Activity-Based Protein Profiling of Arenavirus-Host Interactions

 描述（由申请方提供）：沙粒病毒是重要的人类病原体，其中几种引起出血热（HF）疾病，在其流行地区构成重要的公共卫生问题。此外，世界范围内分布的原型沙粒病毒LCMV是一种被忽视的人类病原体的临床意义。此外，沙粒病毒是一种可信的生物防御威胁。没有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，这只是部分有效。因此，对于开发新的抗沙粒病毒治疗剂存在未满足的需求，这将通过表征有助于病毒增殖的沙粒病毒-宿主细胞蛋白质相互作用来促进。基于活性的蛋白质谱分析（ABPP）是一种新的方法，允许监测 病毒感染对宿主蛋白质组功能状态的影响，以鉴定可能影响细胞生理学、病毒繁殖和发病机制的新型病毒-宿主蛋白质相互作用。这项探索性R21应用的目标是利用丝氨酸水解酶（SH）的ABPP来鉴定新型沙粒病毒-宿主相互作用，以及靶标和候选药物，以开发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)我们已经观察到LCMV感染细胞中SH活性的改变，并且一些SH抑制剂表现出抗LCMV活性。我们的目标（SA）是：SA 1。鉴定具有抗沙粒病毒活性的SH抑制剂。我们将检验特定的SH对沙粒病毒生命有贡献的假设 循环，并且它们的抑制将干扰病毒增殖。我们将使用一种新的基于细胞的测定来筛选250种SH抑制剂的文库，以鉴定具有抗LCMV活性的SH抑制剂。将选择具有高抑制效力（EC 90 ≤ 5 µM）和治疗指数（TI = CC 50/EC 90）≥ 30的候选药物用于旨在确定分子靶点和作用机制的研究。SA 2.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来确定选定的命中对LCMV的细胞进入、RNA合成和出芽以及对人类健康影响最大的两种HF沙粒病毒Lassa和朱宁的影响。SA 3.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抑制剂与特异性宿主SH相互作用并抑制特异性宿主SH的假设。选择的命中物将用光反应性二氮杂环丙烯基团官能化以共价修饰相互作用的蛋白质靶标和生物正交炔柄以使生物素能够“点击化学”，以促进富集和随后的使用多维蛋白质鉴定技术（MudPIT）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。

 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.