Screening Assays for Small Molecules Targeting Oncogenic eIF4E Expression

描述(申请人提供)：真核细胞翻译起始因子4E(EIF4E)在人类癌症中经常过表达，并通过选择性地驱动对癌细胞生长和生存至关重要的基因(如c-myc、VEGF、bcl2)的翻译而促进癌症的发展和进展。在过去的几十年里，越来越多的证据表明，下调eIF4E在癌细胞中的表达可以成为癌症治疗干预的有效策略。事实上，eIF4E反义寡核苷酸已经被证明可以抑制肿瘤生长，对宿主动物没有毒性，并已进入临床试验。然而，由于缺乏可用于高通量药物筛选的可靠方法，目前还没有能够抑制癌细胞eIF4E表达的小分子可用于临床测试。这项应用是对NIH计划公告PA-10-213“用于探针和治疗前发现的高通量筛查分析的开发”的响应，将满足发现针对癌细胞中eIF4E表达的药物先导的高通量筛查(HTS)分析的迫切需求。具体地说，我们的目标是利用新兴的细胞工程技术来开发一种创新的检测方法，其中整合在天然eIF4E基因座上的生物发光报告在内源性eIF4E启动子的控制下表达。这种分析有望克服目前的报告分析的局限性，这些分析大多基于克隆的转基因启动子，而这些启动子通常缺乏必要的末端/内含子顺式调控元件，而这些元件存在于外国染色质环境中。因此，这种创新的方法将忠实地复制内源性eIF4E基因对化学处理的反应，从而在高通量寻找抑制eIF4E表达的小分子方面更加可靠。因此，该项目的具体目标是：(1)建立在天然eIF4E基因座上含有报告基因的细胞系，用于高通量药物筛选；(2)建立二次检测方法，用于识别假阳性并确定进一步药物开发的筛选重点。这项拟议研究的完成将带来一种创新的HTS检测方法，可用于开发通过抑制eIF4E表达来治愈癌症的治疗剂。因此，拟议中的研究将影响数百万癌症患者的健康。这项应用还将为一种创新的药物发现策略提供概念证明，该策略可用于搜索针对癌症中异常表达的任何给定基因的药物，从而显着推进靶向癌症治疗。 公共卫生相关性： 该项目的目标是开发一种创新的筛选试验，用于发现针对eIF4E的治疗剂--eIF4E是一种在人类癌症中普遍过度表达的蛋白质。最终，拟议研究的完成将导致新癌症疗法的开发，从而使数百万癌症患者受益。

DESCRIPTION (provided by applicant): The eukaryotic translation initiation factor 4E (eIF4E) is frequently overexpressed in human cancers and contributes to cancer development and progression by selectively driving translation of genes that are essential for cancer cell growth and survival (e.g., c-myc, VEGF, BCL-2). In the past decades accumulating evidence has demonstrated that down-regulation of eIF4E expression in cancer cells can be an effective strategy for therapeutic intervention of cancer. Indeed, eIF4E antisense oligonucleotides have been shown to inhibit tumor growth without toxicity to host animals, and have entered clinical trials. However, because of the lack of a reliable assay that can be used in high-throughput drug screening, no small molecule that can inhibit eIF4E expression in cancer cells is currently available for clinical testing. This application is in response to the NIH Program Announcement PA-10-213, "Development of Assays for High-Throughput Screening for Use in Probe and Pre-therapeutic Discovery", and will address the imperative need of a high-throughput screening (HTS) assay for discovery of drug leads that target eIF4E expression in cancer cells. Specifically, our objective is to utilize emerging cell-engineering technologies to develop an innovative assay in which a bioluminescent reporter integrated in the native eIF4E gene locus is expressed under control of the endogenous eIF4E promoter. Such an assay is expected to overcome limitations of current reporter assays that are mostly based on cloned, transgenic promoters, which often lack essential distal/intronic cis-regulatory elements while residing in foreign chromatin environments. Therefore, the innovative assay would faithfully reproduce responses of the endogenous eIF4E gene to chemical treatments and thus be more reliable in high-throughput search for small molecules inhibitory for eIF4E expression. Accordingly, the specific aims of this project are: (1) to develop cell lines harboring a reporter gene in the native eIF4E gene locus for high-throughput drug screening, (2) to develop secondary assays for identification of false positives and prioritization of screening hits for further drug development. Completion of the proposed research will result in an innovative HTS assay useful for developing therapeutic agents that can cure cancer through inhibiting eIF4E expression. Therefore, the proposed studies will impact health of millions of people afflicted with cancer. This application will also provide a proof of concept for an innovative drug-discovery strategy that can be applied to search for agents targeting any given gene aberrantly expressed in cancer thereby significantly advancing targeted cancer therapy. PUBLIC HEALTH RELEVANCE: The objective of this project is to develop an innovative screening assay for use in discovery of therapeutic agents targeting eIF4E - a protein commonly overexpressed in human cancers. Ultimately, completion of the proposed research will lead to the development of novel cancer therapies, and thereby benefit millions of people afflicted with cancer.