Inhibitors of Epstein-Barr Virus Nuclear Protein 1 Mediated Latent Infection

描述（由申请人提供）：eb病毒（EBV）潜伏感染导致几乎所有eb病毒相关的发病率和死亡率，包括感染性单核细胞增多症早期的淋巴细胞增殖、艾滋病和其他免疫功能低下状态的淋巴细胞增生性疾病、eb病毒相关的淋巴瘤、霍奇金病和鼻咽癌。EBV基因组在所有潜伏感染细胞中作为非整合的多拷贝片段持续存在。EBV发作在分裂细胞中的持续性依赖于EBV编码的核抗原1蛋白（EBNA1）。EBNA1与EBV片段体的特定位点结合，增强片段体的初始复制、转录和持久性。由于EBNA1对所有分裂细胞和恶性细胞中EBV发作体的持续存在至关重要，因此本应用程序的主要目的是鉴定可以抑制EBNA1介导的发作体持续存在的化合物。为了实现这一目标，我们建议：(1)进行筛选，以确定在体内中断EBNA1- orip依赖性片段体转录和持久性的化合物，在体外中断EBNA1二聚化和与同源DNA结合的化合物，以及在硅中与EBNA1结合的化合物。(2)鉴定鉴定的化合物对B淋巴母细胞中EBNA1-oriP依赖性片段体的转录和持久性、EBV转化淋巴母细胞样细胞（LCL）的生长以及LCL诱导的裸鼠淋巴瘤和NPC肿瘤的生物学和生化作用。(3)利用生物化学、生物物理和结构方法确定EBNA1结合中生物活性化合物作用的位点。利用这些知识，最有效地进行结构活性修饰，以提高化合物的活性和特异性。(4)利用反向遗传学技术鉴定EBNA1 DBD中的关键残基，提高筛选敏感性，为硅口袋选择、化合物修饰和化合物相互作用分析提供信息。公共卫生相关性：eb病毒（EBV）在HIV感染或其他免疫功能低下的人群中积极引起恶性淋巴细胞增生性疾病，如伯基特淋巴瘤、其他淋巴瘤、霍奇金病和几乎所有鼻咽癌。由于EBV编码的核抗原1 （EBNA1）蛋白对EBV基因组在这些恶性细胞中的持久性至关重要，因此我们提出了鉴定和改善抗EBNA1化合物并阻止EBV基因组持久性的实验。这些化合物可能阻止或停止EBV相关的肿瘤细胞生长并导致肿瘤细胞死亡。

DESCRIPTION (provided by applicant): Epstein-Barr Virus (EBV) latent infections cause almost all EBV associated morbidity and mortality including lymphoblast proliferation early in Infectious Mononucleosis, Lymphoproliferative Diseases in people with AIDS and other immune compromised states, and EBV associated Lymphomas, Hodgkin's Disease, and Nasophryngeal Carcinoma. The EBV genome persists in all latently infected cells as a non-integrated multi-copy episome. The persistence of EBV episomes in dividing cells is dependent on the EBV encoded nuclear antigen 1 protein (EBNA1). EBNA1 binds to a specific site in the EBV episome and enhances episome initial replication, transcription, and persistence. Since EBNA1 is essential for the persistence of EBV episomes in all dividing and malignant cells, the central objective of this application is to identify compounds that can inhibit EBNA1 mediated episome persistence. To achieve that objective, we propose to: (1) Undertake screens to identify compounds that interrupt EBNA1-oriP dependent episome transcription and persistence in vivo, compounds that interrupt EBNA1 dimerization and binding to cognate DNA in vitro, and compounds that bind to EBNA1 in silico. (2) Identify the biological and biochemical effects of the identified compounds on EBNA1-oriP dependent episome transcription and persistence in B lymphoblasts, on EBV transformed lymphoblastoid cell (LCL) growth, and on LCL induced Lymphoma and NPC tumors in nude mice. (3) Determine the sites of bioactive compound effects in EBNA1 binding, using biochemical, biophysical, and structural approaches. Use this knowledge to most effectively undertake structure activity modifications to improve compound activity and specificity. (4) Use reverse genetics to identify the critical residues in EBNA1 DBD that can improve screening sensitivity and inform in silico pocket selection, compound modification, and compound interaction analyses. PUBLIC HEALTH RELEVANCE: Epstein-Barr Virus (EBV) actively causes malignant lymphoproliferative diseases in HIV infected or otherwise immune compromised people, Burkitt Lymphoma, other Lymphomas, Hodgkin's Disease, and almost all Nasopharyngeal Carcinomas. Since the EBV-encoded nuclear antigen 1 (EBNA1) protein is essential for the persistence of the EBV genome in these malignant cells, we propose experiments that identify and improve anti-EBNA1 compounds and prevent EBV genome persistence. These compounds are likely to prevent or halt EBV associated tumor cell growth and cause tumor cell death.