Novel Peptide Fusion Inhibitors for the Treatment of COVID-19

项目总结 新冠肺炎是由吸入最新的冠状病毒(CoV)SARS-CoV-2进入肺部和呼吸道引起的 上皮细胞特别容易感染这种病毒。大量证据表明，血管紧张素 转换酶2(ACE2)与SARS-CoV-2刺突蛋白(S1)的S1亚基结合，触发选择性 蛋白水解性裂解，释放S2亚基。S2经历了广泛的构象变化，形成了6- 在S2的七个重复序列(HR)-1和HR-2结构域之间的螺旋束(6-HB)，最终导致 病毒颗粒与细胞膜融合，随后病毒进入。基于病毒致病机制的研究 进入，并得到ACE2·S1界面和S2的6-Hb络合物的结晶学研究的支持，巨大的 目前正在努力开发基于多肽的疗法来针对这两个事件：相互作用 SARS-CoV-2与ACE2受体的结合，病毒颗粒与细胞膜的融合。我们有 发现分化良好的原代呼吸道上皮细胞长期暴露在烟草烟雾中 时间段增强了ACE2的活性并增加了重组S1的结合，这可能解释了 吸烟者对新冠肺炎的易感性增加。S1中的受体结合域(RBD)是高度 变异区，2020年底出现多个高传染性SARS-CoV-2变异株； 因此，针对这一地区可能不是抗病毒开发的理想选择。相比之下，S2的HR区域 6-Hb复合体中HR-1和HR-2结构域的亚基和相互作用模式高度保守 在多种冠状病毒中，这使其成为开发广谱抗病毒药物的最佳靶点。EK1是一种多肽 那。本申请的目标是开发以S2亚基的HR1结构域为靶点的新型多肽 抑制SARS-CoV-2及其他几种冠状病毒的膜融合和假病毒感染。这些 多肽应作为治疗新冠肺炎和随后的冠状病毒感染的广谱冠状病毒抗病毒药物。 我们建议评估几种多肽在肺部恶劣环境中的蛋白分解稳定性，因为 SARS-CoV-2的主要进入途径，包括具有增强螺旋约束的装订多肽和N-帽多肽。我们 将使用吸烟者的人肺分泌物来测量体外多肽的蛋白分解稳定性 和不吸烟的人。我们将使用原代呼吸道上皮细胞来询问多肽抑制的能力 融合和SARS-CoV-2假病毒感染对健康和烟雾暴露的呼吸道培养物的影响。的功效。 这些多肽最终将在动物模型中进行评估。这项研究将探讨螺旋线的可行性。 抑制病毒融合和抑制病毒进入呼吸道上皮细胞的模拟物作为一种新的有效治疗方法 新冠肺炎。

PROJECT SUMMARY COVID-19 is caused by inhalation of the latest coronavirus (CoV) SARS-CoV-2 into the lungs, and airway epithelia are particularly susceptible to uptake this virus. Extensive evidence indicates that angiotensin converting enzyme 2 (ACE2) binds to the S1 subunit of the SARS-CoV-2 Spike protein (S1), triggering selective proteolytic cleavage that liberates the S2 subunit. S2 undergoes extensive conformational changes to form a 6- helix bundle (6-HB) between Heptad Repeat (HR)-1 and HR-2 domains of S2, which ultimately results in the fusion of the viral particle with the cell membrane and subsequent viral entry. Based on the mechanism of viral entry, and supported by crystallography studies of the ACE2•S1 interface and the 6-HB complex of S2, enormous efforts are currently under way to develop peptide-based therapeutics to target both events: the interaction of SARS-CoV-2 Spike with ACE2 receptor, and the fusion of the viral particle to the cell membrane. We have discovered that exposure of well-differentiated, primary airway epithelial cultures to tobacco smoke for extended periods of time enhances ACE2 activity and increases binding of recombinant S1, which might explain the increased susceptibility of smokers to COVID-19. The Receptor Binding Domain (RBD) in S1 is part of a highly mutable region, as revealed by the appearance of multiple highly infectious SARS-CoV-2 variants in late 2020; thus, targeting this region might not be ideal for antiviral development. In contrast, the HR regions of the S2 subunit and the interaction mode of HR-1 and HR-2 domains within the 6-HB complex are highly conserved among various CoVs, which makes it an optimal target to develop broad-spectrum antivirals. EK1 is a peptide that. The goal of this application is to develop novel peptides that target the HR1 domain of the S2 subunit to inhibit membrane fusion and pseudovirus infection of SARS-CoV-2 as well as several other CoVs. These peptides should serve as broad-spectrum CoV antivirals for the treatment of COVID-19 and subsequent COVIDs. We propose to evaluate the proteolytic stability of several peptides in the hostile environment of the lung, as the main entry way of SARS-CoV-2, including stapled and N-capped peptides with enhanced helical constraint. We will measure the proteolytic stability of the peptides ex vivo using human lung secretions obtained from smokers and non-smokers. We will use primary airway epithelial cells to interrogate the ability of the peptides to inhibit fusion and SARS-CoV-2 pseudovirus infection to healthy and smoke-exposed airway cultures. The efficacy of these peptides will be ultimately evaluated in animal models. This study will address the feasibility of helical mimics to inhibit viral fusion and suppress viral entry into airway epithelia as a novel effective treatment against COVID-19.