A High-Throughput Assay for Probes of the Flavivirus RNA Guanylyltransferase

描述（由申请人提供）：黄病毒感染（如登革热、黄热病和西尼罗河病毒）是世界范围内主要的医学和社会经济问题，但目前尚无有效的抗病毒治疗方法来治疗黄病毒感染。因此，必须确定有效的、选择性的和具有成本效益的抗病毒化合物。该项目的总体目标是发现黄病毒RNA盖顶酶（CE）的GTase活性位点的新型抑制剂，这些抑制剂可以作为治疗黄病毒感染的有效广谱化疗药物。CE在病毒rna的5'端产生帽状结构，这是病毒基因组有效翻译所必需的，也是病毒生长所必需的。GTase将鸟苷单磷酸从GTP转移到基因组RNA的5'端，从而产生cap 0结构。CE破坏GTP结合可抑制病毒复制，表明该功能对病毒生存能力至关重要。我们最近发表了GTP结合位点含有GTase活性位点，这增加了人们对GTP结合位点作为潜在药物靶点的兴趣。我们设计并成功实施了一种简单快速的体外高通量测定方法，以鉴定干扰GTP结合的化合物。在本申请中，我们建议通过在MLPCN内进行额外的HTS来扩展我们之前的筛选，目的是识别和优化化学上多样化的CE gtp结合抑制剂，以确定用于药物开发的先导化合物。最佳抑制剂（在宽度，效力和药物样特性方面）将测试其在细胞培养中抑制GTase酶活性，细胞毒性和抗病毒活性的能力。该项目将导致快速有效地鉴定黄病毒复制抑制剂，最终目标是描述具有药物样特性的先导化合物，适合用于治疗黄病毒感染的临床前开发。

DESCRIPTION (provided by applicant): Infection by flaviviruses such as dengue, yellow fever, and West Nile is a major medical and socio-economic problem worldwide, yet effective antiviral therapeutics to treat flavivirus infection are not currently available. As such, it is imperative that potent, selective, and cost-effective antiviral compounds be identified. The overall goal of this project is the discovery of novel inhibitors of the guanylyltransferase (GTase) active site of the flavivirus RNA capping enzyme (CE) that could serve as effective broad-spectrum chemotherapeutic agents for the treatment of flavivirus infection. The CE generates the cap structure at the 5' end of viral RNAs that is required for efficient translation of the viral genome and is essential for viral growth. The GTase transfers a guanosine monophosphate from GTP to the 5' end of the genomic RNA to produce the cap 0 structure. Disruption of GTP binding by the CE inhibits viral replication, indicating that this function is critical to viral viability. We recently published that the GTP binding site harbors the GTase active site, increasing interest in the GTP-binding site as a potential drug target site. We have designed and successfully implemented a simple and rapid in vitro high-throughput assay to identify compounds that interfere with GTP binding by the CE. In this application we propose to expand on our previous screening by performing additional HTS within the MLPCN with the goal of identifying and optimizing a chemically diverse set of CE GTP-binding inhibitors in order to identify lead compounds for drug development. The best inhibitors (in terms of breadth, potency and drug-like characteristics) will be tested for their ability to inhibit GTase enzymatic activity, cytotoxicity, and antiviral activity in cell culture. This project will result in the rapid and efficient identification of inhibitors of flavivirus replication with the ultimate goal of describing lead compounds with drug-like properties suitable for preclinical development for the treatment flavivirus infection. PUBLIC HEALTH RELEVANCE: Disease resulting from mosquito-borne flavivirus infection causes significant human morbidity and mortality throughout the world, yet there currently are no effective therapeutics to treat infected patients. The goal of this project is to identify and characterize broadly active antiviral compounds targeting the guanylyltransferase active site of the conserved flavivirus capping enzyme.