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（由申请方提供）：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%。由于LASV和朱宁病毒（JUNV）的致病株具有气溶胶传播性和较高的发病率和死亡率，因此构成生物恐怖主义风险，并被列入卫生与公众服务部高度优先的A类物剂名单。由于这些病毒对国家安全构成威胁，目前还没有获得许可的预防性疫苗来保护民众。目前的抗沙粒病毒疗法仅限于使用非特异性抗病毒利巴韦林（Rib），其仅部分有效且与显著的副作用相关。缺乏快速和定量检测LASV和JUNV致病株增殖的测定方法，以及处理它们的生物安全控制水平4（BSL 4）实验室条件的要求，对开发HTS活动以鉴定候选抗病毒药物构成了巨大障碍。为了克服这个问题，我们已经产生了重组淋巴细胞性脉络丛脑膜炎病毒（rLCMV）表达报告基因的利益。这些病毒可以安全地用于基于细胞的筛选，其中可以在宽松的生物安全条件下基于报告基因活性容易地评估病毒增殖。我们建议使用这种方法，首先筛选LCMV增殖的抑制剂，然后鉴定具有抗高致病性LASV和JUNV的抗病毒活性的化合物。该提案的具体目标是：具体目标1。开发一种基于HT细胞的检测方法，以鉴定LCMV复制抑制剂。我们将使用携带GFP报告基因的三节段LCMV（r3 LCMV/GFP）来开发适合HTS的基于细胞的测定。将优化试验参数，以获得与HTS开发相容的Z'值。将使用已知的抗竞技场病毒药物来验证该测定，并确保我们的筛选平台可以识别在病毒周期的不同步骤中起作用的复制抑制剂。特殊目标2.筛选50，600个小分子的文库。我们将使用优化的测定来筛选高度多样化的化学库，其中包括破坏蛋白质-蛋白质相互作用的分子，针对靶向激酶优化的药效团，以及具有已知药理学特征的化合物库（Lopac）。EC 50值d 5 μ M和治疗指数（TI）e 30的候选人将被提升至SA 3。具体目标3。命中效价和特异性的验证和表征。将通过一系列二次试验评价命中，以确认活性，确定特异性并收集有关这些抑制剂作用机制的重要信息。将评估最有效的命中，以确定它们阻断LCMV复制的阶段。将在病毒产率降低测定中测试一小组化合物（<30），以在BSL 4实验室环境下鉴定具有抗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先导化合物将用于药物化学SAR优化，以获得效价、特异性和口服生物利用度。将在沙粒病毒感染的豚鼠模型中测试改进的化合物，并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阶段，以支持未来的人体试验。这些研究可能导致开发急需的抗病毒药物，用于治疗高致病性沙粒病毒感染。PHS 398/2590（Rev.06/09）