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

项目摘要： 该SBIR的目标是发现一种小分子抑制剂，以EB病毒（EBV）编码的ZTA 蛋白质，以防止EB病毒相关的癌症和免疫系统疾病。EB病毒是一种人类疱疹病毒， 潜伏基因表达和溶解基因表达之间的关系，以逃避免疫检测并促进致癌转化 不同的细胞类型。EB病毒被世界卫生组织归类为I类致癌物，据估计， 每年约有20万新的癌症病例。EBV相关的癌症是多种多样的，并且包括胃癌的亚型。 癌、鼻咽癌、伯基特淋巴瘤、霍奇金淋巴瘤、NK/T细胞淋巴瘤和 各种非霍奇金淋巴瘤，特别是在免疫功能低下的人群。EB病毒是主要的 传染性单核细胞增多症的病原体，以及多发性硬化症（MS）的主要病毒风险因素 和其他自身免疫性疾病。病毒编码的蛋白质ZTA（也称为BZLF 1、ZEBRA和Z）是一种蛋白质。 对EBV裂解周期基因表达至关重要的氧化还原敏感的DNA结合转录激活因子， 病毒生产裂解性病毒的周期性循环是EBV驱动的恶性肿瘤和自身免疫的关键组成部分。 ZTA及其激活的下游病毒和细胞基因与免疫性疾病有关 在癌症发生和免疫病理学中的作用。因此，ZTA是治疗干预的有吸引力的靶标。 ZTA的DNA结合结构域已经在结构上和生物化学上进行了表征，并提供了理想的 靶向小分子抑制EBV裂解生命周期。 我们已经开发了用于检测ZTA DNA结合的稳健的生物化学和基于细胞的测定法，并使用 用于传统高通量筛选（HTS）的生物化学测定。我们用一个 不相关的DNA结合蛋白，以消除DNA嵌入剂，并测试了最具选择性的化合物，使用 表面等离子体共振（SPR）以测定与靶蛋白的直接结合。我们现在建议使用 改进的计数器筛选测定来表征来自先前筛选工作的命中，扩展了我们的 筛选天然产物和针对传染病设计的合成化合物的额外库 目标的天然产物具有有利的特性，例如对靶蛋白质具有更高的刚性 像ZTA这样的活跃位点之外的表面。我们将使用药物化学来定义化学基团 我们的命中的结构-活性关系，并确定一个合适的先导化合物，进一步发展。我们 目标是生产在生物化学测定中具有纳摩尔效力的先导化合物， 基于细胞的测定，以及对对照反筛选的大于10倍的选择性。在第二阶段，我们将 把我们的先进成果发展成临床前的主要候选人该SBIR计划的最终目标是 开发一种新型的小分子治疗剂来阻断EBV裂解周期及其相关疾病。

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.