Characterization of 3.47, a NPC1-targeting inhibitor of Ebola GP-mediated entry

2014-15年西非爆发的埃博拉病毒(EBOV)规模和持续时间都是史无前例的， 强调需要更深入地了解知情人士病毒生命周期的多个方面 发展急需的治疗方法。此前的研究发现，3.47是一种可逆的小分子 以受体Neimann-Pick C1(NPC1)为靶标的典型扎伊尔1976 Mainga EBOV的抑制剂， 导致胆固醇积聚并阻断表面糖蛋白(GP)与含有NPC1的晚期蛋白的相互作用 内体/溶酶体膜。尽管已经阐明了进入途径中的多个步骤， 到目前为止，3.47如何阻止EBOV GP介导的进入的确切机制尚未确定。 本研究方案的目标是：(1)识别和定位不同敏感度的ebov到3.47 使用病毒学和基因技术进行抑制，(2)使用AFP鉴定NPC1上3.47的结合位点 化学生物学与蛋白质生物化学相结合；(3)3.47作用机理分析 用生化方法研究3.47对GP介导的NPC1相互作用和细胞周期的影响 GP2触发。 这项提案的完成将使人们对一种抑制剂的作用机制有一个了解 EBOV GP介导的进入。确定3.47的作用机制不仅可以揭示3.47的作用机制 EBOV gp-NPC1相互作用的分子机制，但也为EBOV gp如何逃逸提供了线索 针对进入途径中的关键一步的潜在疗法。

The 2014-15 outbreak of Ebola virus (EboV) in West Africa was one of unprecedented size and duration, highlighting the need for a deeper understanding of multiple aspects of the viral lifecycle for the informed development of critically needed therapeutics. Previous studies identified 3.47, a reversible small molecule inhibitor of the prototypical Zaire 1976 Mayinga EboV that targets the receptor Neimann-Pick C1 (NPC1), causes cholesterol buildup and blocks the interaction of the surface glycoprotein (GP) to NPC1-containing late endosomal/lysosomal membranes. Although multiple steps in the entry pathway have already been elucidated, to date the precise mechanism of how 3.47 blocks EboV GP-mediated entry has yet to be determined. The goals of this research proposal are: (1) To identify and map EboVs with different sensitivities to 3.47 inhibition using virological and genetic techniques, (2) To identify the binding site of 3.47 on NPC1 using a combination of chemical biology and protein biochemistry and (3) To analyze the mechanism of 3.47 action using biochemical assays to interrogate the effect of 3.47 on the GP-mediated steps of NPC1 interaction and GP2 triggering. The completion of this proposal will provide an understanding of the mechanism of action of an inhibitor of EboV GP-mediated entry. Determining the mechanism of action of 3.47 can not only reveal insights into the molecular mechanisms of EboV GP-NPC1 interaction, but also provide clues to how the EboV GP can escape potential therapeutics that target a critical step in the entry pathway.