High throughput screening of orthosteric inhibitors of flavivirus protease

黄病毒蛋白酶正构抑制剂的高通量筛选

基本信息

批准号：
10318299
负责人：
HONGMIN LI
金额：
$ 56.34万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-02-15 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10318299
关键词：
Acquired Immunodeficiency Syndrome Active Sites Address Affect Affinity Animals Antiviral Agents Antiviral Therapy Arboviruses Area Binding Biological Assay Biological Availability Biological Models Cell Culture Techniques Cells Charge Cleaved cell Clinical Complement Complex Dengue Dengue Hemorrhagic Fever Dengue Virus Development Development Plans Disease Disease Outbreaks Drug Kinetics Drug Targeting Ensure Enzyme Inhibitor Drugs Enzyme Interaction Enzymes Flavivirus Flavivirus Infections Fluorescence Resonance Energy Transfer Generations Genes Goals Growth Hepatitis C Infection Japanese Encephalitis Vaccines Japanese encephalitis virus Lead Libraries Life Luciferases Mass Vaccinations Meningitis Microcephaly Modeling Multienzyme Complexes Mus Mutagenesis Mutation Outcome Study Peptide Hydrolases Pharmaceutical Preparations Polyproteins Populations at Risk Prevention Protease Inhibitor Proteins Public Health Renilla Luciferases Replicon Research Resistance Resources Site Solid Structure Structure-Activity Relationship System Technology Testing Therapeutic Tick-Borne Encephalitis Virus Toxic effect Toxicity Tests Vaccinated Vaccines Variant Viral Viral Genome Viral Physiology Viral Proteins Virus Virus Assembly Virus Replication West Nile Encephalitis West Nile virus Work Yellow fever virus ZIKA Zika Virus anti-viral efficacy arthropod-borne base burden of illness combat drug development drug discovery efficacy evaluation fighting high throughput screening human disease human pathogen in vivo inhibitor/antagonist innovation nanomolar novel public health priorities screening small molecule libraries therapeutic target therapy design tool vaccine development viral resistance virology

项目摘要

Project Summary/Abstract: Many flaviviruses such as Dengue virus, Zika virus, West Nile virus, and Yellow Fever virus cause significant human diseases. However, no clinically approved antiviral therapy is available for treatment of flavivirus infections. Therefore, the development of vaccines and antiviral agents for prevention and treatment of flavivirus infections is a clear public health priority. The long- range goal of this proposal is to address this need by developing a new system to identify, screen and validate a class of potential flavivirus inhibitors, and successfully identify and analyze candidate compounds that show promise in disrupting viral survival. During flaviviral infection of the host cell, a single viral polyprotein is synthesized from the viral genome. This polyprotein is cleaved into its component proteins by a combination of host cell proteases and a two-component viral protease, encoded in genes NS2B and NS3. The function of this viral protease is indispensable for virus assembly and replication. The objectives of this project are to develop innovative high throughput screening strategies to identify and characterize compounds that orthosterically inhibit the function of the critical protease NS2B- NS3. To achieve project objectives, in Specific Aim 1, we will develop and perform novel high- throughput screening primary, secondary, and tertiary assays capable of screening large chemical libraries to identify orthosteric inhibitors. In Specific Aim 2, after optimizing these assays, we will perform large-scale HTS to screen a compound library of >400,000 compounds for potential protease inhibitor candidates, employing five levels of screening to fully eliminate false positives. In Specific Aim 3, we will evaluate the efficacy of the resulting candidate compounds for their capacity to block protease activity, and determined their modes of actions. Lead compounds against the protease will be tested for cellular toxicity, reduction of flavivirus titer in cell culture, resistant variants, and limited structure-activity relationship. We will ultimately test the toxicity, pharmacokinetics, and efficacy in live animals for the most potent compounds. We anticipate the outcome of this study will be the generation of crucial new information on flavivirus inhibitor-enzyme interaction at structurally significant sites. This creates the potential for identifying novel classes of flaviviral inhibitors, suitable for addressing the global public health need for new antiviral agents to fight flaviviruses.

项目摘要/摘要：许多黄病毒，例如登革热病毒，寨卡病毒，西尼罗河病毒和黄热病病毒引起严重的人类疾病。但是，没有临床批准的抗病毒疗法可用用于治疗黄病毒感染。因此，疫苗和抗病毒剂的发展对于预防和治疗黄病毒感染是明显的公共卫生优先事项。长期该建议的范围目标是通过开发一个新系统来识别，筛选和验证一类潜在的黄病毒抑制剂，并成功识别和分析候选化合物，这些化合物在破坏病毒存活方面有望。在宿主细胞的黄素感染期间，从病毒中合成单个病毒多蛋白基因组。该多蛋白通过宿主细胞的组合裂解到其成分蛋白中蛋白酶和两个分量的病毒蛋白酶，编码在基因NS2B和NS3中。功能这种病毒蛋白酶在病毒组装和复制中是必不可少的。目标的目标项目将制定创新的高通量筛选策略来识别和表征正向抑制关键蛋白酶NS2B-的功能的化合物 NS3。为了实现项目目标，在特定的目标1中，我们将开发和执行新颖的高级 - 吞吐量筛查主要，次要和第三次测定，能够筛选大化学文库以鉴定定位抑制剂。在特定的目标2中，在优化了这些测定，我们将执行大规模的HTS筛选> 400,000种化合物的复合库对于潜在的蛋白酶抑制剂候选物，采用五个级别的筛查以完全消除误报。在特定目标3中，我们将评估最终候选人的功效它们可以阻止蛋白酶活动的能力并确定其作用模式的化合物。针对蛋白酶的铅化合物将用于细胞毒性，黄素降低细胞培养，抗性变体和有限的结构活性关系中的滴度。我们最终会测试活动物中最有效化合物的毒性，药代动力学和功效。我们预计这项研究的结果将是有关关键的新信息的产生黄病毒抑制剂 - 酶的相互作用在结构意义的位点。这会产生潜力用于识别新颖的黄素抑制剂，适合针对全球公众新的抗病毒药物来对抗黄素病毒的健康需求。