A New Targeting Approach to Inhibit Budding of the Ebola Virus

抑制埃博拉病毒萌芽的新靶向方法

基本信息

批准号：
9763445
负责人：
Robert Virgil Stahelin
金额：
$ 22.64万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-14 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9763445
关键词：
Affinity Amino Acid Sequence Amino Acids Animal Model Animals Antibodies Antibody Therapy Biological Assay Biological Models Biophysics Cell Survival Cell membrane Cells Chemicals Clinical Communicable Diseases Computer Analysis Data Dimerization Disease Outbreaks Drug Targeting Ebola virus Equilibrium Escape Mutant FDA approved Family Fatality rate Filoviridae Infections Filovirus Frankfurt-Marburg Syndrome Virus Fright Generations Genes Glycoproteins Human In Vitro Laboratories Lead Life Cycle Stages Lipid Bilayers Lipid Binding Lipids Malignant Neoplasms Mammalian Cell Mediating Methods Modeling Mutate Mutation N-terminal Patients Penetration Peptides Pharmacology Plasma Cells Preventive measure Process Production Property Proteins Public Health Recording of previous events Structural Protein Structure Testing Therapeutic Vaccine Clinical Trial Vaccine Therapy Vaccines Viral Viral Matrix Proteins Virion Virus Virus Assembly Virus Replication Virus-like particle Western Africa alpha helix base chemical synthesis cost design dimer experimental study insight lead candidate monomer pandemic disease protein protein interaction protein structure small molecule therapeutic target therapeutic vaccine tool virus envelope

项目摘要

Abstract: Lipid-enveloped viruses replicate and bud from host cell membranes where they acquire their lipid coat. Understanding the budding processes of several viruses has had significant impact on elucidating the viral life cycle and identifying therapeutic targets. Filoviruses have a filamentous lipid- envelope and despite being discovered more than 30 years ago, not much is known on how they assemble and bud from the host cell plasma membrane. Filoviruses, which include Ebola virus (EBOV), have a high fatality rate and there is still a lack of FDA approved therapeutics or vaccines for treatment. Moreover, the EBOV glycoprotein, the prime target of antibody and vaccine therapy undergoes a high rate of mutation in animal and human studies and escape mutant of glycoprotein have been found as EBOV is passaged through animal models. Filoviruses encode seven genes including the viral matrix protein VP40, which regulates budding from the host cell. VP40 as the only filovirus protein expressed in mammalian cells is sufficient to produce virus like particles (VLPs) nearly indistinguishable from live virions. Thus, VP40 has served as a model to study viral budding outside of BSL-4 laboratories. VP40 has been shown to be a dimer, which is mediated by a-helical interactions in its N-terminal domain (NTD). Mutation of residues in the NTD of VP40 that mediate dimerization is sufficient to abrogate viral budding in model systems. To date, little is known about how VP40 monomer/dimer equilibrium and biophysics of oligomer assembly are regulated as well as if VP40 is a viable drug target in the viral life cycle. The central hypothesis of this R21 proposal is that generation of a new chemical toolkit based upon stapled a-helical peptides can be used to study VP40 assembly and inhibit VP40 dimerization. In specific aim 1, we will design and synthesize lead candidate stapled a-helical peptides that target the VP40 dimer interface. We will elucidate the optimal amino acid sequences and chemical linker of stapled a-helical peptides using computational analysis. We hypothesize that optimization of the stapled helices can be performed to block VP40 dimer formation in vitro and in cells. We will use computational analysis and a rapid chemical synthesis method to generate lead candidates for quantitative analysis. Specific aim 2 will investigate the mechanism by which stapled a- helical peptides interact with VP40 and inhibit VP40 dimerization and budding of VLPs. Quantitative assays of VP40 dimer formation, VP40 lipid-binding, and budding of VLPs will be assessed to decipher the ability of lead compounds to inhibit dimer formation and subsequent budding. Taken together, these studies should produce new and important mechanistic insight into the viability of VP40 as a drug target and a better biophysical understanding of the properties that govern VP40 assembly.

摘要：脂包膜病毒从宿主细胞膜上复制并出芽，并在那里获得它们的病毒。了解几种病毒的出芽过程对病毒产生了重大影响。阐明病毒生命周期并确定治疗靶点。丝状病毒具有丝状脂质- 尽管 30 多年前就被发现了，但人们对它们的组装方式知之甚少丝状病毒，包括埃博拉病毒 (EBOV)，具有很高的病毒感染率。死亡率高，而且仍然缺乏 FDA 批准的治疗方法或疫苗。埃博拉病毒糖蛋白是抗体和疫苗治疗的主要靶标，在随着埃博拉病毒的传代，动物和人类研究发现了糖蛋白的逃逸突变体通过动物模型，丝状病毒编码 7 个基因，包括病毒基质蛋白 VP40。 VP40 是哺乳动物细胞中唯一表达的丝状病毒蛋白，可调节宿主细胞的出芽。足以产生与活病毒体几乎无法区分的病毒样颗粒（VLP）。 VP40 已被证明是在 BSL-4 实验室之外研究病毒出芽的模型。二聚体，由其 N 末端结构域 (NTD) 中的 aα 螺旋相互作用介导。介导二聚化的 VP40 的 NTD 足以消除模型系统中的病毒出芽。关于 VP40 单体/二聚体平衡和寡聚体组装的生物物理学如何进行知之甚少 R21 的中心假设是 VP40 是否是病毒生命周期中可行的药物靶标。建议基于钉合α-螺旋肽生成新的化学工具包，可用于研究VP40组装并抑制VP40二聚化在具体目标1中，我们将设计并合成先导化合物。我们将阐明针对 VP40 二聚体界面的候选钉合 aα-螺旋肽。我们使用计算分析研究了钉合α-螺旋肽的氨基酸序列和化学接头。促进了可以对钉合螺旋进行优化以阻断体外 VP40 二聚体的形成我们将在细胞中使用计算分析和快速化学合成方法来产生铅。具体目标 2 将研究钉合 a- 的机制。螺旋肽与 VP40 相互作用并抑制 VP40 二聚化和 VLP 的出芽。将评估 VP40 二聚体形成、VP40 脂质结合和 VLP 出芽的能力，以破译综上所述，这些研究应该能够抑制二聚体形成和随后的出芽。对 VP40 作为药物靶点的可行性产生新的、重要的机制见解，并更好地对控制 VP40 组装特性的生物物理学理解。