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

项目摘要 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 新型冠状病毒病（COVID-19）引发了一场前所未有的全球大流行。感染激增需要新的 对快速变化的病毒有效的治疗剂。抑制病毒进入宿主的抗病毒药物 细胞已被证明对具有类似发病机制的其它病毒有效。 这项高度合作的研究的长期目标是开发有效的肽 SARS-CoV-2感染抑制剂，通过阻断刺突蛋白的结构重排发挥作用 病毒进入宿主细胞所必需的。对于SARS-CoV-2感染的发生，病毒表面刺突（S）蛋白， 同源三聚体重排形成能量有利的融合后状态。在这种融合后构象中，两个 螺旋结构域，N-末端（HRN）和C-末端（HRC）七肽重复，结合形成6-螺旋束 （6HB）。从HRC衍生的肽可以抑制6 HB的形成，从而抑制SARS-CoV-2感染。 然而，完全由α-氨基酸残基组成的传统肽非常容易受到 蛋白水解降解，这需要频繁和高剂量。盖尔曼实验室与 哥伦比亚大学的病毒学家Anne Moscona教授和Matteo Porotto教授已经证明了该位点- 骨架修饰的选择性掺入与胆固醇缀合结合，可以降低 蛋白水解敏感性，同时保持高抗病毒效力。我们的研究小组最近发现， 在生物学试验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 针对SARS-CoV-2变种、SARS-CoV-1和MERS。在此基础上，我提出的项目寻求 开发含有骨架修饰的脂肽， 蛋白水解Aim 1将产生SARS-CoV-2（和其他冠状病毒）的有效抑制剂， 修饰和抵抗蛋白水解。目的2：评价抑制剂与6 HB形成的稳定性 候选人和天然SARS-CoV-2 HRN。目标3将阐明关键结构之间的相互作用， HRC模仿和本地HRN。 我的假设是，将骨架修饰位点选择性地掺入到基于HRC的设计中， 增加体内抗病毒活性和半衰期，提高治疗效果。拟议的研究，其中 将在威斯康星州大学的Sam Gellman教授的指导下进行，将为我提供 在大分子X射线晶体学、分子设计、蛋白质工程和表达方面的经验， 病毒学通过结构导向工程和复杂的多管齐下的分析， 通过实施，这些努力可以产生针对COVID-19的有效泛变体疗法。

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.