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

被包裹的病毒通过病毒和细胞膜的融合进入并感染细胞。融合步骤是一个 治疗干预的潜在目标，就像它是通过一些中和来抑制的靶子一样 抗体。我们提出了四个目标：(1)从登革病毒包膜蛋白在ITS中的结构 三聚体，融合后构象，我们已经开发出一种用于小分子抑制剂的高通量筛选 病毒入侵的可能性。我们将进一步鉴定我们最近发现的一组化合物(针对登革热病毒 类型2)在培养中抑制病毒感染性，我们将筛选新的文库来鉴定更多的化合物 脚手架。我们将研究这些化合物对其他黄病毒的抑制程度，从其他 登革病毒血清型和西尼罗河病毒(WNV)。(2)我们发现，来自所谓的多肽 登革热病毒E蛋白的“茎”段抑制病毒的感染性。抑制作用与细胞的亲和力相关 三聚体，低pH诱导的E蛋白胞外区构象的多肽，与概念一致 它们抑制了聚变反应中的“拉链”步骤。我们将确定多肽的结构- 并研究出这些多肽抑制进入的机制。(3)我们有 发展(最初针对流感病毒)遵循个体、单一病毒遗传学的融合检测方法 核聚变事件。我们将利用这一检测方法来研究登革热和西尼罗河病毒的机制，并将其与 小分子和多肽抑制融合的结构和机制。我们亦会研究 靶-膜脂成分对融合过程的影响。(4)我们将发展更高的- 单病毒粒子融合分析的吞吐量格式。最终目标是将该方法应用于筛查 融合抑制剂和分析中和抗体。

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.