Identification of Novel Broad Spectrum Influenza Virus Inhibitors

描述(申请人提供)：迫切需要针对流感病毒感染的广谱疗法来解决流感大流行的问题，流感大流行是全球公共卫生的主要威胁。干扰病毒进入是控制病毒感染的一种新颖而有吸引力的治疗策略。这种方法的原理的证据来自艾滋病毒抑制剂恩福韦肽(T-20)。我们的目标是发现非肽性小分子，以抑制禽流感H5N1和其他潜在的大流行流感病毒的进入。我们的策略是针对包膜糖蛋白血凝素(HA)，它通过受体结合和与宿主细胞融合来介导流感病毒的进入。HA是与副粘病毒的HIV gp120和F蛋白一样的I类融合蛋白。I类病毒融合蛋白在融合过程中经历了一系列的构象重排，最终形成融合发夹结构。这种结构在融合过程中促进了病毒和细胞包膜的并列，并由蛋白质相互作用维持。副粘病毒的小分子进入抑制剂已经被确定干扰这种融合发夹结构的形成。由于HA和其他I类融合蛋白中存在相似的结构，这些结果支持了我们以HA为靶点作为药物发现策略的假设。针对这一保守位点的抑制剂将对多种亚型有效，包括一种新出现的大流行毒株。我们将使用一种表达HA(H5亚型)的伪型病毒作为替代模型来模拟HA介导的进入，并在BSL2条件下筛选进入抑制剂。在初步研究中，我们开发并鉴定了一种敏感的伪型病毒试验，用于筛选HA抑制剂。在这个STTR项目的第一阶段，我们将优化快速筛选结构多样化小分子的大型(&gt；100,000)文库的检测方法。HITS将由我们的合作者、伊利诺伊大学芝加哥分校的Lijun Rong博士以盲法确认，并将由西奈山医学院的Adolfo Garcia-Sastre博士在增强型BSL 3实验室中评估其对活H5禽流感病毒的抗流感活性。确认的命中将根据它们的频谱与其他子类型进行评估，并根据它们的作用机制确定优先级。这些新的流感治疗药物将阻止病毒进入，并抑制病毒与细胞接触所产生的细胞毒性。由于保守结构域的微小偏差将阻止病毒与内体膜融合，预计它们也将具有较低的抗药性发展发生率。在第二阶段，我们将通过合理的药物设计计划来开发最有希望的支架，并将在动物模型中测试先导化合物的有效性和毒性。最活跃、毒性最小的化合物将进入IND使能研究。公共卫生相关性：流感是一种高度传染性的急性呼吸道疾病，以每年反复流行和周期性的全球大流行为特征。疫苗目前是预防流感病毒感染的主要策略，只有当它们与流行的病毒类型匹配时才有效(S)，并且不能针对新出现的大流行毒株(S)提前开发。我们的目标是开发一种抗流感疗法，通过靶向包膜蛋白血凝素(HA)的保守融合和受体结合域来防止病毒入侵，并对所有亚型都有效，包括一种新出现的大流行毒株。

DESCRIPTION (provided by applicant): Broad-spectrum therapeutics against influenza virus infections are critically needed to address the problem of influenza pandemics, a major threat to the public health globally. Interfering with virus entry is a novel and attractive therapeutic strategy to control virus infection. Proof of principle of this approach has come from the HIV inhibitor enfuvirtide (T-20). Our goal is to discover nonpeptidic small molecules that will inhibit entry of avian influenza H5N1 and other potentially pandemic influenza viruses. Our strategy is to target envelope glycoprotein hemagglutinin (HA), which mediates influenza virus entry through receptor binding and fusion with host cells. HA is class I fusion protein like the HIV Gp120 and F protein of paramyxoviruses. The class I virus fusion proteins undergo a series of conformational rearrangements during fusion that leads to fusion hairpin structure. This resultant structure promotes the juxtaposition of the viral and cellular envelopes during fusion and is sustained by protein protein interactions. Small molecule entry inhibitors of paramyxoviruses have been identified that interfere with the formation of this fusion hairpin structure. Because analogous structures are present in HA and other class I fusion proteins, the results support our hypothesis of targeting HA as a strategy for drug discovery. Inhibitors targeting this conserved site will be active against multiple subtypes, including a newly emerged pandemic strain. We will use a pseudotype virus expressing HA (H5 subtype), which has been developed as a surrogate model, to mimic HA mediated entry and screen for entry inhibitors under BSL2 conditions. In preliminary studies, we have developed and characterized a sensitive pseudotype virus assay for screening HA inhibitors. In Phase I of this STTR project, we will optimize the assay for rapid screening of a large (>100,000) library of structurally diverse small molecules. Hits will be confirmed in a blind fashion by our collaborator Dr Lijun Rong, University of Illinois at Chicago and will be evaluated for their anti-influenza activity against live H5 avian influenza viruses in an enhanced BSL 3 laboratory by Dr Adolfo Garcia-Sastre, at Mount Sinai Medical College. Confirmed hits will be evaluated for their spectrum against other subtypes and prioritized based on their mechanism of action. These novel influenza therapeutics will block virus entry and suppress cellular cytotoxicity resulting from virus-cell contact. They will also be expected to have a low incidence of resistance development since minor deviations in the conserved domains would prevent fusion of virus with endosomal membrane. In Phase II, we will progress the most promising scaffolds through a rational drug design program, and will test lead compounds for efficacy and toxicity in animal models. The most active compound, with the least toxicity, will advance to IND enabling studies. PUBLIC HEALTH RELEVANCE: Influenza is a highly infectious acute respiratory disease, characterized by recurrent annual epidemics and periodic major worldwide pandemics. Vaccines, currently the primary strategy for protection against influenza virus infection, are only effective if they match the circulating virus type(s) and cannot be developed in advance against new emerging pandemic strain(s). Our goal is to develop an anti-influenza therapeutic that will prevent virus entry by targeting the conserved fusion and receptor binding domains of envelope protein hemagglutinin (HA), and will be active against all subtypes, including a newly emerging pandemic strain.