Validation of Src kinases as Anti-Dengue Viral Targets

验证 Src 激酶作为抗登革热病毒靶点

基本信息

批准号：
7533684
负责人：
Priscilla Li-ning Yang
金额：
$ 37.62万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-06-15 至 2013-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7533684
关键词：
Affect Alleles Antiviral Agents Area Biochemical Pathway Biochemical Reaction Biological Biological Assay Biology Capsid Cell Line Cells Chronic Myeloid Leukemia Class Clinical Treatment Clinical Trials Collaborations Complement Country Culicidae Dana-Farber Cancer Institute Dasatinib Data Dengue Dengue Hemorrhagic Fever Dengue Virus Digit structure Disease Drug resistance Environment Epidemic Event Growth Human Human poliovirus Imatinib In Vitro Individual Infection Intervention Laboratories Lead Life Life Cycle Stages Mass Spectrum Analysis Measures Mediating Modeling Molecular Monitor Mus Mutation Outcome Pathogenesis Pathway interactions Pharmaceutical Preparations Phase Phase II Clinical Trials Phenocopy Phosphorylation Phosphotransferases Play Polioviruses Process Production Proteins Public Health RNA Interference RNA Viruses Rate Regulation Research Resistance Risk Role SRC gene Series Serotyping Signal Pathway Signal Transduction Signal Transduction Pathway Solid Neoplasm Testing Therapeutic Time Today United States Food and Drug Administration Up-Regulation Vaccines Validation Viral Viral Proteins Viremia Virion Virus Virus Assembly Virus Diseases Virus Replication Withdrawal Work cellular targeting design fitness gain of function in vivo inhibitor/antagonist insight kinase inhibitor loss of function member mutant prevent protein function recombinant virus research study small molecule small molecule libraries src-Family Kinases tool transmission process

项目摘要

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.

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 亿人生活在存在流行病传播风险的地区。尽管如此，目前还没有有效对抗登革热病毒的抗病毒药物，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，以识别抑制剂和相关细胞靶标，其活性对于病毒复制至关重要。由于它们在信号转导和蛋白质功能调节中的核心作用，我们选择激酶作为一类细胞靶标，可以成功地利用它们来开发有效的抗登革热疗法。从最初的一系列筛选中，我们发现 Src 家族激酶 (SFK) 抑制剂在体外是 DENV 的有效抑制剂。达沙替尼（一种双重 Src-Abl 激酶抑制剂，已获得 FDA 批准用于治疗伊马替尼耐药的慢性粒细胞性白血病）和 AZD0530（一种双重 Src-Abl 抑制剂，目前正处于治疗实体瘤的 II 期临床试验中）导致在个位数微摩尔浓度下，病毒滴度降低超过 100 倍。此外，SFK 抑制剂对代表四种登革热血清型的登革热病毒分离株具有活性，但对脊髓灰质炎病毒（一种不相关的 RNA 病毒）没有活性。 c-Src 的 RNAi“敲低”表型复制了 SFK 抑制剂的抑制作用。总的来说，我们的结果表明，这些 Src 抑制剂针对的病毒过程是高度保守的，并且大多数 DENV 分离株应该对这些化合物的抗病毒作用敏感。此外，耐药性应该不太可能出现，因为这些化合物针对的是对病毒必需但对宿主非必需的细胞因子。我们现在建议结合体外和体内实验来验证 SFK 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 毒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，在特定目标 2 中，我们将进行磷酸化蛋白质组学分析实验，旨在识别登革热病毒感染细胞时发生的蛋白质磷酸化变化，以及这些变化中的哪一个是取决于 Src 激酶活性。利用体外积累的数据，我们将进行实验，以评估 Src 激酶抑制剂在 DENV 感染和发病机制的小鼠模型中降低病毒滴度和改善登革出血热样疾病的功效。公共卫生相关性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，但目前尚无有效对抗登革热病毒的抗病毒药物，也没有保护性疫苗。我们希望鉴定出类似药物的小分子，它们可以通过与宿主细胞中的靶标相互作用来抑制病毒。然后，这些小分子可用于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。