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（由适用提供）：体育症病毒是重要的人类病原体，其中几种引起出血热（HF）疾病，在其内在地区构成了重要的公共卫生问题。此外，全球范围内的原型体育病毒LCMV是一种被忽视的临床意义的人类病原体。此外，体育症病毒代表了可靠的生物陷入威胁。没有FDA许可的疫苗，当前的抗嗜动病毒疗法仅限于利巴韦林的使用，这仅是部分有效的。因此，对于开发新型抗动脉粥样硬病毒疗法的发展，这将是通过甲状腺病毒宿主宿主细胞蛋白相互作用的表征来制备的，这有助于病毒繁殖。基于活动的蛋白质分析（ABPP）是一种新型方法，可以监视 病毒感染对宿主蛋白功能状态的影响，以鉴定可能影响细胞生理，病毒传播和发病机理的新型VIRU宿主蛋白相互作用。这种探索性R21应用的目的是使用丝氨酸水解酶（SHS）的ABPP来识别新型的体育症病毒 - 宿主相互作用以及靶标和候选药物，以制定策略来打击Arenavirus感染。我们专注于SHS，因为：1）SHS代表哺乳动物蛋白质组的1％，并参与许多物理和病理过程。 2）我们已经观察到在LCMV感染的细胞和一些SH抑制剂暴露的抗LCMV活性中的SH活性改变。我们的具体目标（SA）是：SA1。鉴定具有抗动脉粥样硬病活性的SH抑制剂。我们将检验以下假设，即特定的SH有助于体育馆的生命 循环和它们的抑制作用会干扰病毒繁殖。我们将使用一种新型的基于细胞的评估来筛选250个SH抑制剂的库，以识别具有抗LCMV活性的库。将选择具有较高抑制效力（EC90≤5µM）和治疗指数（Ti = CC50/EC90）≥30的候选者用于定义分子靶标和作用机理的研究。 SA2。选定命中的验证和表征。我们将检验以下假设：抗LCMV活性的SH抑制剂抑制病毒生命周期的特定步骤。我们将使用既定的基于细胞的Assas来确定选定的命中对细胞进入，LCMV的RNA合成和萌芽以及LASSA和JUNIN的影响。 SA3。鉴定具有抗蛛病毒活性的SH抑制剂的靶标。我们将检验以下假设，即具有抗动脉粥样硬病毒活性的SH抑制剂相互作用并抑制特定的宿主SH。选定的命中将与光反应重氮嘧啶基一起功能化，以共价修改相互作用的蛋白质靶标和生物正交酒精手柄，以实现“单击化学”，以支持生物素，以支持富集和随后的质谱分析，并使用多维蛋白质鉴定技术（Mudpit）进行质谱分析。我们将使用完整的遗传和药物方法来验证已确定的靶标对体育症病毒生命周期的贡献。