Fusion Inhibitors of Influenza Virus

流感病毒融合抑制剂

• 批准号: 7564248
• 负责人: MING LUO
• 金额: $ 35.91万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-21 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7564248
• 关键词: Affinity; Antiviral Agents; Binding Sites; Chimeric Proteins; Cocrystallography; Extravasation; Glycoproteins; Hemagglutinin; In Vitro; Knowledge; Lead; Libraries; Lipids; Liposomes; Maps; Mediating; Membrane Fusion; Membrane Glycoproteins; Methods; Modeling; Modification; Molecular Conformation; Mutate; Mutation; Outcome; Pathway interactions; Peptide Hydrolases; Process; Proteolysis; Recombinants; Resistance; Series; Site; Staging; Structure; Surface; Testing; Variant; Viral; Virus; Virus Replication; base; chemical synthesis; design; influenzavirus; inhibitor/antagonist; mutant; novel; prevent; public health relevance; research study; small molecule

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.

描述（由申请人提供）：糖蛋白介导的膜融合是进入包膜病毒的常见机制。病毒表面上的糖蛋白通常以亚稳态构型存在，在融合过程中经历了戏剧性的重折叠。小分子化合物对这种融合的抑制是描述糖蛋白介导的膜融合机制的独特方法，并有可能寻找新型的抗病毒药物。在我们的初步研究中，发现一类化合物抑制了在皮摩尔浓度下流感病毒的血凝集素（HA）介导的膜融合。为了进一步研究抑制HA介导的膜融合的机制，该提案中提出了三个目的：AIM 1。结构研究。初步研究中的三个实验表明，这些化合物针对其抑制活性靶向HA。在此目标中，HA的共晶化将与一组抑制剂进行，将解决它们的结构。晶体结构将显示这些化合物的确切结合位点，并可能揭示这些化合物可能引起的结构变化。目标2。体外融合研究。 HA介导的脂质体融合将在抑制剂存在下进行研究。将定义抑制剂融合抑制的步骤。此外，重组HA将用不同的蛋白酶处理以绘制构象敏感区域。抑制剂将包括在蛋白水解研究中，以定义抑制剂在哪些区域的影响最大。目标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如何变化构象以介导膜融合。另一种类型的抗性突变体可能是改变抑制剂结合位点的突变体。由于缺乏对抑制剂结合位点的知识，因此先前未发现此类突变体。 AIM 1的结果将在这方面有所帮助。我们还将根据AIM 1的结果设计位点特异性突变体。AIM4。化学合成。从铅化合物得出的扩展库已合成。这些化合物包含相似的总体结构，但具有截然不同的亲和力。对化合物的进一步修改可能有两个目的。首先，新化合物可能由于取代基的变化而导致不同的结构变化。如果我们解决了一系列结构变化，这些结构是由不同抑制剂引起的结构变化，则可以对触发膜融合所需的构象变化的结构变化的途径进行建模。其次，我们可以通过设计新的抑制剂来测试我们对HA构象变化是否正确的理解。公共卫生相关性：黑凝集素是流感病毒的主要表面糖蛋白。它是负责进入流感病毒的融合蛋白。我们发现了一种新的有效抑制剂，这些抑制剂抑制了血凝素介导的膜融合。实验旨在研究这些化合物抑制的机制。结果将有助于设计新型融合抑制剂，这些融合抑制剂可能成为流感病毒潜在的抗病毒药物。