Validation of Src kinases as Anti-Dengue Viral Targets

验证 Src 激酶作为抗登革热病毒靶点

• 批准号: 7634494
• 负责人: Priscilla Li-ning Yang
• 金额: $ 37.71万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 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/7634494
• 关键词: Affect; Alleles; Antiviral Agents; Area; Biochemical Pathway; Biochemical Reaction; Biological; Biological Assay; Biology; Capsid; Cell Line; Cells; Chronic Myeloid Leukemia; 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; FDA approved; 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; Regulation; Research; Resistance; Risk; Role; SRC gene; Series; Serotyping; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Solid Neoplasm; Testing; Therapeutic; Time; 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; public health relevance; recombinant virus; research study; small molecule; small molecule libraries; src-Family Kinases; therapy development; tool; transmission process

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25亿人生活在流行病传播风险地区。尽管如此，目前还没有有效的抗病毒药物来对抗登革热病毒，也没有保护性疫苗。我们开发抗DENV治疗剂的策略是筛选具有已知生物活性的小分子文库，以鉴定其活性对病毒复制至关重要的抑制剂和相关细胞靶标。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疗法的细胞靶点。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。达沙替尼（一种FDA批准用于治疗伊马替尼耐药的慢性髓性白血病的双重Src-Abl激酶抑制剂）和AZD 0530（一种用于治疗实体瘤的II期临床试验的双重Src-Abl抑制剂）在个位数微摩尔浓度下均可使病毒滴度降低100倍以上。此外，SFK抑制剂对代表四种登革热血清型中的每一种的登革热病毒分离株有活性，但对脊髓灰质炎病毒（一种不相关的RNA病毒）无活性。c-Src的RNAi“敲低”表型模仿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，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，由于这些化合物靶向对病毒必需但对宿主非必需的细胞因子，因此不太可能出现耐药性。我们现在建议使用体外和体内实验的组合来验证SFKs作为抗DENV靶点。首先，我们将进行体外实验，以定量和定性表征SFK抑制剂（包括AZD 0530和达沙替尼）对多种DENV毒株和多种细胞系的作用。其次，为了理解SFK信号转导在DENV生物学中的作用，在具体目标2中，我们将进行磷酸蛋白质组学分析实验，旨在鉴定登革病毒感染细胞后发生的蛋白质磷酸化的变化以及这些变化中的哪一个依赖于Src激酶活性。使用体外积累的数据，我们将进行实验以评估Src激酶抑制剂在DENV感染和发病机制的鼠模型中降低病毒滴度和改善登革出血热样疾病的功效。 虽然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。我们希望能找到类似药物的小分子，通过与宿主细胞中的靶点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宿主相互作用，并开发治疗登革热病毒感染的疗法。