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

SARS-CoV-2 蛋白酶抗性脂肽抑制剂的结构指导设计

• 批准号: 10679139
• 负责人: Ariel Jade Kuhn
• 金额: $ 6.87万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679139
• 关键词: 2019-nCoV; Address; Amino Acid Substitution; Amino Acids; Animal Model; Antiviral Agents; Biodistribution; Biological Assay; Biological Availability; C-terminal; COVID-19; COVID-19 therapeutics; Cell membrane; Cells; Cessation of life; Chemicals; Cholesterol; Circular Dichroism; Collaborations; Coronavirus; Crystallization; Crystallography; Data; Development; Dose; Drug Kinetics; Economics; Engineering; Etiology; Exhibits; Fluorescence Polarization; Foundations; Goals; HIV; Half-Life; Hybrids; In Vitro; Infection; Learning; Length; Mediating; Membrane Fusion; Membrane Proteins; Middle East Respiratory Syndrome; Modification; Molecular; Molecular Conformation; N-terminal; Para-Influenza Virus Type 3; Pathogenesis; Peptide Hydrolases; Peptides; Positioning Attribute; Predisposition; Productivity; Property; Prophylactic treatment; Protein Engineering; Proteins; Proteolysis; Research; Research Personnel; Resistance; Resolution; SARS coronavirus; SARS-CoV-2 infection; SARS-CoV-2 inhibitor; SARS-CoV-2 variant; Side; Site; Structure; Surface; Techniques; Therapeutic Agents; Treatment Efficacy; Universities; Vaccines; Variant; Vertebral column; Viral; Viral Physiology; Virus; Virus Diseases; Virus Inhibitors; Vulnerable Populations; Wisconsin; Work; X ray diffraction analysis; X-Ray Crystallography; analog; career; coronavirus disease; design; experience; improved; in vivo; inhibitor; insight; interdisciplinary approach; mimicry; novel; novel strategies; novel therapeutics; pandemic disease; protein expression; viral entry inhibitor; virology

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.

项目摘要 严重的急性呼吸综合征2型冠状病毒（SARS-COV-2），冠状病毒的病因 疾病（Covid-19）刺激了前所未有的全球大流行。感染激增必要新的 有效防止快速变化病毒的治疗剂。抑制病毒进入宿主的抗病毒药 细胞已被证明对具有类似发病机理的其他病毒有效。 这项高度协作的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尖峰蛋白的结构重排起作用 病毒进入宿主细胞所需。为了使SARS-COV-2感染发生，病毒表面尖峰（S）蛋白A，A 同型聚合物，重新布置以形成基本上有利的后置换状态。在这个陷入后构象中，两个 螺旋结构域，N末端（HRN）和C末端（HRC）七点重复，辅助形成6螺旋束 （6hb）。源自HRC的肽可以抑制6HB的形成，从而抑制SARS-COV-2感染。 但是，完全由α-氨基酸残基组成的常规肽非常容易受到 蛋白水解降解，经常和高给药。 Gellman Lab与 病毒学家Anne Moscona教授和哥伦比亚大学的Matteo Porotto教授证明了该地点 - 选择性合并主链修饰，结合胆固醇制备，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种脂蛋白肽 可以在生物学测定和动物模型中抑制SARS-COV-2感染，并且这些抑制剂是有效的 反对SARS-COV-2变体，SARS-COV-1和MERS。在这个基础的基础上，我拟议的项目寻求 开发含有骨链修饰的脂肪肽 蛋白水解。 AIM 1将产生含有主链的SARS-COV-2（和其他冠状病毒）的潜在抑制剂 修饰并抵抗蛋白水解。 AIM 2将评估抑制剂之间6HB形成的稳定性 候选人和本地SARS-COV-2 HRN。 AIM 3将阐明关键的结构相互作用 HRC模拟和本地HRN。 我的假设是，现场选择性的骨干修改为基于HRC的设计的行业将 增加体内抗病毒活性和半衰期，从而提高治疗效率。拟议的研究，这是 将在威斯康星大学的Sam Gellman教授的指导下进行 具有大分子X射线晶体学，分子设计，蛋白质工程和表达以及 病毒学。通过结构引导的工程和复杂和多管齐下的测定 实施，这些努力可能会为Covid-19产生有效的泛变体疗法。