Broad Spectrum Antivirals Against Biodefense Select Agents That Activate RNase L

项目总结(见说明)： 我们在这个项目中的目标是开发和研究广谱抗病毒药物，这些药物通过激活 核糖核酸酶L，抗病毒天然免疫的主要介体。在自然界中，核糖核酸酶L是在与 不寻常的5‘-磷酸化，2’-5‘连接的寡腺苷酸盐(2-5A)在干扰素(干扰素)治疗和治疗后积累 被病毒感染的细胞。核糖核酸酶L通过切割病毒和细胞RNA直接限制病毒复制。在……里面 此外，核糖核酸酶L通过刺激干扰素-β的产生来放大和维持先天免疫。 然而，尽管2-5A是核糖核酸酶L的有效激活剂，因此具有抗病毒天然免疫功能，但它具有 这使得它不适合在体内用作抗病毒药物。2-5A在几分钟内在血清和 细胞，不通过细胞膜，是促凋亡的。早期的两个导联显示出广谱 对RNA和DNA病毒都有抗病毒活性，但缺乏细胞毒性，这表明它们可能有 与IFN相比，副作用更少。在我们之前的工作中，包括由MRCE New支持的研究 机会基金，我们已经确定了一系列具有药物样核糖核酸酶L的小分子激活剂 属性，基于使用一种新的分析方法高通量筛选化学文库。在本计划中 项目资助我们对核糖核酸酶L的研究将与西尼罗河病毒和登革热病毒的调查相结合 (RP1)；克里米亚-刚果出血热病毒和埃博拉病毒(RP6)；辛德比斯病毒，罗斯河病毒， 委内瑞拉马脑炎病毒和基孔肯雅病毒(RP7)；裂谷热病毒，SARS冠状病毒和 甲型流感病毒(核心D)。我们的具体目标是：(1)识别和表征下一代 抗病毒药物我们将进行补充方法，包括进一步的高通量筛选 (NTS)合成小分子和天然产品的运动与迭代设计轮次相结合 和综合体。此外，我们还将探索将肽核酸(PNA)作为潜在的核糖核酸酶L 激活剂。HITS的生化效力将用纯化的重组核糖核酸酶L(2)测定。 使用x射线结晶学获得关于化合物结合的详细结构信息，我们将获得和 解决与分离的配体结合域(ANK)结合的HITS的共晶结构 核糖核酸酶的结构域原子结构将导致基于结构的药物设计和合成 与核糖核酸酶L亲和力增强和抗病毒活性增强的化合物。(3)衡量和比较 在细胞培养和小鼠体内，我们将测定化合物的抗病毒活性和毒性。 对广泛的RNA和DNA病毒病原体进行效力和剂量范围的毒性筛选 在两种细胞培养系统和野生型小鼠中，与RNaseL缺陷小鼠进行比较。

PROJECT SUMMARY (See instructions): Our GOAL in this project is to develop and study broad-spectrum antiviral agents that function by activating RNase L, a principal mediator of antiviral innate immunity. In nature, RNase L is activated upon binding to unusual 5'-phosphorylated, 2'-5' linked oligoadenylates (2-5A) that accumulate in interferon (IFN)-treated and virus-infected cells. RNase L directly restricts viral replication by cleaving viral and cellular RNAs. In addition, RNase L amplifies and perpetuates innate immunity through stimulating the production of IFN-p. However, while 2-5A is a potent activator of RNase L, and thus of antiviral innate immunity, it has properties that render it unsuitable for use as an antiviral agent in vivo. 2-5A is degraded within minutes in sera and in cells, does not transit the cell membrane and is pro-apoptotic. Two early leads showed broad-spectrum antiviral activities against both RNA and DNA viruses, but lacked cytotoxicity suggesting that they might have fewer adverse effects than IFNs. In our previous work, including studies supported by MRCE New Opportunities grants, we have identified a series of small molecular activators of RNase L with drug-like properties, based on high throughput screening of chemical libraries using a novel assay. In this Program Project Grant our studies on RNase L will integrate with investigation on West Nile virus and dengue virus (RP1); Crimean-Congo hemorrhagic fever virus and Ebola virus (RP6); Sindbis virus, Ross river virus, Venezuelan equine encephalitis virus and Chikungunya virus (RP7); Rift Valley Fever virus, SARS-CoV and influenza A virus (Core D). Our SPECIFIC AIMS are: (1) To identify and characterize the next generation of antiviral agents we will perform complementary approaches including further high throughput screening (NTS) campaigns of synthetic small molecules and natural products coupled with iterative rounds of design and synthesis. In addition, we will explore the use of peptide nucleic acids (PNAs) as potential RNase L activators. Biochemical potency of the hits will be determined with purified, recombinant RNase L. (2) To obtain detailed structural information about compound binding using x-ray crystallography, we will obtain and solve the structures of co-crystals of the hits bound to the isolated ligand binding domain (ANK) and other domains of RNase L. The atomic structures will lead to structure-based drug design and synthesis of compounds with increased affinity for RNase L and enhanced antiviral activity. (3) To measure and compare the antiviral activities and toxicities of compounds in cell culture and in mice we will determine antiviral potencies and perform dose-ranging toxicity screens against a wide-range of RNA and DNA viral pathogens in both cell culture systems and in wild type mice in comparison with mice deficient in RNase L.