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

黄病毒 RNA 鸟苷基转移酶探针的高通量测定

• 批准号: 8204514
• 负责人: Brian Geiss
• 金额: $ 3.68万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-06 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8204514
• 关键词: Active Sites; Amino Acids; Antiviral Agents; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biological Assay; Categories; Cell Culture Techniques; Cells; Characteristics; Chemicals; Code; Computer Simulation; Culicidae; Dengue; Dengue Virus; Development; Disease; Drug Delivery Systems; Enzymes; Evaluation; Flavivirus; Flavivirus Infections; Foundations; GTP Binding; Genomics; Goals; Growth; Guanosine; Guanosine Monophosphate; Guanosine Triphosphate; Human; In Vitro; Infection; Laboratories; Lead; Life; Medical; Methyltransferase; Modeling; Monitor; Morbidity - disease rate; Mutation; N-terminal; National Institute of Allergy and Infectious Disease; Patients; Pharmaceutical Preparations; Positioning Attribute; Property; Proteins; Publishing; RNA; RNA Caps; Replicon; Resolution; Screening procedure; Site; Solid; Structure; Structure-Activity Relationship; Testing; Therapeutic; Translations; Validation; Viral; Viral Genome; Virus; West Nile virus; Yellow Fever; Yellow fever virus; analog; base; chemotherapeutic agent; cost effective; cytotoxic; cytotoxicity; design; drug development; guanylyltransferase; high throughput screening; human morbidity; human mortality; in vitro activity; in vivo; inhibitor/antagonist; interest; mRNA guanylyltransferase; mortality; novel; pathogen; pre-clinical; public health relevance; scaffold; small molecule; socioeconomics; therapy development; viral RNA

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.

描述（由申请人提供）：黄病毒（如登革热、黄热病和西尼罗河病毒）的感染是世界范围内的主要医学和社会经济问题，但目前还没有有效的抗病毒治疗药物来治疗黄病毒感染。因此，必须鉴定有效的、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加帽酶（CE）的鸟苷酰转移酶（GTase）活性位点的新型抑制剂，其可以作为用于治疗黄病毒感染的有效广谱化疗剂。CE在病毒RNA的5'端产生帽结构，其是病毒基因组的有效翻译所需的并且是病毒生长所必需的。GTP酶将鸟苷一磷酸从GTP转移到基因组RNA的5'末端，以产生帽0结构。通过CE破坏GTP结合抑制病毒复制，表明该功能对病毒活力至关重要。我们最近发表的GTP结合位点窝藏的GTP酶活性位点，增加了兴趣的GTP结合位点作为一个潜在的药物靶点。我们已经设计并成功地实现了一个简单而快速的体外高通量测定，以确定干扰GTP结合的CE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，目的是鉴定和优化化学上多样化的CE GTP结合抑制剂，以鉴定用于药物开发的先导化合物。将检测最佳抑制剂（在广度、效力和药物样特征方面）在细胞培养物中抑制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性质的先导化合物，适用于治疗黄病毒感染的临床前开发。 公共卫生关系：由蚊子传播的黄病毒感染引起的疾病在全世界引起显著的人类发病率和死亡率，然而目前没有有效的治疗剂来治疗感染的患者。本项目的目标是鉴定和表征广泛活性的抗病毒化合物，靶向保守的黄病毒加帽酶的鸟苷酰转移酶活性位点。