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

描述（由申请方提供）：几种沙粒病毒引起人类出血热（HF）疾病，证据表明，世界范围内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，沙粒病毒构成生物防御威胁。没有获得许可的抗沙粒病毒疫苗可用，目前的抗沙粒病毒治疗仅限于使用利巴韦林，其仅部分有效且与严重副作用相关。我们的长期目标是了解HF沙粒病毒发病机制的分子基础，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S1 P细胞蛋白酶之间的相互作用可能有助于HF沙粒病毒的发病机制，S1 P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）沙粒病毒GPC的S1 P介导的加工对于产生感染性子代和病毒繁殖至关重要，和2）S1 P在涉及正常细胞生理学所需过程的调节的几种转录因子的活化中起关键作用，并且沙粒病毒感染的细胞中GP的高表达水平可能干扰S1 P的正常活性，从而导致细胞生理学改变。我们的具体目标是：1.沙粒病毒GP-S1 P相互作用的生物化学和功能表征。沙粒病毒GPC和S1 P共定位于细胞内，但GP表达不改变S1 P的正常亚细胞分布。我们推测沙粒病毒GP在感染细胞中的表达可以干扰参与脂质代谢调节和ER应激反应的S1 P细胞底物的加工和活化，从而有助于沙粒病毒的发病机制。为了验证这一假设，我们将表征GP-S1 P复合物的形成和稳定性，并评估GPC和细胞底物蛋白之间可能的竞争，以结合S1 P蛋白酶。同样，我们将使用基于细胞的测定来揭示GP诱导的对正常S1 P细胞功能的干扰，这可能有助于沙粒病毒的发病机制。2.沙粒病毒GPC的S1 P介导的加工的小分子抑制剂的鉴定。我们将使用两种不同的HTS测定形式筛选沙粒病毒GPC的S1 P介导的切割的候选抑制剂的组合文库：1）基于源自LASV GP的S1 P识别位点的分子内淬灭荧光（IQF）肽的体外测定和2）基于细胞的测定，监测高尔基体相关的S1 P活性作为读数。选择的具有高抑制效力（低EC 90）和低细胞毒性（高CC 50）的候选物将进行反筛选方案，以选择特异性抑制S1 P介导的LASV和JUNV GP加工同时对S1 P的细胞靶标加工具有最小影响的候选物。3.确定所选候选化合物的抗病毒潜力。我们将测试治疗指数10（TI= CC 50/EC 50）的候选化合物将表现出真正的抗病毒活性的假设。为此，我们将确定这些选定的化合物通过特异性靶向致病性沙粒病毒拉沙和朱宁病毒的S1 P介导的GPC加工来抑制培养细胞中病毒增殖的能力。抑制剂逃逸突变体在抗病毒治疗中造成了普遍问题，因此我们将评估对S1 P介导的GPC裂解抑制剂耐药的病毒变体的出现是否对我们提出的抗病毒策略造成了重大障碍。公共卫生相关性：几种沙粒病毒，主要是拉沙热病毒，在人类中引起严重的出血热（HF）疾病，并且有证据表明，世界范围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，沙粒病毒的武器化形式作为生物恐怖主义制剂构成严重威胁。没有获得许可的抗沙粒病毒疫苗可用，目前的抗沙粒病毒疗法仅限于使用利巴韦林，其仅部分有效且通常与严重副作用相关。因此，开发更好的抗病毒策略来对抗致病性沙粒病毒与人类健康高度相关，这是一项由于对沙粒病毒致病机制的现有有限知识而进一步复杂化的任务。我们的建议旨在研究GP-S1 P相互作用对沙粒病毒发病机制的贡献，并全面评估抑制S1 P介导的病毒GPC加工作为对抗沙粒病毒感染的新型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.