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

EB病毒（EBV）潜伏感染几乎引起所有EBV相关的发病率 和死亡率，包括传染性单核细胞增多症早期的淋巴母细胞增殖， 艾滋病患者和其他免疫功能低下者的恶性增殖性疾病 与EBV相关的淋巴瘤、霍奇金病和鼻咽 Carcinoma. EB病毒基因组在所有潜伏感染的细胞中以非整合的形式存在， 多拷贝附加体。EBV游离体在分裂细胞中的持续存在依赖于 EBV编码核抗原1蛋白（EBNA 1）。EBNA 1结合到一个特定的位点， EBV附加体并增强附加体的初始复制、转录和持久性。 由于EBNA 1对于EBV游离体在所有分裂和增殖中的持续存在是必不可少的， 恶性细胞，该提案的中心目标是鉴定可以 抑制EBNA 1介导的附加体持久性。为实现这一目标，我们建议： (1)进行筛选以鉴定中断EBNA 1-oriP依赖性的化合物 附加体转录和体内持久性，中断EBNA 1的化合物 体外二聚化和与同源DNA的结合，以及与EBNA 1结合的化合物 计算机模拟。(2)确定已识别化合物的生物学和生物化学效应 在B淋巴母细胞中依赖EBNA 1-oriP的附加体转录和持续存在， EBV转化的淋巴母细胞（LCL）生长，以及LCL诱导的淋巴瘤 和裸鼠NPC肿瘤。(3)确定生物活性化合物作用的部位， EBNA 1结合，使用生物化学，生物物理和结构方法。使用此 知识，以最有效地进行结构活动修改， 化合物活性和特异性。(4)使用反向遗传学来识别关键的 EBNA 1 DBD中的残基，可以提高筛选灵敏度，并通过计算机信息 袋选择、化合物修饰和化合物相互作用分析。

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 proposal 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.