Fusion Inhibitors of Influenza Virus

描述(由申请人提供)：糖蛋白介导的膜融合是包膜病毒进入的一种常见机制。病毒表面的糖蛋白通常以亚稳态构象存在，在融合过程中经历戏剧性的折叠。用小分子化合物抑制这种融合是阐明糖蛋白介导的膜融合的机制以及潜在地寻找新型抗病毒药物的唯一途径。在我们的初步研究中，发现一类化合物在皮摩尔浓度下对流感病毒血凝素(HA)介导的膜融合具有抑制作用。为了进一步研究HA介导的膜融合的抑制机制，本方案提出了三个目的：目的1.结构研究。初步研究中的三个实验表明，这些化合物针对HA的抑制活性。在这一目标中，HA将与一组抑制剂进行共结晶，并解决它们的结构问题。晶体结构将显示这些化合物的确切结合位置，并可能揭示这些化合物可能引起的结构变化。目的2.体外融合研究。HA介导的脂质体融合将在抑制剂存在的情况下进行研究。将定义抑制剂抑制融合的步骤。此外，重组HA将被不同的酶处理，以定位构象敏感区域。这些抑制剂将被包括在蛋白质分解研究中，以确定抑制剂对哪个区域的影响最大。目的3.突变研究。了解这类化合物抑制机制的另一种方法是选择抗性突变体。对抗性突变体的研究将有助于我们了解HA是如何改变构象来介导膜融合的。另一种类型的抗药性突变可能是那些改变抑制剂结合位点的突变。由于缺乏对抑制剂结合位点的了解，这些突变体以前没有被鉴定出来。目标1的结果将在这方面帮助我们。我们还将根据目标1.目标4.化学合成的结果设计定点突变体。从先导化合物衍生的扩展文库已经被合成。这些化合物包含相似的整体结构，但具有非常不同的亲和力。化合物的进一步修饰可能有两个目的。首先，由于取代基的不同，新化合物可能会引起不同的结构变化。如果我们解决一系列由不同抑制剂引起从小到大结构变化的结构，我们就可以模拟触发膜融合所需构象变化的结构变化路径。其次，我们可以通过设计新的抑制剂来测试我们对HA构象变化的理解是否正确。公共卫生相关性：血凝素是流感病毒的主要表面糖蛋白。它是一种融合蛋白，负责流感病毒的进入。我们发现了一类新的抑制血凝素介导的膜融合的有效抑制剂。通过实验研究了这些化合物的缓蚀机理。这一结果将有助于设计新型融合抑制剂，可能成为潜在的流感病毒抗病毒药物。

DESCRIPTION (provided by applicant): Glycoprotein mediated membrane fusion is a common mechanism for entry of enveloped viruses. The glycoprotein on the viral surface is usually present in a metastable conformation that undergoes a dramatic refolding during the fusion process. Inhibition of such fusion by small molecule compounds is a unique approach to delineate the mechanism of glycoprotein mediated membrane fusion, and to potentially search for novel antiviral drugs. In our preliminary studies, a class of compounds was found to inhibit membrane fusion mediated by hemagglutinin (HA) of influenza viruses at picomolar concentrations. To further study the mechanism of inhibition of HA-mediated membrane fusion, three aims are presented in this proposal: Aim 1. Structural studies. Three experiments in preliminary studies suggested that the compounds target HA for their inhibitory activities. In this aim, cocrystallization of HA will be carried out with a panel of inhibitors and their structures will be solved. The crystal structures will show the exact binding site for these compounds and may reveal what structural changes may have been caused by these compounds. Aim 2. in vitro fusion studies. HA mediated fusion of liposomes will be studied in the presence of inhibitors. Steps of fusion inhibition by the inhibitors will be defined. Further more, recombinant HA will be treated with different proteases in order to map the conformationally sensitive regions. The inhibitors will be included in the proteolysis study to define on which region the inhibitors have the most impact. Aim 3. Mutational studies. Another approach to understand the mechanism of inhibition by this class of compounds is to select resistant mutants. Studies on the resistant mutants will help us to understand how HA changes conformation to mediate membrane fusion. Another type of resistant mutants could be those that alter the binding site for inhibitors. Such mutants were not identified previously because of lack of knowledge on inhibitor binding sites. The result from aim 1 will help us in this aspect. We will also design site-specific mutants based on the outcome of Aim 1. Aim 4. Chemical synthesis. Expanded libraries derived from the lead compounds have been synthesized. These compounds contain a similar overall structure, but with very different affinities. Further modification of the compounds may serve two purposes. First, new compounds may cause different structural changes due to variations in substituents. If we solve a series of structures that have structural changes from small to large as caused by different inhibitors, we can model the pathway of structural changes that trigger the conformational change required for membrane fusion. Second, we can test if our understanding of the conformational change in HA is correct or not by designing new inhibitors. PUBLIC HEALTH RELEVANCE: Hemagglutinin is the major surface glycoprotein of influenza viruses. It is a fusion protein responsible for entry of influenza virus. We have discovered a new class of potent inhibitors that inhibit membrane fusion mediated by hemagglutinin. Experiments are designed to study the mechanism of inhibition by these compounds. The outcome will help the design of novel fusion inhibitors that may become potential antiviral drugs of influenza viruses.