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

包膜病毒通过病毒膜和细胞膜的融合进入并感染细胞。融合步骤是 它是治疗干预的潜在靶点，正如它是一些中和剂抑制的靶点一样。 抗体的我们提出了四个目标：（1）从登革病毒包膜蛋白的结构出发， 三聚体，融合后构象，我们已经开发了一种高通量筛选小分子抑制剂 病毒入侵的迹象我们将进一步描述我们最近发现的一组化合物（用于登革热病毒 2型），抑制病毒感染的文化，我们将筛选新的图书馆，以确定其他化合物 脚手架我们将研究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)我们已经发现，来自所谓的 登革病毒E蛋白的“茎”段抑制病毒感染性。抑制作用与蛋白质的亲和力相关。 肽的三聚体，低pH诱导构象的E蛋白胞外域，符合概念 它们抑制了聚变反应中的“拉链”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（最初用于流感病毒）一种融合试验， 聚变事件我们将使用这种方法来研究登革热和西尼罗河病毒的机制，并将其与登革热和西尼罗河病毒之间的关系联系起来。 小分子和肽抑制融合的结构和机制。我们亦会研究 靶膜脂质组成对融合过程的影响。 (4)我们将建立更高的- 单病毒体融合测定的通量形式。最终目的是将该方法应用于筛选 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.