Discovery of influenza virus polymerase inhibitors

项目摘要 在这项申请中，我们用新的靶点来解决流感药物的需求，特别是我们建议 发现针对流感病毒聚合酶复合体的抗病毒化合物。目前只有一家 临床上有用的一类流感药物；神经氨酸酶抑制剂。依赖单一的药物类别是 有问题，因为耐药病毒的出现可能会使这些药物无效，就像已经发生的那样 金刚烷类药物以病毒M2蛋白为靶点。事实上，对奥司他韦耐药性的担忧是一种 口服神经氨酸酶抑制剂是最广泛使用的流感处方药，它推动了流感的呼声 具有新靶点和新机制的药物。病毒聚合酶是所有 病毒，并已被证明是开发有效的抗病毒药物的极好靶点，但迄今为止还没有 聚合酶抑制剂已被批准用于流感治疗。在初步研究中，我们进行了一项 使用基于细胞的分析高通量筛选了900,000个化合物文库，并确定了744个匹配 流感的抗病毒活性。我们建议使用一种新的体外流感聚合酶检测来快速鉴定 在这些有效的命中中有聚合酶抑制剂。这种独特的分析方法捕捉到了所有必要的步骤 最终的RNA产物的形成，从而使我们能够识别出抑制 核酸内切酶活性、mRNA帽结合、RNA结合和RNA合成。具体的化验将是 被用来阐明抗病毒机制，我们还将进行研究，以选择抗药性作为 识别目标和确定抵抗障碍的手段。我们的总体目标是确定一个或多个 流感病毒聚合酶抑制剂是有效的，在多种甲型流感中表现出抗病毒广度 病毒亚型和乙型流感病毒。这些化合物为抗病毒提供了一个令人兴奋的起点 但也将作为正在进行的确定流感病毒聚合酶功能的努力的探针。

Project Summary In this application we address the need for influenza drugs with novel targets, and specifically we propose to discover antiviral compounds that target the influenza virus polymerase complex. Currently there is only one class of influenza drug that is clinically useful; the neuraminidase inhibitors. Relying on a single drug class is problematic as the emergence of resistant virus could render these drugs ineffective as has already happened with the adamantane drugs targeting the viral M2 protein. In fact, concerns about resistance to oseltamivir, an oral neuraminidase inhibitor that is the most widely prescribed influenza drug, are driving the call for influenza drugs with new targets and mechanisms. The viral polymerase is an essential catalytic component of all viruses, and has proven to be an excellent target for development of potent antiviral drugs, but as yet no polymerase inhibitors have been approved for influenza treatment. In preliminary studies we have performed a high-throughput screen of a 900,000 compound library using a cell-based assay and identified 744 hits with influenza antiviral activity. We propose to use a novel in vitro influenza polymerase assay to rapidly identify polymerase inhibitors amongst these validated hits. This unique assay captures all the essential steps leading to formation of the final RNA product and thus will allow us to identify novel compounds that inhibit endonuclease activity, mRNA cap-binding, RNA-binding, and RNA synthesis. Specific assays will be employed to elucidate the antiviral mechanism and we will also perform studies to select for resistance as a means to identify the target and determine the barrier to resistance. Our overall goal is to identify one or more influenza virus polymerase inhibitors that are potent and display antiviral breadth across multiple influenza A virus subtypes and influenza B virus. Such compounds present an exciting starting point for antiviral development but will also serve as probes in ongoing efforts to define influenza virus polymerase function.