Targeting the processing of the arenavirus glycoprotein for anti-viral therapy.

描述(申请人提供)：几种阿拉伯病毒引起人类出血热(HF)疾病，有证据表明，全球分布的原型阿拉伯病毒LCMV是一种被忽视的具有临床意义的人类病原体。此外，新城疫病毒还构成了生物防御威胁。目前还没有获得许可的抗ArenaVirus疫苗，目前的抗ArenaVirus疗法仅限于利巴韦林的使用，利巴韦林只有部分疗效，并与严重的副作用相关。我们的长期目标是了解HF arenaVirus发病的分子基础，并开发更好的抗病毒策略来对抗这些病原体。我们认为，病毒前体糖蛋白(GPC)和S1P细胞蛋白酶之间的相互作用可能参与了HF ArenaVirus的致病过程，S1P是一个有吸引力的抗病毒靶点。我们的假设基于以下发现：1)S1P介导的GPC的加工对感染后代的产生和病毒的繁殖至关重要，2)S1P在几个转录因子的激活中起关键作用，这些转录因子参与调节正常细胞生理所需的过程，并且在ArenaVirus感染细胞中高水平的GP表达可能干扰S1P的正常活性，从而导致细胞生理改变。我们的具体目标是：1.研究沙门氏菌GP-S1P相互作用的生化和功能特性。ArenaVirus GPC和S1P在细胞内共定位，但GP的表达不改变S1P的正常亚细胞分布。我们推测，ArenaVirus GP在感染细胞中的表达可以干扰S1P细胞底物的处理和激活，参与脂代谢调节和内质网应激反应，从而参与ArenaVirus的发病。为了验证这一假设，我们将表征GP-S1P复合体的形成和稳定性，并评估GPC和细胞底物蛋白之间可能存在的与S1P蛋白酶结合的竞争。同样，我们将使用基于细胞的分析来揭示GP对正常S1P细胞功能的干扰，这可能有助于ARENA病毒的发病。2.S1P介导的ArenaVirus GPC加工的小分子抑制剂的鉴定我们将使用两种不同的HTS分析格式来筛选S1P介导的ArenaVirus GPC切割候选抑制剂的组合库：1)基于来自LASV GP S1P识别位点的分子内猝灭荧光生成(IQF)多肽的体外分析，以及2)监测高尔基体相关S1P活性的细胞分析。具有高抑制效力(低EC90)和低细胞毒性(高CC50)的选定候选基因将受到反筛选协议的影响，以选择特定抑制S1P介导的LASV和JUNV GP处理的候选基因，同时对S1P的细胞靶标的处理影响最小。3.选定候选化合物的抗病毒潜力的测定。我们将测试这样的假设，即治疗指数为10(TI=CC50/EC50)的候选化合物将显示出真正的抗病毒活性。为此，我们将通过专门针对致病ArenaVirus Lassa和Junin病毒的GPC的S1P介导的处理来确定这些选定的化合物抑制病毒在培养细胞中增殖的能力。抑制-逃逸突变是抗病毒治疗中的一个普遍问题，因此我们将评估对S1P介导的GPC裂解抑制剂产生的病毒变异是否会对我们提出的抗病毒策略构成重大障碍。公共卫生相关性：几种阿拉伯病毒，主要是拉沙热病毒，可引起人类严重出血热(HF)疾病，有证据表明，全球分布的原型性阿拉伯病毒LCMV是一种被忽视的具有临床意义的人类病原体。此外，武器化形式的阿拉伯病毒作为生物恐怖主义的媒介构成严重威胁。目前还没有获得许可的抗ArenaVirus疫苗，目前的抗ArenaVirus疗法仅限于利巴韦林的使用，利巴韦林只有部分疗效，而且往往与严重副作用有关。因此，开发更好的抗病毒策略来对抗致病性ArenaVirus与人类健康高度相关，这项任务由于现有对ArenaVirus致病机制的有限了解而进一步复杂化。我们的建议旨在研究GP-S1P相互作用在ArenaVirus致病机制中的作用，并全面评估抑制S1P介导的病毒GPC处理作为对抗ArenaVirus感染的一种新的抗病毒策略的可行性。

DESCRIPTION (provided by applicant): Several arenaviruses cause hemorrhagic fever (HF) disease in humans, and evidence indicates that the worldwide-distributed prototypic arenavirus LCMV is a neglected human pathogen of clinical significance. In addition, arenaviruses pose a biodefense threat. No licensed anti-arenavirus vaccines are available, and current anti-arenavirus therapy is limited to the use of ribavirin, which is only partially effective and associated with severe side effects. Our long-term goal is to understand the molecular bases of HF arenavirus pathogenesis and to develop better antiviral strategies to combat these pathogens. We propose that the interaction between the virus precursor glycoprotein (GPC) and the S1P cellular protease may contribute to the pathogenesis of HF arenaviruses, and that S1P represents an attractive antiviral target. Our hypotheses are based on the following findings: 1) S1P-mediated processing of arenavirus GPC is critical for production of infectious progeny and virus propagation, and 2) S1P plays a critical role in the activation of several transcription factors implicated in the regulation of processes required for normal cell physiology, and high expression levels of GP in arenavirus infected cells may interfere with the normal activity of S1P, thus leading to altered cell physiology. Our specific aims are: 1. Biochemical and functional characterization of the arenavirus GP-S1P interaction. Arenavirus GPC and S1P co-localize within cells, but GP expression does not alter the normal sub-cellular distribution of S1P. We hypothesize that expression of arenavirus GP in infected cells can interfere with the processing and activation of S1P's cellular substrates involved in lipid metabolism regulation and ER stress response, thus contributing to arenaviral pathogenesis. To test this hypothesis we will characterize the formation and stability of the GP-S1P complex, and assess a possible competition between GPC and cellular substrate proteins for binding to the S1P protease. Likewise, we will use cell-based assays to uncover GP-induced perturbations on the normal S1P's cellular functions that could contribute to arenavirus pathogenesis. 2. Identification of small molecule inhibitors of S1P-mediated processing of arenavirus GPC. We will screen combinatorial libraries for candidate inhibitors of S1P-mediated cleavage of arenavirus GPC using two distinct HTS assay formats: 1) In vitro assays based on intra-molecular quenched fluorigenic (IQF) peptides derived from the S1P recognition sites of LASV GP and 2) cell-based assays monitoring Golgi- associated S1P activity as a readout. Selected candidates with high inhibitory potency (low EC90) and low cell toxicity (high CC50) will be subjected to counter screen protocols to select candidates that specifically inhibit S1P-mediated processing of LASV and JUNV GPs while having minimal effect on the processing of S1P's cellular targets. 3. Determination of the anti-viral potential of selected candidate compounds. We will test the hypothesis that candidate compounds with therapeutic index 10 (TI= CC50/EC50) will exhibit bona fide antiviral activity. For this we will determine the ability of these selected compounds to inhibit virus multiplication in cultured cells by specifically targeting S1P-mediated processing of GPCs of the pathogenic arenaviruses Lassa and Junin viruses. Inhibitor-escape mutants pose a general problem in antiviral therapy and thereby we will assess whether the emergence of viral variants resistant to inhibitors of S1P-mediated cleavage of GPC pose a significant obstacle to our proposed antiviral strategy. PUBLIC HEALTH RELEVANCE: Several arenaviruses, chiefly Lassa fever virus, cause severe hemorrhagic fever (HF) disease in humans, and evidence indicates that the worldwide-distributed prototypic Arenavirus LCMV is a neglected human pathogen of clinical significance. In addition, weaponized forms of arenaviruses pose a serious threat as agents of bioterrorism. No licensed anti-arenavirus vaccines are available, and current anti-arenavirus therapies are limited to the use of ribavirin, which is only partially effective and often associated with severe side effects. Therefore, the development of better antiviral strategies to combat pathogenic arenaviruses is highly relevant to human health, a task that is further complicated by the existing limited knowledge about the mechanisms underlying arenavirus pathogenesis. Our proposal is designed to examine the contribution of the GP-S1P interaction to arenavirus pathogenesis, and to provide a comprehensive assessment of the feasibility of inhibiting the S1P-mediated processing of the virus GPC as a novel antiviral strategy to combat arenavirus infections.