Assay For Inhibitors of Epstein-Barr Virus

EB 病毒抑制剂的测定

基本信息

批准号：
7846978
负责人：
PAUL M LIEBERMAN
金额：
$ 5.21万
依托单位：
WISTAR INSTITUTE
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 2010-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7846978
关键词：
Acquired Immunodeficiency Syndrome B Cell Proliferation B-Cell Lymphomas B-Lymphocytes BZLF1 protein, Herpesvirus 4, Human Binding Binding Sites Biological Biological Assay Burkitt Lymphoma Carcinogens Cells DNA DNA Binding DNA Binding Domain DNA biosynthesis Data Databases Development Dominant-Negative Mutation EBV-associated disease Electrophoretic Mobility Shift Assay Episome Epstein-Barr Virus Infections Epstein-Barr pathogenesis Fluorescence Polarization Genetic Genome Goals Health Herpesviridae Hodgkin Disease Human Human Herpesvirus 4 Human Herpesvirus 8 Human Papillomavirus Immunosuppression In Vitro Label Lytic Maintenance Malignant - descriptor Malignant Neoplasms Mission Molecular Bank Monitor Nasopharynx Carcinoma National Institute of Allergy and Infectious Disease Oligonucleotides Pathology Phase Production Property Proteins Reagent Relative (related person)Risk Site Small Interfering RNA Southern Blotting Stomach Carcinoma Technology Testing Time Treatment Efficacy United States National Institutes of Health Viral Viral Genome Virus World Health Organization base cell growth cell type cytotoxicity high throughput screening inhibitor/antagonist latent infection meetings neoplastic cell prevent protein E2, Human papilloma virus type 1 public health relevance small molecule

项目摘要

DESCRIPTION (provided by applicant): Project Summary The goal of this R21 is to develop a high-throughput screen for identification of new small molecule inhibitors for Epstein-Barr virus (EBV) latent infection, and its associated pathologies. EBV is a prevalent human herpesvirus that has been classified by the World Health Organization as a human carcinogen. The latent infection is associated with multiple human malignancies, including Burkitt's lymphoma, nasopharyngeal carcinomas, Hodgkin's lymphoma, gastric carcinomas, and immunoblastic B-cell lymphoma's during immunosuppression. Currently, no therapies exist that target latent infection, and therefore it remains impossible to effectively treat or prevent EBV-associated disease. The latent infection depends on one viral encoded protein, EBNA1, which functions in the replication and maintenance of the viral genome. Genetic and biological disruption of EBNA1, including siRNA depletion and dominant negative mutants, block viral latent infection and EBV-dependent B-cell growth. The EBNA1 DNA binding domain has been characterized structurally and biochemically, and serves as an ideal molecule for targeted small molecule inhibition of EBV infection. We have produced preliminary data that validates EBNA1 as a necessary factor for the maintenance of the EBV viral genome, and have demonstrated that we can synthesize highly pure EBNA1 protein that retains its specific DNA-binding activity, and that this binding activity can be monitored in vitro. In this R21 proposal, we will extend these findings to develop a reliable assay for EBNA1 activity that can be used in HTS format to screen for small molecule inhibitors. The risk of this proposal is substantially mitigated by our previous successful development of a similar assay for another EBV regulatory factor (a lytic, rather than a latent phase protein). Public Health Relevance Statement The significance of this proposal to human health will be the development of new therapies for eliminating EBV latent infection and preventing or treating associated malignant pathologies. Episomal maintenance of EBV genomes during latency is essential for the propagation and survival of EBV. Moreover, tumor cells latently infected with EBV typically harbor episomal genomes, suggesting that episomal maintenance is required for EBV pathogenesis. With the exception of HU, no other pharmacological agents have been shown to effectively eliminate EBV episomes from latently infected cells. However, targeting latent genomes with EBNA1 directed siRNA, dominant negatives, or binding site competitors have been shown to be effective inhibitors of EBV-driven B-cell proliferation and viability. The goals of this application are to identify highly specific inhibitors of EBNA1 DNA binding function. These studies may provide important new reagents for manipulation of EBV latent infection, and may also provide new opportunities to interrupt latent infections of EBV-related viruses, including Kaposi's Sarcoma Associated Herpesvirus (KSHV) and human papillomavirus (HPV).

描述（由申请人提供）：项目摘要该R21的目的是开发一个高通量屏幕，以鉴定新的小分子抑制剂，用于爱泼斯坦 - 巴尔病毒（EBV）潜在感染及其相关的病理。 EBV是一种普遍存在的人类疱疹病毒，已被世界卫生组织归类为人类致癌。潜在感染与多种人类恶性肿瘤有关，包括伯基特的淋巴瘤，鼻咽癌，霍奇金的淋巴瘤，胃癌和免疫细胞B细胞淋巴瘤在免疫抑制期间。目前，尚无靶向潜在感染的疗法，因此仍然不可能有效治疗或预防与EBV相关的疾病。潜在感染取决于一种病毒编码的蛋白EBNA1，该蛋白在病毒基因组的复制和维持中起作用。 EBNA1的遗传和生物学破坏，包括siRNA耗竭和显性阴性突变体，阻断病毒潜在感染和EBV依赖性B细胞生长。 EBNA1 DNA结合结构域已在结构和生化上进行了表征，并作为靶向小分子抑制EBV感染的理想分子。我们已经产生了初步数据，将EBNA1验证为维持EBV病毒基因组的必要因素，并证明我们可以合成高度纯净的EBNA1蛋白，该蛋白保留其特定的DNA结合活性，并且可以在体外监测这种结合活性。在此R21提案中，我们将扩展这些发现，以开发可靠的EBNA1活性测定法，该测定可用于HTS格式以筛选小分子抑制剂。我们先前成功开发了对另一个EBV调节因子（裂解的，而不是潜在的相蛋白）的类似测定法，从而大大减轻了该提案的风险。公共卫生相关性声明该提案对人类健康的重要性将是开发消除EBV潜在感染以及预防或治疗相关的恶性病理学的新疗法。延迟期间EBV基因组的偶发性维持对于EBV的传播和存活至关重要。此外，受EBV感染的肿瘤细胞通常携带偶发性基因组，表明EBV发病机理需要偶发性维持。除HU外，尚无其他药理学剂可有效消除潜在感染细胞的EBV发作。然而，用EBNA1定向siRNA，主导的负负值或结合位点竞争者靶向潜在基因组是有效的EBV驱动B细胞增殖和生存能力的有效抑制剂。该应用的目标是确定EBNA1 DNA结合函数的高度特异性抑制剂。这些研究可能会为操纵EBV潜在感染提供重要的新试剂，还可以为中断与EBV相关病毒的潜在感染提供新的机会，包括Kaposi的肉瘤相关疱疹病毒（KSHV）和人类乳头虫病毒（HPV）。