Novel Screen to Identify Arenavirus Inhibitors

描述(申请人提供)：拉沙热病毒(LASV)和阿雷纳病毒家族的其他成员是重要的人类病原体，可导致出血热，死亡率超过20%。由于LASV和Junin病毒(JUNV)的气溶胶可传播性以及高发病率和高死亡率，它们构成了生物恐怖主义风险，并被列入卫生与公众服务部(DHHS)高度优先的A类药物名单。突显这些病毒构成的国家安全威胁的是，目前还没有获得许可的预防性疫苗来保护人口。目前的抗病毒疗法仅限于使用非特异性抗病毒利巴韦林(Rib)，这种药物只有部分有效，且有显著的副作用。缺乏快速、定量检测LASV和JUNV致病毒株增殖的方法，以及生物安全遏制级别4(BSL4)实验室条件的要求，给HTS活动的发展带来了巨大的障碍，以确定候选抗病毒药物。为了克服这个问题，我们产生了表达感兴趣的报告基因的重组淋巴细胞性脉络膜脑膜炎病毒(RLCMV)。这些病毒可以安全地用于基于细胞的筛选，在放松的生物安全条件下，可以很容易地根据报告基因的活性来评估病毒的增殖。我们建议使用这种方法首先筛选LCMV增殖的抑制剂，然后鉴定对高致病性LASV和JUNV具有抗病毒活性的化合物。这项建议的具体目的是：具体目的1。建立一种基于HT细胞的检测方法，以确定LCMV复制的抑制物。我们将使用携带GFP报告基因的三节段LCMV(r3LCMV/GFP)来建立一种适合HTS的细胞检测方法。测试参数将进行优化，以达到与HTS发展相适应的Z‘值。已知的抗ARENA病毒药物将用于验证该分析，并确保我们的筛选平台可以识别在病毒周期的不同步骤发挥作用的复制抑制物。特殊目标2.筛选包含50,600个小分子的文库。我们将使用优化的方法来筛选一个高度多样化的化学库，其中将包括破坏蛋白质-蛋白质相互作用的分子，优化的靶向蛋白激酶的药效团，以及一个具有已知药理特征的化合物库(LOPAC)。EC50值d 5？M和治疗指数(TI)e 30的候选人将晋级为SA3。具体目标3.确认和表征HIT的效力和特异性。HITS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这些抑制剂作用机制的重要信息。将对大多数有效的命中进行评估，以确定它们阻止LCMV复制的阶段。一小部分化合物(&lt；30)将在病毒产量减少试验中进行测试，以确定在BSL4实验室环境下对JUNV和LASV具有抗病毒活性的化合物。在第二阶段的提案中，具有最好性能的化学引线将用于药物化学SAR的有效性、特异性和口服生物利用度的优化。改进后的化合物将在豚鼠ArenaVirus感染模型中进行测试，并将产生抗药性菌株以确定其作用机制。最好的化合物将被推进到临床前开发，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来治疗高致病性ArenaVirus感染。PHS 398/2590(06/09版)页面续格式页面

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