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， 核糖核酸酶L，抗病毒天然免疫的主要介质。在自然界中，RNase L在结合至 在干扰素（IFN）治疗和 病毒感染的细胞RNA酶L通过切割病毒和细胞RNA直接限制病毒复制。在 此外，RNase L通过刺激IFN-β的产生来放大和维持先天免疫。 然而，虽然2-5A是RNase L的有效激活剂，因此是抗病毒先天免疫的有效激活剂，但它具有以下性质： 这使得它不适合用作体内抗病毒剂。2-5A在血清中几分钟内降解， 细胞，不通过细胞膜并且是促凋亡的。两条早期线索显示 对RNA和DNA病毒都有抗病毒活性，但缺乏细胞毒性，这表明它们可能具有 比IFN的副作用少。在我们以前的工作中，包括MRCE New支持的研究， 机会赠款，我们已经确定了一系列小分子的RNase L激活剂与药物样 性质，基于使用新测定法对化学文库进行高通量筛选。在这个程序中 项目资助我们对RNase L的研究将与西尼罗河病毒和登革热病毒的研究相结合 (RP1)克里米亚-刚果出血热病毒和埃博拉病毒（RP 6）;辛德毕斯病毒，罗斯河病毒， 委内瑞拉马脑炎病毒和基孔肯雅病毒（RP 7）;裂谷热病毒、SARS-CoV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，我们将进行补充的方法，包括进一步的高通量筛选 (NTS)合成小分子和天然产品的活动，加上迭代的设计 和合成。此外，我们将探索使用肽核酸（PNAs）作为潜在的RNase L 活化剂。将使用纯化的重组RNase L测定命中物的生物化学效价。(2)到 通过使用X射线晶体学获得关于化合物结合的详细结构信息， 解决了与分离的配体结合结构域（ANK）结合的命中物的共晶体的结构和其他 RNase L.结构域原子结构将导致基于结构的药物设计和合成 对RNase L具有增加的亲和力和增强的抗病毒活性的化合物。(3)衡量和比较 我们将测定化合物在细胞培养物和小鼠中的抗病毒活性和毒性， 效力，并对广泛的RNA和DNA病毒病原体进行剂量范围毒性筛选 在两种细胞培养系统和野生型小鼠中与RNase L缺陷小鼠相比，

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.