肺纤维化的机制并发展治疗治疗是迫切需要减少当前大流行相关死亡的公共卫生。通过对急性供体急性共核肺液的纤维蛋白原和凝血酶原水平的蛋白质组学分析，我们发现与健康供体相比，我们发现纤维蛋白原和凝血酶原水平显着升高。我们使用原代人支气管肺泡上皮细胞作为模型，我们发现SARS-COV-2感染了原代肺细胞，而不是其他SARS-COV-2易感细胞（包括VERO和293T细胞），在没有许多血浆凝结因子的情况下诱导了纤维蛋白。 SARS-COV-2感染诱导的许多纤维蛋白纤维起源于肺部细胞。这些感染的肺细胞诱导的纤维蛋白凝结发生在包括Omicron在内的所有SARS-COV-2伪病毒以及测试的循环复制型SARS-COV-2菌株中。它们与那些凝血酶血块没有区别，可以被药理直接凝血酶抑制剂抑制。重要的是，被感染的肺细胞引发了4个急性中的3个，但没有恢复的纤维蛋白凝结，但没有恢复健康或健康的BALF。我们建议病毒感染激活的细胞表面成员表达的跨膜丝氨酸蛋白酶直接激活凝血酶蛋白以进行纤维蛋白凝结，这是从一种跨膜蛋白酶ST14的脱落来响应感染的跨膜蛋白酶ST14的，并且既可以重组催化剂蛋白蛋白酶蛋白酶蛋白酶蛋白酶蛋白酶蛋白蛋白酶蛋白酶蛋白蛋白酶蛋白蛋白蛋白酶蛋白酶蛋白酶呈proth proth proth proth proth prothip prothip octivication。我们的发现表明，由SARS-COV-2感染触发的肺细胞介导的纤维化与血浆凝结无关，并表明需要将治疗性治疗聚焦于呼吸道气道而不是血管来减轻共染色的肺纤维化。