Discovery of inhibitors of EBV lytic cycle inducing protein ZTA for therapeutic development

项目总结： 这项SBIR的目标是发现一种针对EB病毒(EBV)编码的ZTA的小分子抑制剂 预防EBV相关癌症和免疫疾病的蛋白质。EB病毒是一种人类疱疹病毒， 潜伏和裂解基因表达之间逃避免疫检测和促进致癌转化 不同类型的细胞。EBV被世界卫生组织归类为I类致癌物质，估计与 每年约有20万新的癌症病例。EB病毒相关的癌症是多样的，包括胃癌的亚型 癌症，鼻咽癌，伯基特淋巴瘤，霍奇金淋巴瘤，NK/T细胞淋巴瘤，以及 各种非霍奇金淋巴瘤，特别是在免疫功能低下的人群中。EBV是主要的 传染性单核细胞增多症的病原体，以及多发性硬化(MS)的主要病毒危险因素 以及其他自身免疫性疾病。病毒编码的蛋白质ZTA(也称为BZLF1、Zebra和Z)是一种 氧化还原敏感的DNA结合转录激活剂，对EBV裂解周期基因的表达和 病毒生产。裂解病毒的周期性循环是EBV驱动的恶性肿瘤和自身免疫性疾病的关键组成部分。 免疫疾病，以及ZTA及其激活的下游病毒和细胞基因，已被牵连 在致癌和免疫病理学方面。因此，ZTA是治疗干预的一个有吸引力的靶点。 ZTA的dna结合域已经从结构和生化上进行了表征，并提供了一个理想的 靶向抑制EBV裂解生命周期的小分子。 我们已经开发了用于检测ZTA DNA结合的可靠的生化和基于细胞的分析方法，并使用 用于传统高通量筛选(HTS)的生化分析。我们使用了一种 不相关的DNA结合蛋白来消除DNA嵌入物并测试最具选择性的化合物 表面等离子共振(SPR)确定与目标蛋白的直接结合。我们现在建议使用 改进的计数器筛查分析，以表征之前筛查工作中的命中结果，扩大我们的 对天然产物和专为传染病设计的合成化合物的额外文库进行筛选 目标。天然产物具有靶向蛋白质刚性高等优点 活性部位之外的表面，如ZTA。我们将使用药物化学来定义化学载体 研究我们的HITS的构效关系，并为进一步开发寻找合适的先导化合物。我们的 目标是生产在生物化学分析中具有纳摩尔效力、低微摩尔活性的先导化合物 以细胞为基础的分析，与对照计数器筛选相比，选择性超过10倍。在第二阶段，我们将 将我们的高级HITS发展成为临床前的领先候选者。该SBIR计划的最终目标是 开发一种新的小分子治疗剂来阻断EBV裂解循环及其相关疾病。

Project Summary: The goal of this SBIR is to discover a small molecule inhibitor to the Epstein-Barr Virus (EBV)-encoded ZTA protein to prevent EBV-associated cancer and immune disorders. EBV is a human herpesvirus that switches between latent and lytic gene expression to escape immune detection and promote oncogenic transformation of diverse cell types. EBV is classified as a class I carcinogen by the WHO and estimated to be responsible for ~200,000 new cancer cases per year. EBV-associated cancers are diverse, and include subtypes of gastric carcinoma, nasopharyngeal carcinoma, Burkitt’s lymphoma, Hodgkin’s lymphoma, NK/T cell lymphoma, and various non-Hodgkin’s lymphomas particularly among immunocompromised populations. EBV is the major causative agent of infectious mononucleosis, and the predominant viral risk factor for multiple sclerosis (MS) and other auto immune diseases. The viral-encoded protein, ZTA (also known as BZLF1, ZEBRA, and Z) is a redox-sensitive DNA-binding transcriptional activator that is essential for EBV lytic cycle gene expression and viral production. The periodic cycling of lytic virus is a critical component for EBV-driven malignancy and auto- immune disease, and ZTA and the downstream viral and cellular genes that it activates, have been implicated in carcinogenesis and immune pathology. Therefore, ZTA is an attractive target for therapeutic intervention. The DNA-binding domain of ZTA has been characterized structurally and biochemically, and provides an ideal target for small molecule inhibition of EBV lytic life cycle. We have developed robust biochemical and cell-based assays for detection of ZTA DNA binding and used the biochemical assays for traditional high throughput screening (HTS). We screened the HTS hits using an unrelated DNA binding protein to eliminate DNA intercalators and tested the most selective compounds using surface plasmon resonance (SPR) to determine direct binding to the target protein. We now propose to use improved counter screening assays to characterize the hits from the previous screening efforts, expand our screening to natural products and an additional library of synthetic compounds designed for infectious disease targets. Natural products have advantageous characteristics such as higher rigidity for targeted protein surfaces outside of active sites like that of ZTA. We will use medicinal chemistry to define chemophore structure-activity relationships of our hits and identify a suitable lead compound for further development. Our goal is to produce lead compounds with nanomolar potency in biochemical assays, low micromolar activity in cell-based assays, and greater than 10-fold selectivity against control counter-screens. In Phase 2, we will develop our advanced hits into a pre-clinical lead candidate. The ultimate goal of this SBIR program is to develop a novel small molecule therapeutic agent to block the EBV lytic cycle and its associated diseases.