Structure-Guided Design of Peptidomimetic Inhibitors of Paramyxoviral Fusion

副粘病毒融合的拟肽抑制剂的结构引导设计

• 批准号: 9396856
• 负责人: Victor Kenneth Outlaw
• 金额: $ 5.71万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9396856
• 关键词: Affinity; Amides; Amino Acids; Antiviral Agents; Automobile Driving; Binding; Biochemical; Biochemistry; Biological Assay; Biological Availability; Bronchiolitis; C-terminal; Cell membrane; Cellular Membrane; Cessation of life; Childhood; Chimeric Proteins; Clinical; Collaborations; Complex; Croup; Crystallization; Development; Disadvantaged; Elements; Encephalitis; Evaluation; Exhibits; Family; Fatality rate; Fluorescence Polarization; Foundations; Future; Goals; Health; Hendra Virus; Hospitalization; Human; Infection; Infectious Agent; Interdisciplinary Study; Lead; Letters; Mediating; Membrane Fusion; Methods; Modification; Molecular; Molecular Biology; Molecular Conformation; N-terminal; Nipah Virus; Organic Chemistry; Para-Influenza Virus Type 3; Paramyxoviridae; Paramyxovirus; Pathogenicity; Pathway interactions; Peptides; Physiological; Pneumonia; Protein Inhibition; Proteolysis; Research; Resistance; Resistance to infection; Respiratory syncytial virus; Roentgen Rays; Structure; Techniques; Therapeutic; Thermodynamics; Toxic effect; Transmembrane Domain; Universities; Vertebral column; Viral; Viral Fusion Proteins; Virus; Virus Diseases; Virus Inhibitors; X-Ray Crystallography; combat; design; effective therapy; functional mimics; in vivo; inhibitor/antagonist; insight; member; novel; parainfluenza virus; pathogen; peptide analog; peptidomimetics; structural biology; virology

Project Summary The Paramyxoviridae family of viruses includes many pathogens that negatively impact global human health. Human parainfluenza viruses (HPIVs) and respiratory syncytial virus (RSV), are a leading cause of childhood croup, bronchiolitis, and pneumonia cases, leading to tens of thousands of hospitalizations and deaths annually in the U.S.14 Hendra virus (HeV) and Nipah virus (NiV) are emerging infectious agents with high fatality rates due to infection-induced encephalitis.16,17 Despite the demand for therapeutics to combat these pathogens, few effective treatment options are clinically available; existing treatments are somewhat limited by concerns of toxicity or poor efficacy. For this reason, viral fusion has emerged as a compelling target for the development of novel antiviral therapeutics. Paramyxoviral fusion is mediated by a viral fusion (F) protein, which when activated, forms an elongated transient intermediate and exposes two conserved heptad repeat regions (CHR and NHR). The CHR and NHR segments then come together to form a six-helix-bundle (6HB) structure, thermodynamically driving fusion. Segments of paramyxoviral CHR domains from each virus have been shown to bind the transient intermediate of that virus and inhibit viral fusion.9 However, peptides (e.g., 1 and 2; See Fig. 2 of Research Strategy for corresponding sequences) derived from the CHR of HPIV3, have been shown to exhibit activity against several members of the paramyxoviral family.10,11 Despite the conserved fusion mechanism, paramyxoviral F proteins possess distinct primary sequences; therefore, the ability of a single peptide to disrupt the fusion machinery within multiple viruses is highly puzzling. The proposed research focuses on (1) structural characterization of the interactions between broad spectrum antiviral peptides and viral fusion proteins and (2) the design, synthesis, and evaluation of peptidomimetic inhibitors with unnatural amide backbones to mimic those interactions. I will use co-crystallization techniques to pursue x-ray structures of 1 and 2 bound to their viral targets in HPIV3, RSV, NiV, and HeV F. Already, these efforts have led to crystal structures of 1 bound to PIV3 NHR and 2 bound to RSV NHR, which reveal atomic-level insights that will guide future inhibitor development. I will then use structure-guided design principles to incorporate β- and g-amino acid residues into the sequences of 1 and 2 to create foldamers that structurally and functionally mimic the diverse secondary structural elements observed in preliminary structures of the α-peptides. Peptidomimetics will be evaluated for binding affinity to paramyxoviral NHR domains using competitive fluorescence polarization and thermal shift assays. With the guidance of Prof. Anne Moscona, a molecular virologist at Columbia University (See Letter of Collaboration), lead peptidomimetics will be evaluated for inhibition of fusion mediated by PIV3-, RSV-, and NiV F and efficacy against infection by wild-type PIV3 and RSV, or pseudotyped NiV.

项目摘要 副粘病毒科病毒家族包括许多对全球人类造成负面影响的病原体 健康。人类副流感病毒(HPIV)和呼吸道合胞病毒(RSV)是导致 儿童哮喘、毛细支气管炎和肺炎病例，导致数万人住院和死亡 亨德拉病毒(HEV)和尼帕病毒(NIV)是每年在美国流行的高致死性传染病病原体。 16、17尽管需要治疗药物来对抗这些病原体， 临床上几乎没有有效的治疗选择；现有的治疗方法在一定程度上受到以下问题的限制 毒性或疗效差。出于这个原因，病毒融合已经成为发展的一个引人注目的目标 新型抗病毒治疗药物。副粘病毒融合是由病毒融合(F)蛋白介导的，当激活该蛋白时， 形成一个拉长的瞬时中间体，并暴露出两个保守的七肽重复区域(ChR和NHR)。 然后，ChR和NHR片段聚集在一起，形成热力学上的六螺旋-束(6HB)结构 推动核聚变。来自每种病毒的副粘病毒CHR结构域片段已被证明结合了瞬时 9然而，多肽(例如，1和2；见研究的图2 相应序列的策略)衍生自HPIV3的CHR，已被证明具有活性 针对副粘病毒家族的几个成员。10，11尽管融合机制保守， 副粘病毒F蛋白具有不同的初级序列；因此，单个多肽能够破坏 多种病毒之间的融合机制非常令人费解。 建议的研究集中在(1)广度之间相互作用的结构特征 光谱抗病毒多肽和病毒融合蛋白；(2)设计、合成和鉴定 具有非天然酰胺骨架的模拟肽抑制剂来模拟这些相互作用。我将使用共结晶 在HPIV3、RSV、NIV和HEV F中追踪与其病毒靶标结合的1和2的X射线结构的技术。 这些努力已经导致了与PIV3 NHR结合的1和与RSV NHR结合的2的晶体结构，其中 揭示原子级的洞察力，将指导未来的抑制剂开发。然后，我将使用结构导向设计 将β-和g-氨基酸残基结合到1和2的序列中以产生可 在结构和功能上模拟了在初步结构中观察到的各种二级结构元素 α多肽。将评估模拟多肽与副粘病毒NHR结构域的结合亲和力 竞争荧光偏振法和热位移法。在安妮·莫斯科纳教授的指导下， 哥伦比亚大学的分子病毒学家(见合作信函)，将对领先的多肽仿制学进行评估 抑制PIV3-、RSV-和Niv F介导的融合以及对抗野生型PIV3和 RSV，或伪型Niv.