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

• 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
• 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

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.

项目摘要/摘要 我们的实验室在 肺部影响A病毒（IAV）感染。在此提案中，我们试图将这些发现扩展到SARS-COV-2 因为我们有强有力的理由相信SARS-COV-2（例如IAV）会激活坏死。我们已经确定了 SARS-COV-2可能引发坏死性的一种机制，并提出这种坏死的基础 在严重的Covid-19疾病中观察到肺泡细胞死亡和炎症性的“细胞因子风暴”。重要的是， 坏死性可以由专用的RIPK3激酶抑制剂靶向，开辟了一个新的和意外的 covid-199的治疗切入点，特别是，我们发现SARS-COV-2非结构蛋白 包含一个功能性rhim基序，这对于传播坏死信号传导至关重要。在所有已知细胞类型中， 当激酶RIPK3与其他含有Rhim的Rhim-Rhim相互作用时，将开始坏死性。 蛋白质。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引发坏死性。因此，我们假设COV-2蛋白中的rhim允许它与 RIPK3激活坏死。确实，我们发现SARS-COV-2蛋白与RIPK3接合并激活 人类细胞中坏死性。负责的确切机制仍然未知。我们还发现所有人 三个致病性COV（SARS-COV，MERS-COV和SARS-COV-2）在该蛋白质中具有RHIM，而没有 在人适应的菌株中（HKU-1，CO43，NL63和229E）具有一种。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 单个氨基酸从非BAT RIPK3变化。这种变化显着衰减坏死信号传导， 表明坏死性机制在蝙蝠中是有缺陷或非功能性的。基于这些和其他 观察结果，我们假设SARS-COV-2和相关致病性COV激活了人类的坏死作用 肺上皮，通过涉及含有COV-2 RHIM的蛋白质和RIPK3的Rhim-Rhim相互作用，以及 这种坏死性启动和放大器在严重的肺部损伤和感染中 19。我们进一步提出，蝙蝠中该死的坏死信号传导使它们能够携带病原体（TO TO 人类）COV没有明显的过度炎性后果。在此提案中，我们将研究 SARS-COV激活人类细胞的坏死性，如果这种坏死是体内的新治疗机会 通过评估SARS-COV-2小鼠模型中的FDA批准和新的高效力RIPK3抑制剂 感染。我们还开发了一种带有蝙蝠RIPK3多态性的敲击鼠标，并将测试是否 与对照组相比，该小鼠中SARS-COV-2引发的肺病理受损。成功 这些研究的完成将提供有关对机制和进化生物学的开创性见解 SARS-COV-2发病机理中的坏死信号传导，并具有重要的影响。 严重的Covid-19。