Necroptosis in SARS-CoV-2 pathogenesis, evolution, and therapy

SARS-CoV-2 发病机制、进化和治疗中的坏死性凋亡

基本信息

批准号：
10433040
负责人：
SIDDHARTH BALACHANDRAN
金额：
$ 28万
依托单位：
RESEARCH INST OF FOX CHASE CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10433040
关键词：
2019-nCoV Alveolar Cell Amino Acids Biology COVID-19 COVID-19 test Cell Death Cells Chiroptera Disease Epithelial Epithelial Cells Evolution FDA approved Genetic Polymorphism Human Inflammatory Influenza A virus Knock-in Mouse Laboratories Laboratory Study Left Lung Middle East Respiratory Syndrome Coronavirus Mus Nonstructural Protein Pathogenesis Pathogenicity Pathology Pathway interactions Phosphotransferases Proteins Public Health Pulmonary Inflammation Pulmonary Pathology RIPK1 gene RIPK3 gene Role SARS coronavirus SARS-CoV-2 infection SARS-CoV-2 pathogenesis Series Severity of illness Signal Transduction Testing Therapeutic Variant Viral Pathogenesis Virulent Virus Virus Diseases base cell type cytokine release syndrome experimental study feasibility testing helicase in vivo inhibitor insight kinase inhibitor lung injury mortality mouse model novel therapeutics pathogenic virus prevent sensor severe COVID-19

项目摘要

PROJECT SUMMARY/ABSTRACT Our laboratory has recently implicated necroptosis as a pathogenic and targetable host pathway during pulmonary influenza A virus (IAV) infections. In this proposal, we seek to extend these findings to SARS-CoV-2 because we have strong reason to believe that SARS-CoV-2, like IAV, activates necroptosis. We have identified a mechanism by which SARS-CoV-2 may trigger necroptosis, and propose that such necroptosis underlies the alveolar cell death and inflammatory ‘cytokine storm’ observed in severe COVID-19 disease. Importantly, necroptosis can be targeted by dedicated RIPK3 kinase inhibitors, opening up a new and unanticipated therapeutic entry-point for COVID-19. Specifically, we have discovered that a SARS-CoV-2 nonstructural protein contains a functional RHIM motif that is essential for propagating necroptosis signaling. In all known cell types, necroptosis is initiated when the kinase RIPK3 engages in RHIM-RHIM interactions with other RHIM-containing proteins. For example, during IAV infection, the RHIM in RIPK3 interacts with the RHIM in the IAV sensor protein ZBP1 to trigger necroptosis. We thus hypothesized that the RHIM in the CoV-2 protein allows it to interact with RIPK3 to activate necroptosis. Indeed, we found that the SARS-CoV-2 protein engages RIPK3 and activates necroptosis in human cells. The precise mechanism responsible remains unknown. We have also found that all three pathogenic CoVs (SARS-CoV, MERS-CoV, and SARS-CoV-2) have a RHIM in this protein, whereas none of the human-adapted strains (HKU-1, CO43, NL63, and 229E) possess one. Finally, we have found that bats, the likely natural hosts of SARS-CoV-2 and other pathogenic CoVs, encode a variant of RIPK3 which contains a single amino acid change from non-bat RIPK3. This change significantly dampens necroptosis signaling, suggesting that the necroptosis machinery is defective or non-functional in bats. Based on these and other observations, we hypothesize that SARS-CoV-2 and allied pathogenic CoVs activate necroptosis in human pulmonary epithelia, via a RHIM-RHIM interaction involving the CoV-2 RHIM-containing protein and RIPK3, and that such necroptosis initiates and amplifies the lung injury and inflammation seen in severe cases of COVID- 19. We further propose that dampened necroptosis signaling in bats allows them to harbor pathogenic (to humans) CoVs without apparent hyper-inflammatory consequences. In this proposal, we will examine how SARS-CoV activates necroptosis in human cells, and if such necroptosis is a new therapeutic opportunity in vivo by evaluating FDA-approved and new, high potency RIPK3 inhibitors in a mouse model of SARS-CoV-2 infection. We have also developed a knock-in mouse harboring the bat RIPK3 polymorphism, and will test if SARS-CoV-2-initiated lung pathology is dampened in this mouse, compared to controls. The successful completion of these studies will provide pioneering insight into the mechanism and evolutionary biology of necroptosis signaling in SARS-CoV-2 pathogenesis and stand to have important ramifications for the treatment of severe COVID-19.

项目概要/摘要我们的实验室最近发现坏死性凋亡是一种致病性和可靶向的宿主途径在本提案中，我们寻求将这些发现扩展到 SARS-CoV-2。因为我们有充分的理由相信 SARS-CoV-2 与 IAV 一样，我们已经确定会激活坏死性凋亡。 SARS-CoV-2 可能引发坏死性凋亡的机制，并提出这种坏死性凋亡是重要的是，在严重的 COVID-19 疾病中观察到肺泡细胞死亡和炎症“细胞因子风暴”。专门的 RIPK3 激酶抑制剂可以针对坏死性凋亡，开辟新的、意想不到的途径具体而言，我们发现了 SARS-CoV-2 非结构蛋白。包含功能性 RHIM 基序，该基序对于在所有已知细胞类型中传播坏死性凋亡信号至关重要。当激酶 RIPK3 参与 RHIM-RHIM 与其他含有 RHIM 的相互作用时，就会启动坏死性凋亡例如，在 IAV 感染期间，RIPK3 中的 RHIM 与 IAV 传感器蛋白中的 RHIM 相互作用。 ZBP1 触发坏死性凋亡，因此我们培育了 CoV-2 蛋白中的 RHIM 使其能够与病毒相互作用。 RIPK3 激活坏死性凋亡事实上，我们发现 SARS-CoV-2 蛋白与 RIPK3 结合并激活。我们还发现，人类细胞坏死性凋亡的确切机制仍然未知。三种致病性 CoV（SARS-CoV、MERS-CoV 和 SARS-CoV-2）的该蛋白中含有 RHIM，而没有一种人类适应菌株（HKU-1、CO43、NL63 和 229E）拥有一个。最后，我们发现蝙蝠， SARS-CoV-2 和其他致病性 CoV 的可能天然宿主，编码 RIPK3 的变体，其中包含非蝙蝠 RIPK3 的单个氨基酸变化显着抑制了坏死性凋亡信号传导，基于这些和其他，表明蝙蝠的坏死性凋亡机制有缺陷或不起作用。根据观察，我们发现 SARS-CoV-2 和相关致病性 CoV 会激活人类坏死性凋亡肺上皮细胞，通过涉及 CoV-2 含 RHIM 的蛋白和 RIPK3 的 RHIM-RHIM 相互作用，以及这种坏死性凋亡会引发并加剧肺损伤，并在严重的新冠肺炎病例中出现炎症。 19. 我们进一步提出，蝙蝠中坏死性凋亡信号的减弱使它们能够隐藏致病菌（以人类）冠状病毒没有明显的过度炎症后果在这项提案中，我们将研究如何。 SARS-CoV 激活人体细胞的坏死性凋亡，如果这种坏死性凋亡是体内新的治疗机会通过在 SARS-CoV-2 小鼠模型中评估 FDA 批准的新型高效 RIPK3 抑制剂我们还开发了一种携带蝙蝠 RIPK3 多态性的敲入小鼠，并将测试是否存在这种情况。与对照组相比，该小鼠中 SARS-CoV-2 引发的肺部病理学受到抑制。这些研究的完成将为我们提供关于机制和进化生物学的开创性见解。 SARS-CoV-2 发病机制中的坏死性凋亡信号传导并对治疗产生重要影响严重的 COVID-19。