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

项目概要： 该 SBIR 的目标是发现 Epstein-Barr 病毒 (EBV) 编码的 ZTA 的小分子抑制剂 蛋白质可预防 EBV 相关癌症和免疫疾病。 EBV 是一种人类疱疹病毒 潜伏基因表达和裂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。 EBV 被 WHO 列为 I 类致癌物，估计与 每年约有 200,000 个新癌症病例。 EBV 相关癌症多种多样，包括胃癌亚型 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，尤其是免疫功能低下人群中的非霍奇金淋巴瘤。 EBV是主要 传染性单核细胞增多症的病原体，以及多发性硬化症 (MS) 的主要病毒危险因素 和其他自身免疫性疾病。病毒编码蛋白 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，对于 EBV 裂解周期基因表达至关重要 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、ZTA 及其激活的下游病毒和细胞基因都与此有关 在癌发生和免疫病理学方面。因此，ZTA 是治疗干预的一个有吸引力的靶点。 ZTA 的 DNA 结合域已在结构和生物化学上进行了表征，并提供了理想的 EBV 裂解生命周期的小分子抑制靶点。 我们开发了强大的生化和细胞检测方法来检测 ZTA DNA 结合，并使用 用于传统高通量筛选 (HTS) 的生化检测。我们使用以下方法筛选了 HTS 命中 不相关的 DNA 结合蛋白可消除 DNA 嵌入剂，并使用以下方法测试了最具选择性的化合物 表面等离子共振 (SPR) 以确定与靶蛋白的直接结合。我们现在建议使用 改进的反筛选测定来表征先前筛选工作的命中率，扩大我们的 筛选天然产物和专为传染病设计的额外合成化合物库 目标。天然产物具有优势特性，例如目标蛋白质具有更高的刚性 活性位点外部的表面，如 ZTA 的表面。我们将使用药物化学来定义化学团 我们的命中的结构-活性关系，并确定适合进一步开发的先导化合物。我们的 目标是生产在生化分析中具有纳摩尔效力的先导化合物，在生物化学分析中具有低微摩尔活性 基于细胞的测定，与对照反筛选相比选择性超过 10 倍。在第二阶段，我们将 将我们的先进产品发展成为临床前的主要候选药物。该 SBIR 计划的最终目标是 开发一种新型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.