Novel Screen to Identify Arenavirus Inhibitors

鉴定沙粒病毒抑制剂的新屏幕

• 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
• 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

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（由适用提供）：LASSA发烧病毒（LASV）和其他体育症病毒家族的其他成员是重要的人类病原体，可能导致死亡率超过20％的死亡率发作。由于它们的气雾剂可迁移性以及发病率和死亡率升高，因此Junin病毒（JUNV）的LASV和致病菌株构成了生物恐怖风险，并且在卫生和公共服务部（DHHS）的高优先级A类药物列表中。强调这些病毒构成的国家安全威胁，没有持牌的预防疫苗来保护人口。当前的抗动脉粥样硬病毒疗法仅限于使用非特异性抗病毒利巴韦林（RIB），这仅是部分高效的，并且与显着的副作用相关。缺乏测定方法无法快速和定量检测LASV和JUNV致病菌株的多重蛋白，以及生物安全遏制级别4（BSL4）实验室条件的要求，以便在HTS运动中遇到巨大的障碍，以识别HTS运动以识别候选抗病毒药抗体药物。为了克服这个问题，我们已经产生了表达感兴趣的记者基因的重组淋巴细胞性绒毛膜炎病毒（RLCMV）。这些病毒可以安全地用于基于细胞的筛查中，其中可以根据记者基因活性在松弛的生物安全条件下轻松评估病毒繁殖。我们建议使用这种方法作为LCMV乘法抑制剂的第一个筛选，然后鉴定具有抗病毒活性的化合物对高致病性LASV和JUNV的化合物。该提案的具体目的是：特定目的1。开发基于HT细胞的测定法以识别LCMV复制的抑制剂。我们将使用带有GFP报告基因（R3LCMV/GFP）的三部分LCMV来开发可用于HTS的基于细胞的测定。测定参数将被优化，以实现HTS开发兼容的Z值。已知的抗ARANA病毒药物将用于验证测定法，并确保我们的筛查平台可以鉴定出在病毒周期不同步骤的复制抑制剂。特定目标2。筛选50,600个小分子的库。我们将使用优化的测定法来筛选一个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，优化用于靶向激酶的药算体以及具有已知药物剖面（LOPAC）的化合物库。 EC50值D 5€和治疗指数（TI）E 30的候选者将提高到SA3。特定目的3。验证和特异性的验证和表征。将通过一系列辅助测试来评估命中率，以确认活动，确定特异性并收集有关这些抑制剂作用机理的重要信息。将评估大多数潜在的命中，以确定它们阻止LCMV复制的阶段。将在病毒降低测定中测试一小部分化合物（<30），以鉴定在BSL4实验室环境下针对JUNV和LASV的抗病毒活性的化合物。在II期提案中，具有最佳特性的化学铅将用于医学化学优化，以实现效力，特异性和口服生物利用度。改进的化合物将在Arenaviru感染的豚鼠模型中进行测试，并将产生耐药菌株以确定作用机理。最好的化合物将推进临床前开发，以支持未来的人类试验。这些研究可能导致急需的抗病毒药以治疗高度致病性的体育症病毒感染。 PHS 398/2590（修订版06/09）页面延续格式页面