Validation of Src kinases as Anti-Dengue Viral Targets

描述（由申请人提供）：登革热病毒（DENV）是当今影响人类的最重要的蚊媒病毒性疾病，估计有25亿人生活在有流行传播风险的地区。尽管如此，目前还没有针对登革热病毒有效的抗病毒药物，也没有保护性疫苗。我们开发抗denv疗法的策略是筛选已知生物活性的小分子文库，以确定抑制剂和相关的细胞靶标，其活性对病毒的复制至关重要。由于它们在信号转导和蛋白质功能调节中的中心作用，我们选择激酶作为一类可能成功开发有效的抗登革热治疗药物的细胞靶标。从最初的一系列筛选中，我们发现Src家族激酶抑制剂（SFK）在体外是DENV的有效抑制剂。dasatinib是一种双重Src-Abl激酶抑制剂，已被fda批准用于治疗伊马替尼耐药的慢性骨髓性白血病，AZD0530是一种双重Src-Abl抑制剂，正在进行II期临床试验，用于治疗实体肿瘤，在个位数微摩尔浓度下，病毒滴度降低超过100倍。此外，SFK抑制剂对代表登革热四种血清型的登革热病毒分离株都有活性，但对脊髓灰质炎病毒（一种不相关的RNA病毒）没有活性。RNAi“敲低”c-Src的表型反映了SFK抑制剂的抑制作用。总之，我们的研究结果表明，这些Src抑制剂靶向的病毒过程是高度保守的，大多数DENV分离株应该对这些化合物的抗病毒作用敏感。此外，由于这些化合物针对的细胞因子对病毒是必需的，但对宿主不是必需的，因此不太可能产生耐药性。我们现在建议通过体外和体内实验的结合来验证SFKs作为抗denv靶点。首先，我们将进行体外实验，定量和定性地表征SFK抑制剂（包括AZD0530和达沙替尼）对多种DENV菌株和多种细胞系的作用。其次，为了了解SFK信号转导在DENV生物学中的作用，在Specific Aim 2中，我们将进行磷酸化蛋白质组学分析实验，旨在确定在感染登革热病毒的细胞时发生的蛋白质磷酸化变化，以及这些变化中哪些依赖于Src激酶活性。利用在体外积累的数据，我们将在DENV感染和发病机制的小鼠模型中进行实验，评估Src激酶抑制剂在降低病毒滴度和改善登革热出血热样疾病方面的功效。尽管登革热病毒是当今影响人类的最重要的蚊媒病毒性疾病，但目前没有针对登革热病毒有效的抗病毒药物，也没有保护性疫苗。我们想要确定一种小的、类似药物的分子，这种分子可以通过与宿主细胞中的靶标相互作用来抑制病毒。这些小分子可以用来研究登革热病毒如何与其宿主相互作用，并开发治疗登革热病毒感染的疗法。

DESCRIPTION (provided by applicant): Dengue virus (DENV) is the most important mosquito-borne viral disease affecting humans today, with an estimated 2.5 billion living in areas at risk for epidemic transmission. Despite this, there are currently no antiviral drugs effective against dengue virus nor is there a protective vaccine. Our strategy to develop anti-DENV therapeutics is to screen libraries of small molecules of known biological activity in order to identify inhibitors and the associated cellular targets whose activity is essential for replication of the virus. Due to the centrality of their role in signal transduction and regulation of protein function, we selected kinases as a class of cellular targets that might be successfully exploited to develop effective anti-dengue therapeutics. From an initial series of screens, we found that inhibitors of Src family kinases (SFK) are potent inhibitors of DENV in vitro. Both dasatinib, a dual Src-Abl kinase inhibitor that is FDA-approved for the treatment of imatinib-resistant Chronic Myelogenous Leukemia, and AZD0530, a dual Src-Abl inhibitor that is in phase II clinical trials for the treatment of solid tumors, cause a greater than 100-fold reduction in viral titers at single-digit micromolar concentrations. SFK inhibitors are, moreover, active against dengue viral isolates representative of each of the four dengue serotypes but not against poliovirus, an unrelated RNA virus. RNAi "knockdown" of c-Src phenocopies the inhibitory effect of the SFK inhibitors. Collectively, our results suggest that the viral process targeted by these Src inhibitors is highly conserved and that most DENV isolates should be susceptible to the anti-viral effects of these compounds. In addition, drug- resistance should be less likely to arise since these compounds target a cellular factor that is essential for the virus but non-essential for the host. We now propose to validate SFKs as anti-DENV targets using a combination of in vitro and in vivo experiments. First, we will perform in vitro experiments to quantitatively and qualitatively characterize the effects of SFK inhibitors (including AZD0530 and dasatinib) against multiple DENV strains and in multiple cells lines. Second, in order to understand the role of SFK signal transduction in DENV biology, in Specific Aim 2 we will perform phosphoproteomic profiling experiments aimed at identifying changes in protein phosphorylation that occur upon infection of cells with dengue virus as well as which of these changes is dependent upon Src kinase activity. Using the data accumulated in vitro, we will perform experiments to evaluate the efficacy of Src kinase inhibitors in reducing viral titers and ameliorating dengue-hemorrhagic fever-like disease in a murine model of DENV infection and pathogenesis. PUBLIC HEALTH RELEVANCE Although dengue virus is the most important mosquito-borne viral disease affecting humans today, there are currently no antiviral drugs effective against dengue virus, nor is there a protective vaccine. We would like to identify small, drug-like molecules that can inhibit the virus by interacting with targets in the host cell. These small molecules can then be used to study how dengue virus interacts with its host and to develop therapies for the treatment of dengue virus infections.