Discovery of small molecules to block fusion and entry of dengue and other env vi

发现小分子以阻止登革热和其他环境病毒的融合和进入

• 批准号: 8233434
• 负责人: STEPHEN COPLAN HARRISON
• 金额: $ 31.92万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8233434
• 关键词: Affinity; Area; Biological Assay; Cell fusion; Cellular Membrane; Child health care; Complex; Dengue; Dengue Virus; E protein; Event; Flavivirus; Goals; Human; Individual; Infection; Libraries; Membrane Fusion; Membrane Lipids; Methods; Molecular Conformation; New England; Occupations; Peptides; Process; Reaction; Research; Screening procedure; Serotyping; Structure; Therapeutic Intervention; Vaccines; Viral; Virion; Virus; Virus Inhibitors; West Nile virus; Work; biodefense; conformer; env Gene Products; high throughput screening; influenzavirus; inhibitor/antagonist; neutralizing antibody; pathogen; peptide structure; scaffold; small molecule; stem; viron; virus envelope

Enveloped viruses enter and infect cells by fusion of viral and cellular membranes. The fusion step is a potential target for therapeutic intervention, just as it is the taret of inhibition by some neutralizing antibodies. We propose four aims, (1) From the structure of the dengue virus envelope protein in its trimeric, postfusion conformation, we have developed a high throughput screen for small molecule inhibitors of viral entry. We will further characterize a set of compounds we have recently identified (for dengue virus type 2) that inhibit viral infectivity in culture, and we will screen new libraries to identify additional compounds scaffolds. We will examine the extent to which these compounds inhibit other flaviviruses, starting with other dengue virus serotypes and West Nile virus (WNV). (2) We have found that peptides from the so-called "stem" segment of the dengue virus E protein inhibit viral infectivity. Inhibition correlates with affinity of the peptides for the trimeric, low-pH induced conformer of the E protein ectodomain, consistent with the notion that they inhibit the "zipping up" step in the fusion reaction. We will determine the structures of peptide- ectodomain complexes and work out the mechanism by which these peptides inhibit entry. (3) We have developed (initially for influenza virus) a fusion assay that follows the kenetics of individual, single viron fusion events. We will use this assay to study dengue and WNV mechanisms and to correlate structure and mechanism for inhibitition of fusion by small molecules and peptides. We will also study the influence of target-membrane lipid composition on the fusion process. (4) We will develop a higher- throughput format for the single-virion fusion assay. The ultimate goal is to apply the method for screening fusion inhibitors and analyzing neutralizing antibodies.

有包膜病毒通过病毒和细胞膜的融合进入并感染细胞。融合步骤是 治疗干预的潜在目标，就像它是某些中和抑制的目标一样 抗体。我们提出四个目标，（1）从登革热病毒包膜蛋白的结构出发 三聚体，融合后构象，我们开发了小分子抑制剂的高通量筛选 病毒进入。我们将进一步表征我们最近鉴定的一组化合物（针对登革热病毒 类型 2）可抑制培养物中的病毒感染性，我们将筛选新的文库以鉴定其他化合物 脚手架。我们将检查这些化合物抑制其他黄病毒的程度，从其他黄病毒开始 登革热病毒血清型和西尼罗河病毒（WNV）。 (2) 我们发现，来自所谓的肽 登革热病毒E蛋白的“茎”段抑制病毒感染性。抑制与亲和力相关 E蛋白胞外域的三聚体、低pH诱导的构象异构体的肽，与概念一致 它们抑制聚变反应中的“拉紧”步骤。我们将确定肽的结构- 胞外域复合物并找出这些肽抑制进入的机制。 (3) 我们有 开发了（最初针对流感病毒）一种遵循个体、单一病毒体动力学的融合测定 融合事件。我们将使用该测定来研究登革热和西尼罗河病毒机制并将其关联起来 小分子和肽抑制融合的结构和机制。我们还将研究 靶膜脂质成分对融合过程的影响。 (4)我们将开发更高的 单病毒粒子融合测定的通量格式。最终目标是应用该方法进行筛选 融合抑制剂和分析中和抗体。