COVID-19 airway inflammation is due to Spike inhibition of CFTR signaling

COVID-19 气道炎症是由于 CFTR 信号的 Spike 抑制所致

基本信息

批准号：
10566710
负责人：
Harvey Bruce Pollard
金额：
$ 66.36万
依托单位：
HENRY M. JACKSON FDN FOR THE ADV MIL/MED
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-15 至 2026-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10566710
关键词：
2019-nCoV ACE2 Affect Air Animal Model Antiviral Agents Apical Automobile Driving Basal Cell Binding Biopsy COVID-19 COVID-19 mortality COVID-19 patient COVID-19 severity Cardiac Glycosides Cell membrane Cell model Cells Cessation of life Chloride Channels Co-Immunoprecipitations Cyclic AMP Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Development Digitoxin Digoxin Disease Dose Endosomes Epithelial Cells Epithelium Failure Genetic Diseases Hamsters Health Priorities Histologic Human Incubated Infection Inflammation Inflammatory Inflammatory Response Liquid substance Lung Modeling Mutation Organ Ouabain Penetration Pharmaceutical Preparations Phenotype Process Proteins RNA analysis Recovery Recycling Regulator Genes Reporting Reproducibility SARS-CoV-2 infection SARS-CoV-2 inhibitor SARS-CoV-2 spike protein Secretory Cell Signal Transduction Symptoms TRADD gene Telomerase Testing Time Viral Virus airway epithelium airway inflammation autosome cell type cytokine release syndrome epithelial Na+ channel gene function global health nanomolar novel prevent protein expression receptor recessive genetic trait severe COVID-19 stem cells tool viral entry inhibitor

项目摘要

Abstract Over 860,000 COVID-19 deaths have occurred in the U.S., and more than 5.5 million deaths have occurred world-wide. Consequently, development of effective antiviral drugs that block infectivity and airway inflammation continue to be a global health priority. Inflammation in the COVID-19 airway is due to increased NFκB and Epithelial Sodium channel (ENaC) signaling. Cystic fibrosis (CF), a genetic disease caused by inactivating mutations in the CFTR gene, also has the same proinflammatory NFκB and ENaC signaling phenotype in the airway. Using a differentiated human epithelial "lung-on-a-chip" platform, we find that ACE2, the receptor protein for the SARS-CoV-2 Spike protein, co-immunoprecipitates with CFTR. Furthermore, exposure of differentiated epithelia to the SARS-COV-2 Spike protein dose-dependently suppresses cyclic AMP-dependent CFTR chloride channel activity and CFTR protein expression. Spike-dependent loss of CFTR also activates TRADD-dependent NFκB signaling and proteolytically activates ENaC. We have also found that Spike-dependent loss of CFTR may be due to failure of endosomal recycling to return apical CFTR to the plasma membrane. Finally, we find that nanoMolar concentrations of cardiac glycoside drugs such as ouabain, digitoxin and digoxin, which competitively inhibit Spike:ACE2 binding, rescue Spike-dependent reduction in CFTR activities. We have therefore hypothesized that binding of SARS-CoV-2 Spike protein to ACE2 in the lung causes loss of CFTR signaling and activation of proinflammatory NFκB and ENaC signaling. To further test this hypothesis we propose the following Specific Aims: SA #1: To define the mechanism by which SARS-CoV-2 Spike protein reduces CFTR channel activity and CFTR protein levels. We will determine the mechanism by which ACE2 interacts with CFTR. We will determine the mechanism by which Spike protein interaction with ACE2 leads to loss of CFTR. SA#2: To define the mechanism by which SARS-CoV-2 Spike protein drives activation of ENaC and NFκB signaling. We will determine the mechanism by which Spike-induced loss of CFTR protein results in activation of TRADD- dependent NFκB ENaC signaling. SA#3: To identify protective mechanisms of cardiac glycosides on cell and animal models of COVID-19 disease. We will test whether cardiac glycoside drugs block native SARS- CoV-2 infection of epithelia and rescue hamster models of COVID-19. . Novelty and Significance: To our knowledge this is the first time COVID-19 airway inflammation has been traced to inhibition of CFTR signaling. Consistently, COVID-19 patients who are also CF carriers, with only one wildtype CFTR gene and only 50% of CFTR function, were recently reported to suffer more severe COVID-19 symptoms and earlier death than normal subjects with COVID-19.

抽象的美国已有超过 86 万人因 COVID-19 死亡，其中超过 550 万人死亡在世界范围内进行了测试，开发出阻断感染和呼吸道的有效抗病毒药物。炎症仍然是全球健康的一个重点，COVID-19 气道炎症的增加是由于该疾病的增加。 NFκB 和上皮钠通道 (ENaC) 信号传导，囊性纤维化 (CF)，一种由遗传性疾病引起的疾病。 CFTR 基因的失活突变，也具有相同的促炎性 NFκB 和 ENaC 信号传导使用分化的人类上皮“芯片肺”平台，我们发现 ACE2， SARS-CoV-2 刺突蛋白的受体蛋白，与 CFTR 进行免疫共沉淀。分化上皮细胞暴露于 SARS-COV-2 Spike 蛋白会剂量依赖性地抑制循环 AMP 依赖性 CFTR 氯离子通道活性和 CFTR 蛋白表达的 Spike 依赖性损失。还激活 TRADD 依赖性 NFκB 信号传导并通过蛋白水解激活 ENaC。 CFTR 的尖峰依赖性损失可能是由于内体回收未能将顶端 CFTR 返回到最后，我们发现纳摩尔浓度的强心苷类药物，例如哇巴因、洋地黄毒苷和地高辛，竞争性抑制 Spike:ACE2 结合，拯救 Spike 依赖性因此，我们开发了 SARS-CoV-2 Spike 蛋白与 CFTR 活性的结合。肺部的 ACE2 导致 CFTR 信号传导丧失并激活促炎性 NFκB 和 ENaC 为了进一步检验这一假设，我们提出以下具体目标： SA #1：定义 SARS-CoV-2 Spike 蛋白降低 CFTR 通道活性和 CFTR 蛋白的机制我们将确定 ACE2 与 CFTR 相互作用的机制。 Spike 蛋白与 ACE2 相互作用导致 CFTR 丢失的机制：定义 SA#2。 SARS-CoV-2 刺突蛋白驱动 ENaC 和 NFκB 信号传导激活的机制。确定 Spike 诱导的 CFTR 蛋白丢失导致 TRADD- 激活的机制依赖 NFκB ENaC 信号传导 SA#3：确定强心苷对细胞的保护机制。我们将测试糖苷类药物是否能阻断天然 SARS- 心脏疾病。上皮细胞的 CoV-2 感染和 COVID-19 的拯救仓鼠模型。新颖性和意义：据我们所知，这是首次发现 COVID-19 气道炎症与 CFTR 信号传导的抑制一致，同时也是 CF 携带者的 COVID-19 患者中只有一名。最近报道野生型 CFTR 基因和仅 50% 的 CFTR 功能遭受更严重的 COVID-19 与正常的 COVID-19 受试者相比，出现症状并更早死亡。