A pilot study for development of inhibitors of influenza virus

摘要 由于高致病性禽流感病毒在各大洲家禽种群中的广泛传播和人类感染病例的增加，全球大流行性流感的威胁正在升级。我们设计了一项计划，以开发针对核蛋白(NP)的新型抗病毒药物。NP在所有流感病毒株中高度保守，是病毒复制所必需的，这使其成为比神经氨酸酶更具吸引力的抗病毒靶点。这项先导研究有两个目标： 目的1.筛选NP抑制剂。我们已经开发了一种96孔板形式的NP核苷酸结合的高通量(HTP)分析方法。将对10,000种不同化合物的文库进行筛选，以确定阻止NP与核苷酸结合的抑制剂。HTP筛选产生的候选病毒将被测试是否抑制NP与核苷酸的组装，以及流感病毒在细胞培养感染中的复制。初步 使用800种化合物的结果表明，在筛选的化合物中，约有1.4%的化合物影响NP与核苷酸的结合。这些化合物的抗病毒研究正在进行中。 目的2.含抑制剂复合体中NP的结构。为了绘制NP与候选缓蚀剂相互作用的详细结构图，我们将通过X射线结晶学确定NP与缓蚀剂化合物共结晶的三维结构。NP与缓蚀剂形成的复合体的晶体结构有助于我们设计更有效的缓蚀剂。 先导研究的目标是产生概念验证结果和NP-抑制剂复合体的结构，以便我们能够进一步提高这类抑制剂的疗效。设计核蛋白抑制剂作为流感病毒的抗病毒药物是以前从未做过的。在先前的工作中，确定了一种有效的流感病毒融合抑制物(EC50=1.0 NM)。除了对抑制剂进行化学修饰外，下一阶段还将进行其他研究，以进一步使用这类化合物，包括：测试H5N1和H2N2菌株的抗生命活性；测试小鼠模型的抗病毒活性；HA与抑制剂的结晶。

Abstract The threat of global pandemic influenza is escalating due to the wide spread of the highly pathogenic avian influenza virus in poultry populations through continents and increasing cases of human infection. We devised a plan to develop novel antivirals targeting the nucleoprotein (NP). NP is highly conserved among all influenza virus strains and is indispensable for virus replication, which makes it a more attractive antiviral target than the neuraminidase. Two aims are proposed for the pilot study: Aim 1. Screen for NP inhibitors. We have developed a high throughput (HTP) assay for NP nucleotide binding in a 96-well plate format. A library of 10,000 diversified compounds will be screened to identify inhibitors that block NP binding to nucleotide. Candidates yielded from the HTP screen will be tested for inhibition of NP assembly with nucleotide, and influenza virus replication in cell culture infection. Preliminary results using 800 compounds showed that about 1.4% of the compounds screened affect NP binding with nucleotide. Antiviral studies of these compounds are in progress. Aim 2. Structure of NP in complex with inhibitors. To map the detailed structure of NP interaction with candidate inhibitors, we will determine the 3D structure of NP cocrystallized with inhibitor compounds by Xray crystallography. The crystal structure of NP in complex with inhibitors will help us in design of more potent inhibitors. The goal of the pilot study is to produce proof-of-concept results and a structure of NP-inhibitor complex so we can further improve the efficacy of this class of inhibitors. Design of nucleoprotein inhibitors as antivirals against influenza virus has never been done before. During the previous effort, a potent fusion inhibitor of influenza viruses (EC50 =1.0 nM) was identified. In addition to chemical modification of the inhibitor, other studies will be carried out in the next period to further emploit this class of compounds, including: Test antivital activities with H5N1 and H2N2 strains; Test antiviral activities in a mouse model; Cocrystallization of HA with inhibitors.