Novel Screen to Identify Arenavirus Inhibitors

鉴定沙粒病毒抑制剂的新屏幕

基本信息

批准号：
8529177
负责人：
JUAN LAMA
金额：
$ 29.97万
依托单位：
RETROVIROX, INC.
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-02-01 至 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8529177
关键词：
Adverse effects Aerosols Africa Antiviral Agents Arenavirus Arenavirus Infections Biological Assay Biological Availability Biological Warfare Bioterrorism Categories Cavia Cell Density Cells Cessation of life Chemicals Containment Detection Development Disease Drug resistance Engineering Ensure Family Family member Fluorescence Future Genes Geographic Locations Goals HIV Infections Health Human Immunocompromised Host In Vitro Individual Infection Junin virus Laboratories Lassa Fever Lassa fever virus Lassa virus Lead Libraries Licensing Life Cycle Stages Luciferases Lymphocytic choriomeningitis virus Modeling Morbidity - disease rate National Security Oral Pharmaceutical Chemistry Pharmaceutical Preparations Phase Phosphotransferases Population Populations at Risk Preventive Principal Investigator Procedures Property Public Health RNA Viruses Reader Readiness Reagent Recombinants Reporter Reporter Genes Research Institute Ribavirin Risk Safety Scientist Signal Transduction Source Specificity Staging System Testing Therapeutic Index Time Toxic effect United States Dept. of Health and Human Services Vaccines Validation Viral Viral Hemorrhagic Fevers Virus Virus Diseases Virus Replication Work base biodefense cell killing cell type clinically significant combat compound 30 congenital infection drug candidate experience high throughput screening improved inhibitor/antagonist interest member microbial mortality neglect novel nucleoside analog pathogen pharmacophore pre-clinical programs protein protein interaction recombinant virus resistant strain screening small molecule small molecule libraries weapons

项目摘要

DESCRIPTION (provided by applicant): Lassa fever virus (LASV) and other members of the arenavirus family are important human pathogens that can cause hemorrhagic fevers with mortality rates over 20%. Because their aerosol transmissibility and elevated morbidity and mortality, LASV and pathogenic strains of Junin virus (JUNV) pose a bioterrorism risk and are in the list of high priority Category A agents from the department of Health and Human Services (DHHS). Underscoring the national security threat posed by these viruses, there are no licensed preventive vaccines to protect the population. Current anti-arenavirus therapies are limited to the use of the non-specific antiviral ribavirin (Rib), which is only partially efficient and associted with significant side effects. The lack of assays to rapidly and quantitatively detect multiplicatin of LASV and JUNV pathogenic strains together with the requirement of bio-safety containment level 4 (BSL4) laboratory conditions to handle them pose great obstacles for the development of HTS campaigns to identify candidate antiviral drugs. To overcome this problem, we have generated recombinant lymphocytic choriomeningitis viruses (rLCMV) expressing reporter genes of interest. These viruses can be safely used in cell-based screens in which virus multiplication can be readily assessed based on reporter gene activity under relaxed bio-safety conditions. We propose to use this approach to first screen for inhibitors of LCMV multiplication, and then identify compounds with antiviral activity against the highly pathogenic LASV and JUNV. The specific aims of this proposal are: Specific Aim 1. To develop an HT cell-based assay to identify inhibitors of LCMV replication. We will use a tri-segmented LCMV carrying a GFP reporter gene (r3LCMV/GFP) to develop a cell-based assay amenable for HTS. Assay parameters will be optimized to achieve Z' values compatible for the development of HTS. Known anti-arena viral drugs will be used to validate the assay and ensure that our screening platform can identify inhibitors of replication acting at different steps of the virus cycle. Speciic Aim 2. Screening of a library of 50,600 small-molecules. We will use the optimized assay to screen a highly diverse chemical library, which will include molecules disrupting protein-protein interactions, pharmacophores optimized for targeting kinases, and a library of compounds with known pharmacological profiles (Lopac). Candidates with EC50 values d 5 ¿M and therapeutic indices (TI) e 30 will be advanced to SA3. Specific Aim 3. Validation and characterization of hit potency and specificity. Hits will be evaluated with a battery of secondary tests to confirm activity, determine specificity and gather important information on the mechanism of action of these inhibitors. Most potent hits will be assessed to determine the stage at which they block LCMV replication. A small set of compounds (<30) will be tested in a virus yield reduction assay to identify compounds with antiviral activity against JUNV and LASV under BSL4 laboratory settings. In a Phase II proposal, chemical leads with the best properties will be used for medicinal chemistry SAR optimization for potency, specificity and oral bioavailability. Improved compounds will be tested in a guinea pig model of arenavirus infection and drug-resistant strains will be generated to determine mechanism of action. The best compounds will be advanced into preclinical development to support future human trials. These studies may lead to the development of much needed antivirals for the treatment of highly pathogenic arenavirus infections. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page

描述（申请人提供）：拉沙热病毒（LASV）和沙粒病毒家族的其他成员是重要的人类病原体，可引起出血热，死亡率超过 20%，因为它们具有气溶胶传播性以及较高的发病率和死亡率，LASV 和致病株。胡宁病毒 (JUNV) 构成生物恐怖主义风险，并被列入美国卫生与公众服务部 (DHHS) 的高度优先 A 类病毒名单。由于这些病毒造成的安全威胁，目前还没有获得许可的预防性疫苗来保护人群，目前的抗沙粒病毒疗法仅限于使用非特异性抗病毒药物利巴韦林 (Rib)，这种药物仅部分有效且具有显着的副作用。缺乏快速、定量检测 LASV 和 JUNV 致病株增殖的检测方法，以及处理它们的生物安全防护 4 级（BSL4）实验室条件的要求，给该病毒的发展带来了巨大障碍。 HTS 致力于识别候选抗病毒药物，为了克服这一问题，我们生成了表达感兴趣的报告基因的重组淋巴细胞脉络膜脑膜炎病毒 (rLCMV)，这些病毒可以安全地用于基于细胞的筛选，其中可以根据细胞增殖情况轻松评估病毒的增殖情况。我们建议使用这种方法首先筛选 LCMV 增殖的抑制剂，然后鉴定对高致病性 LASV 和 JUNV 具有抗病毒活性的化合物。该提案的具体目标是：具体目标 1. 开发基于 HT 细胞的测定法来鉴定 LCMV 复制抑制剂我们将使用携带 GFP 报告基因 (r3LCMV/GFP) 的三段 LCMV 来开发细胞-。适合 HTS 的测定参数将被优化，以实现与 HTS 开发兼容的 Z' 值，将用于验证测定并确保我们的筛选平台可以识别抑制剂。具体目标 2. 筛选 50,600 个小分子文库我们将使用优化的检测方法筛选高度多样化的化学文库，其中包括破坏蛋白质-蛋白质相互作用的分子、药效团。针对激酶进行了优化，以及具有已知药理学特征的化合物库 (Lopac)，其 EC50 值 d 5 ¿ M 和治疗指数 (TI) e 30 将提升至 SA3。命中效力和特异性的验证和表征将通过一系列二次测试进行评估，以确认活性、确定特异性并收集有关命中的重要信息。将评估这些抑制剂的作用机制，以确定它们阻断 LCMV 复制的阶段。将在病毒产量减少测定中测试一小组化合物（<30），以确定具有抗病毒活性的化合物。在 BSL4 实验室环境下的 JUNV 和 LASV 将使用具有最佳特性的化学先导化合物来优化药物化学 SAR 的效力、特异性和口服生物利用度，并在沙粒病毒感染的豚鼠模型中进行测试。将产生耐药菌株以确定作用机制，最好的化合物将进入临床前开发以支持未来的人体试验，这些研究可能会导致开发出用于治疗病原体的急需的抗病毒药物。沙粒病毒感染。 PHS 398/2590（修订版 06/09）页继续格式页