Screen for Inhibitors of La-dependent IRES-mediated Translation in Cancer Cells

描述(由申请人提供)：蛋白质翻译是导致蛋白质表达的一种基本的和高度调控的细胞机制。然而，在癌症等人类疾病中，蛋白质合成往往是失控的。翻译起始通常发生在帽子依赖的机制中，但在应激和癌细胞中，翻译起始可能是通过内部核糖体进入位点(IRES)介导的。IRES介导的翻译解释了一些促肿瘤因子在应激和癌细胞中的表达。辅助RNA结合蛋白，称为IRES反式作用因子(ITAF)，支持IRES介导的翻译。因此，针对刺激IRES介导的促肿瘤因子翻译所需的特定ITAF：IRES相互作用代表了一种创新的治疗方法，并针对通常在癌细胞中激活的细胞机制。我们的长期目标是识别针对特定ITAF：IRES相互作用的小分子抑制剂，以展示它们选择性抑制肿瘤翻译的功能 促进因子，并开发此类分子来开发新型抗癌药物。为了实现这一目标，申请人实验室的研究重点是开发高通量检测方法，以筛选抑制RNA结合蛋白La功能的化合物，La是一种著名的ITAF。申请人最近发表了ITAFLa在不同类型的肿瘤(如口腔癌、宫颈癌)中过表达，刺激细胞增殖，并在 IRES依赖的肿瘤促进因子的翻译，例如协作性癌基因细胞周期蛋白D1。中心假设是La刺激的细胞周期蛋白D1IRES介导的翻译的小分子抑制剂将减少癌细胞的增殖。其基本原理是我们发现癌细胞中La的枯竭降低了Cyclin D1的表达和细胞的增殖。我们的一组强大的初步数据指导我们制定了三个具体目标来挑战我们的假设。在特定目标1：我们的目标是筛选抑制RNA结合蛋白La功能的小分子。在特定目标2中，我们期望展示在初步筛选中确定的化合物的活性和特异性，最后在特定目标3中，我们希望 展示先导化合物在基于细胞的分析中的功能。这种方法是创新的，因为它使用了新的HTS-分析来筛选蛋白质的抑制物：RNA相互作用，它在肿瘤发生中发挥关键作用。这项拟议的研究意义重大，因为识别出抑制La：RNA相互作用的新分子将意味着概念的证明。此外，预计拟议的研究将代表着对现状的实质性偏离，并将阐明作为潜在药物靶点的蛋白质：RNA相互作用的极其广泛和多样化的领域。最终，这些知识可能会被转化为针对肿瘤促进因子在癌症中表达的新治疗策略。

DESCRIPTION (provided by applicant): Protein translation is an essential and highly regulated cellular mechanism leading to the expression of proteins. However, protein synthesis is often miss-regulated in human diseases, such as cancer. Translation initiation occurs normally in a cap-dependent mechanism but in stressed and cancerous cells translation initiation might be mediated via Internal Ribosome Entry Sites (IRES). IRES-mediated translation accounts for the expression of some tumor-promoting factors in stressed and cancerous cells. Auxiliary RNA-binding proteins, referred to as IRES-Trans-Acting Factors (ITAF), support IRES-mediated translation. Thus, targeting specific ITAF:IRES interactions required to stimulate the IRES-mediated translation of tumor-promoting factors represents an innovative therapeutic approach and targets a cellular mechanism often activated in cancerous cells. Our long-term goal is to identify small molecule inhibitors for specific ITAF:IRES interactions, to demonstrate their functionality in repressing selectively the translation of tumor promoting factors and to develop such molecules to novel anti-cancer drugs. To reach this goal the research in the applicant's laboratory focused on the development of high-throughput assays to screen for compounds inhibiting the functionality of the RNA-binding protein La, a well-known ITAF. The applicant recently published that the ITAF La is overexpressed in different types of tumors (e.g. oral, cervical cancer), stimulates cell proliferation and acts as ITAF during IRES-depending translation of tumor-promoting factors, such as the cooperative oncogene cyclin D1. The central hypothesis is that small molecule inhibitors of La-stimulated cyclin D1 IRES-mediated translation will reduce proliferation of cancerous cells. The rationale is that we found that depletion of La in cancerous cells reduce cyclin D1 expression and cell proliferation. Our strong set of preliminary data guided us to develop three Specific Aims to challenge our hypothesis. In Specific Aim 1: we aim to screen for small molecules inhibiting the functionality the RNA-binding protein La. In Specific Aim 2 we anticipated to demonstrate activity and specificity of compounds identified in the primary screen, and finally in Specific Aim 3 we wish to demonstrate the functionality of lead compounds in cell-based assays. The approach is innovative because it uses novel HTS-assays to screen for inhibitors of protein:RNA interactions which playing a critical role in tumorigenesis. The proposed research is significant, because identification of novel molecules inhibiting La:RNA interactions would signify a proof of concept. Furthermore it is anticipated that the proposed research would represents a substantive departure from the status quo and would illuminate the extremely wide and diverse field of protein:RNA interactions as potential drug target. Ultimately, such knowledge might be translated into new therapeutic strategies targeting the expression of tumor-promoting factors in cancer.