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

描述（由申请人提供）：EB病毒（EBV）潜伏性感染引起几乎所有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、艾滋病和其他免疫受损状态患者的淋巴细胞增殖性疾病以及EBV相关的淋巴瘤、霍奇金病和鼻咽癌。EBV基因组作为非整合的多拷贝附加体存在于所有潜伏感染的细胞中。EBV游离体在分裂细胞中的持续存在依赖于EBV编码的核抗原1蛋白（EBNA 1）。EBNA 1与EBV附加体中的特定位点结合，并增强附加体的初始复制、转录和持久性。由于EBNA 1对于EBV附加体在所有分裂细胞和恶性细胞中的持久性是必需的，因此本申请的中心目的是鉴定可以抑制EBNA 1介导的附加体持久性的化合物。为了实现该目标，我们提出：（1）进行筛选以鉴定在体内中断EBNA 1-oriP依赖性附加体转录和持久性的化合物，在体外中断EBNA 1二聚化和与同源DNA结合的化合物，以及在计算机中与EBNA 1结合的化合物。(2)鉴定所鉴定的化合物对EBNA 1-oriP依赖性附加体转录和在B淋巴母细胞中的持久性、对EBV转化的淋巴母细胞样细胞（LCL）生长和对LCL诱导的裸鼠淋巴瘤和NPC肿瘤的生物学和生物化学作用。(3)确定EBNA 1结合的生物活性化合物的影响的网站，使用生物化学，生物物理和结构的方法。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)使用反向遗传学来鉴定EBNA 1 DBD中的关键残基，这些残基可以提高筛选灵敏度，并为计算机口袋选择、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感染者或其他免疫受损者中活跃地引起恶性淋巴组织增生性疾病、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EB病毒编码的核抗原1（EBNA 1）蛋白是必不可少的持久性的EBV基因组在这些恶性细胞，我们提出的实验，确定和改善抗EBNA 1化合物和防止EBV基因组持久性。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.