Broad Spectrum Antivirals Against Biodefense Select Agents That Activate RNase L

点击翻译按钮获取中文摘要

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.