Activity-Based Protein Profiling of Arenavirus-Host Interactions

基于活性的沙粒病毒-宿主相互作用的蛋白质分析

• 批准号: 8970028
• 负责人: Juan C. de la Torre
• 金额: $ 23.69万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-28 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8970028
• 关键词: Adverse effects; Affect; Africa; Alkynes; Antiviral Agents; Area; Arenavirus; Arenavirus Infections; Biological; Biological Assay; Biotin; Calculi; Cell Density; Cell physiology; Cells; Chemistry; Complex; Development; Diazomethane; Disease; Environment; Exhibits; Genetic; Goals; Gold; Health; Human; Hydrolase; Individual; Infection; Junin virus; Label; Lassa Fever; Libraries; Licensing; Life Cycle Stages; Longitudinal Studies; Lymphocytic choriomeningitis virus; Mass Spectrum Analysis; Molecular Target; Monitor; Morbidity - disease rate; Pathogenesis; Pathologic Processes; Physiological Processes; Play; Process; Property; Proteins; Proteome; Public Health; RNA Viruses; RNA chemical synthesis; Readiness; Recombinants; Ribavirin; Role; Serine Hydrolase; Specificity; Technology; Testing; Therapeutic; Therapeutic Index; Time; Toxic effect; Travel; Vaccines; Validation; Viral Hemorrhagic Fevers; Virus; Virus Diseases; Virus Replication; Wit; activity-based protein profiling; base; biodefense; cellular targeting; clinically significant; combat; drug candidate; drug development; inhibitor/antagonist; metropolitan; mortality; neglect; novel; novel strategies; novel virus; pathogen; public health relevance; transforming virus; virus host interaction

 DESCRIPTION (provided by applicant): Arenaviruses are important human pathogens with several of them causing hemorrhagic fever (HF) disease that pose an important public health problem in their endemic regions. In addition, the worldwide-distributed prototypic arenavirus LCMV is a neglected human pathogen of clinical significance. Moreover, arenaviruses represent a credible biodefense threat. There are not FDA-licensed vaccines and current anti-arenavirus therapy is limited to the use of ribavirin, which is only partially effective. Therefore, there is n unmet need for the development of novel anti-arenaviral therapeutics, which will be facilitated by the characterization of arenavirus-host cell protein interactions contributing to virus multiplication. Activity-based protein profiling (ABPP) is a novel approach that permits to monitor the effects of viral infections on the functional state of the host proteome to identify novel viru-host protein interactions that may affect cell physiology, virus propagation and pathogenesis. The goal of this exploratory R21 application is to use ABPP of serine hydrolases (SHs) to identify novel arenavirus-host interactions, as well as targets and candidate drugs, to develop strategies to combat arenavirus infections. We focus on SHs because: 1) SHs represent 1% of the mammalian proteome and are involved in many physiological and pathological processes. 2) We have observed altered SH activities in LCMV-infected cells and some SH inhibitors exhibited anti-LCMV activity. Our specific aims (SA) are: SA1. Identify SH inhibitors with anti-arenaviral activity. We will test the hypothesis that specific SH contribute to the arenavirus life cycle and that their inhibition will interfere with virus multiplication. We will use a novel cell-based assay to screen a library of 250 SH inhibitors to identify those with anti-LCMV activity. Candidates with high inhibitory potency (EC90 ≤ 5 µM) and therapeutic index (TI = CC50/EC90) ≥ 30 will be selected for studies aimed at defining molecular targets and mechanism of action. SA2. Validation and characterization of selected hits. We will test the hypothesis that SH inhibitors with anti- LCMV activity inhibit specific steps of the virus life cycle. We will use established cell-based assays to determine the effects of selected hits on cell entry, RNA synthesis and budding of LCMV, as well as Lassa and Junin the two HF arenaviruses with the highest impact in human health. SA3. Identify targets of SH inhibitors with anti-arenaviral activity. We will test the hypothesis that SH inhibitors with anti-arenavirus activity interact wit and inhibit specific host SH. Selected hits will be functionalized with a photo-reactive diazirine group to covalently modify interacting protein targets and a bio-orthogonal alkyne handle to enable "click chemistry," to biotin to facilitate enrichment and subsequent mass spectrometry analysis using multi-dimensional protein identification technology (MudPIT). We will use complementary genetic and pharmacological approaches to validate the contribution of identified targets to the arenavirus life cycle.

 描述(申请人提供)：ARENAV是重要的人类病原体，其中几种病毒会引起出血热(HF)疾病，在其流行地区构成一个重要的公共卫生问题。此外，全球分布的原型性ArenaVirus LCMV是一种被忽视的人类病原体，具有临床意义。此外，禽流感病毒是一种可信的生物防御威胁。目前还没有FDA许可的疫苗，目前的抗病毒治疗仅限于利巴韦林的使用，利巴韦林只有部分效果。因此，目前还没有得到满足的新型抗病毒治疗药物的开发，这将通过表征促进病毒增殖的病毒与宿主细胞蛋白的相互作用来促进。基于活性的蛋白质图谱(ABPP)是一种新的方法，它允许监测 病毒感染对宿主蛋白质组功能状态的影响，以确定可能影响细胞生理、病毒繁殖和发病机制的新的病毒-宿主蛋白相互作用。这一探索性的R21应用的目标是使用丝氨酸水解酶(SHS)的ABPP来识别新的ArenaVirus-宿主相互作用，以及靶点和候选药物，以开发对抗ArenaVirus感染的策略。我们关注SHS是因为：1)SHS占哺乳动物蛋白质组的1%，参与多种生理和病理过程。2)我们观察到LCMV感染细胞的SH活性发生了变化，一些SH抑制剂具有抗LCMV的活性。我们的具体目标(SA)是：SA1。鉴定具有抗ARENAV活性的SH抑制剂。我们将检验特定的SH对ArenaVirus生命有贡献的假设 循环，它们的抑制将干扰病毒的繁殖。我们将使用一种新的基于细胞的分析来筛选250种SH抑制剂的文库，以确定那些具有抗LCMV活性的化合物。具有高抑制效力(EC90≤5微米)和治疗指数(TI=CC50/EC90)≥30的候选者将被选为旨在确定分子靶标和作用机制的研究。SA2.对选定的命中进行验证和表征。我们将验证具有抗LCMV活性的SH抑制剂抑制病毒生命周期特定步骤的假设。我们将使用已建立的基于细胞的分析方法来确定选定的HIT对LCMV以及Lassa和Junin这两种对人类健康影响最大的HF Arena病毒的细胞进入、RNA合成和萌发的影响。SA3.确定具有抗芳香病毒活性的SH抑制剂的靶点。我们将验证具有抗病毒活性的SH抑制剂与宿主SH相互作用并抑制特定宿主SH的假设。选定的HITS将被功能化，带有一个光反应二氮杂基，以共价修饰相互作用的蛋白质靶标，以及一个生物正交炔把手，以实现“点击化学”，以促进生物素的浓缩和随后使用多维蛋白质识别技术(MudPIT)进行的质谱分析。我们将使用互补的遗传和药理学方法来验证已确定的靶标对ARENA病毒生命周期的贡献。