NMR-based discovery of influenza hemagglutinin probes

描述（由申请人提供）：每年有超过250，000例流感相关死亡。特别值得关注的是H1N1和H5 N1流感病毒株，它们携带H1和H5血凝素（HA），有可能在未来爆发大流行。目前的治疗方法包括达菲（奥司他韦）和瑞乐沙（扎那米韦），它们靶向膜蛋白神经氨酸酶（NA）。不幸的是，在流行的流感病毒株中，耐药性正在增加。例如，2008-2009年H1N1病毒株对达菲表现出约100%的耐药性，因此流感可以被认为是耐药性病原体。因此，迫切需要流感的新靶标。HA在流感进入中起关键作用，因此它是治疗干预的有吸引力的和新的靶标。在流感进入的第一步中，A与细胞受体唾液酸结合。随后，病毒通过受体介导的内吞作用进入细胞。在内体中，所得的低pH触发HA的大的构象变化，其随后导致病毒膜与内体膜融合以允许从内体逃逸。进入抑制剂可以想象靶向受体结合步骤（结合抑制剂）或膜融合步骤（融合抑制剂）。在本项目中，我们将开发基于NMR的H1和H5流感HA小分子文库筛选方法，其长期目标是开发结合和融合抑制剂。具体目标是：（1）发现以中性pH构象结合流感H1和H5 HA的化学探针;（2）发现以低pH构象结合流感H1和H5 HA的化学探针;（3）通过NMR竞争实验鉴定探针结合位点。重要的是，NMR方法可以用于识别和改进小分子治疗剂，表征整个病毒中的探针-包膜相互作用，并应用于其他病毒，如埃博拉病毒，HIV和SARS-CoV。

DESCRIPTION (provided by applicant): There are more than 250,000 influenza-related deaths each year. Of special concern are the influenza H1N1 and H5N1 strains, which harbor H1 and H5 hemagglutinin (HA) and present potential for future pandemic outbreaks. Current treatments include Tamiflu (oseltamivir) and Relenza (zanamivir), which target the membrane protein neuraminidase (NA). Unfortunately, resistance is increasing in circulating influenza strains. For example, the 2008-2009 H1N1 strain exhibited ~100% resistance against Tamiflu, and thus influenza may be considered as a drug-resistant pathogen. As a consequence, new targets in influenza are urgently needed. HA plays a critical role in influenza entry and thus it i an attractive and novel target for therapeutic intervention. In the first step of influenza entry, A binds to the cellular receptor, sialic acid. Subsequently, the virus enters the cell via receptor-mediated endocytosis. In the endosome, the resulting low pH triggers a large conformational change in HA, which subsequently causes the viral membrane to fuse with the endosomal membrane to allow escape from the endosome. Entry inhibitors could conceivably target either the receptor binding step (binding inhibitors) or the membrane fusion step (fusion inhibitors). In this project we will develop NMR-based screening methods of small molecule libraries to influenza H1 and H5 HA with the long-term goal of developing binding and fusion inhibitors. The Specific Aims are: (1) discovery of chemical probes that bind to influenza H1 and H5 HA in the neutral pH conformation; (2) discovery of chemical probes that bind to influenza H1 and H5 HA in the low pH conformation; (3) identify the site of probe binding by NMR competition experiments. Importantly, the NMR methods could be exploited to identify and improve small molecule therapeutics, characterize probe-envelope interactions in whole virus, and be applied to other viruses such as Ebola, HIV and SARS-CoV.