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子传播病毒疾病，估计有25亿居住在有流行病风险的地区。尽管如此，目前尚无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治疗剂的策略是筛选已知生物活性的小分子的文库，以鉴定抑制剂和相关的细胞靶标，其活性对于复制病毒至关重要。由于它们在信号转导和蛋白质功能的调节中的作用的中心性，我们选择了激酶作为一类细胞靶标，这些靶标可能会成功利用，以开发有效的抗登行毒疗法。从最初的一系列筛选中，我们发现SRC家族激酶（SFK）的抑制剂是体外DENV的有效抑制剂。 Dasatinib是一种双重SRC-ABL激酶抑制剂，用于FDA批准用于治疗抗imatinib的慢性髓性白血病和AZD0530，AZD0530是一种双SRC-ABL抑制剂，在II期临床试验中，在实体瘤的治疗中是一个较大的元素，可在100级超过100级降低剂量降低。此外，SFK抑制剂对四种登革热血清型的登革热病毒分离株具有活性，而不是针对无关的RNA病毒脊髓灰质炎病毒。 RNAi的C-SRC表op型SFK抑制剂的抑制作用。总的来说，我们的结果表明，这些SRC抑制剂靶向的病毒过程是高度保守的，并且大多数DENV分离株应敏感这些化合物的抗病毒作用。此外，由于这些化合物的目标是病毒至关重要但对宿主不必要的细胞因子，因此耐药性应较小。现在，我们建议使用体外和体内实验的组合将SFK验证为抗DENV靶标。首先，我们将进行体外实验，以定量和定性地表征SFK抑制剂（包括AZD0530和Dasatinib）对多个DENV菌株以及多个细胞系的影响。其次，为了了解SFK信号转导在DENV生物学中的作用，在特定的目标2中，我们将执行磷蛋白分析实验，旨在识别登革热病毒感染后发生的蛋白质磷酸化的变化以及这些变化依赖于SRC激酶活性。使用在体外积累的数据，我们将执行实验，以评估SRC激酶抑制剂在减少病毒滴度和改善登革热 - 突袭热疾病中的疗效中，在DENV感染和发病机理的鼠模型中。公共卫生相关性尽管登革热病毒是影响人类的最重要的蚊子传播病毒疾病，但目前尚无针对登革热病毒有效的抗病毒药，也没有保护性疫苗。我们想确定可以通过与宿主细胞中靶标相互作用来抑制病毒的小型，类似药物样的分子。然后，这些小分子可用于研究登革热病毒如何与宿主相互作用并开发治疗登革热病毒感染的疗法。