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（由申请人提供）：沙粒病毒是重要的人类病原体，其中几种会引起出血热（HF）疾病，在其流行地区造成重要的公共卫生问题。此外，全球分布的原型沙粒病毒 LCMV 是一种被忽视的具有临床意义的人类病原体。此外，沙粒病毒代表了可靠的生物防御威胁。目前尚无 FDA 许可的疫苗，目前的抗沙粒病毒治疗仅限于使用利巴韦林，而利巴韦林仅部分有效。因此，开发新型抗沙粒病毒疗法的需求尚未得到满足，而沙粒病毒-宿主细胞蛋白相互作用的表征有助于病毒增殖，这将有助于这种疗法的发展。基于活性的蛋白质分析 (ABPP) 是一种新方法，可以监测 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学、病毒传播和发病机制的新型病毒-宿主蛋白质相互作用。这一探索性 R21 应用的目标是利用丝氨酸水解酶 (SH) 的 ABPP 来识别新型沙粒病毒与宿主的相互作用以及靶标和候选药物，以制定对抗沙粒病毒感染的策略。我们关注SHs是因为：1）SHs占哺乳动物蛋白质组的1%，参与许多生理和病理过程。 2）我们观察到感染 LCMV 的细胞中 SH 活性发生改变，并且一些 SH 抑制剂表现出抗 LCMV 活性。我们的具体目标（SA）是：SA1。鉴定具有抗沙粒病毒活性的 SH 抑制剂。我们将检验特定 SH 对沙粒病毒生命有贡献的假设 循环，并且它们的抑制会干扰病毒的增殖。我们将使用一种新型的基于细胞的测定法来筛选 250 种 SH 抑制剂的库，以鉴定具有抗 LCMV 活性的抑制剂。将选择具有高抑制效力 (EC90 ≤ 5 µM) 和治疗指数 (TI = CC50/EC90) ≥ 30 的候选药物进行旨在确定分子靶点和作用机制的研究。 SA2。选定命中的验证和表征。我们将检验具有抗 LCMV 活性的 SH 抑制剂抑制病毒生命周期的特定步骤的假设。我们将使用已建立的基于细胞的测定法来确定选定的命中对 LCMV 以及拉沙病毒和胡宁病毒这两种对人类健康影响最大的 HF 沙粒病毒的细胞进入、RNA 合成和出芽的影响。 SA3。确定具有抗沙粒病毒活性的 SH 抑制剂的靶点。我们将检验具有抗沙粒病毒活性的 SH 抑制剂与特定宿主 SH 相互作用并抑制其的假设。选定的命中将用光反应性二氮丙啶基团进行功能​​化，以共价修饰相互作用的蛋白质靶标，并用生物正交炔烃手柄进行“点击化学”，以生物素进行功能化，以促进使用多维蛋白质识别技术（MudPIT）进行富集和随后的质谱分析。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沙粒病毒生命周期的贡献。