Structure-guided design of protease-resistant, lipopeptide inhibitors of SARS-CoV-2

项目概要 严重急性呼吸综合征冠状病毒 2 型 (SARS-CoV-2)，冠状病毒的病原体 疾病（COVID-19）引发了前所未有的全球大流行。感染激增需要新的 可有效对抗快速变化的病毒的治疗剂。抑制病毒进入宿主的抗病毒药物 细胞已被证明可以有效对抗具有类似发病机制的其他病毒。 这项高度合作的拟议研究的长期目标是开发有效的肽 SARS-CoV-2 感染的抑制剂，通过阻断刺突蛋白的结构重排来发挥作用 病毒进入宿主细胞所需的。对于 SARS-CoV-2 感染的发生，病毒表面刺突 (S) 蛋白是一种 同源三聚体，重新排列以形成能量上有利的融合后状态。在这种融合后构象中，两个 螺旋结构域、N 端 (HRN) 和 C 端 (HRC) 七肽重复序列结合形成 6 螺旋束 (6HB)。来自 HRC 的肽可以抑制 6HB 的形成，从而抑制 SARS-CoV-2 感染。 然而，传统的肽完全由 α-氨基酸残基组成，非常容易受到 蛋白水解降解，需要频繁和高剂量给药。盖尔曼实验室与 哥伦比亚大学病毒学家 Anne Moscona 教授和 Matteo Porotto 教授已经证明， 选择性掺入主链修饰，与胆固醇缀合相结合，可以减少 蛋白水解敏感性，同时保持高抗病毒效力。我们的团队最近发现这样的脂肽 在生物测定和动物模型中可以抑制 SARS-CoV-2 感染，并且这些抑制剂是有效的 对抗 SARS-CoV-2 变种、SARS-CoV-1 和 MERS。在此基础上，我提出的项目寻求 开发含有主链修饰的脂肽，显示出高抗病毒效力并抵抗 蛋白水解。目标 1 将生产包含骨架的 SARS-CoV-2（和其他冠状病毒）的有效抑制剂 修饰并抵抗蛋白水解。目标 2 将评估抑制剂之间 6HB 形成的稳定性 候选者和天然 SARS-CoV-2 HRN。目标 3 将阐明关键的结构相互作用 HRC 模仿者和原生 HRN。 我的假设是，将主链修饰选择性地纳入基于 HRC 的设计中将 增加抗病毒活性和体内半衰期，提高治疗效果。拟议的研究，其中 将在威斯康星大学 Sam Gellman 教授的指导下进行，将为我提供 具有大分子 X 射线晶体学、分子设计、蛋白质工程和表达方面的经验，以及 病毒学。通过结构引导工程和复杂的多管齐下的分析 如果实施的话，这些努力可以为 COVID-19 产生有效的泛变异疗法。

PROJECT SUMMARY Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the etiological agent of coronavirus disease (COVID-19), has spurred an unprecedented global pandemic. Infection surges necessitate new therapeutic agents that are effective against a rapidly changing virus. Antivirals that inhibit viral entry into host cells have proven effective against other viruses with similar mechanisms of pathogenesis. The long-term objective of this highly collaborative proposed research is to develop potent peptides inhibitors of SARS-CoV-2 infection that operate by blocking structural rearrangements of the spike protein required for viral entry into host cells. For SARS-CoV-2 infection to occur, the viral surface spike (S) protein, a homotrimer, rearranges to form an energetically favored postfusion state. In this postfusion conformation, two helical domains, the N-terminal (HRN) and C-terminal (HRC) heptad repeats, associate to form a 6-helix bundle (6HB). Peptides derived from the HRC can inhibit formation of the 6HB, and thus SARS-CoV-2 infection. However, conventional peptides, composed entirely of α-amino acid residues, are highly susceptible to proteolytic degradation, which necessitates frequent and high dosing. The Gellman lab, in collaboration with virologists Prof. Anne Moscona and Prof. Matteo Porotto at Columbia University, has demonstrated that site- selective incorporation of backbone modifications, in combination with cholesterol conjugation, can decrease proteolytic sensitivity while maintaining high antiviral potency. Our team recently found that such lipopeptides can inhibit SARS-CoV-2 infection in biological assays and animal models, and that these inhibitors are effective against SARS-CoV-2 variants, SARS-CoV-1 and MERS. Building on this foundation, my proposed project seeks to develop lipopeptides containing backbone modifications that display high antiviral potency and resist proteolysis. Aim 1 will produce potent inhibitors of SARS-CoV-2 (and other coronaviruses) that contain backbone modifications and resist proteolysis. Aim 2 will evaluate the stability of 6HB formation between inhibitor candidates and the native SARS-CoV-2 HRN. Aim 3 will elucidate critical structural interactions between the HRC mimics and the native HRN. My hypothesis is that site-selective incorporation of backbone modifications into HRC-based designs will increase both antiviral activity and half-life in vivo, improving therapeutic efficacy. The proposed research, which will be conducted under the guidance of Prof. Sam Gellman at the University of Wisconsin, will provide me with experience in macromolecular X-ray crystallography, molecular design, protein engineering & expression, and virology. Through structure-guided engineering and sophisticated and multi-pronged assay implementation, these efforts could generate effective pan-variant therapeutics for COVID-19.