Targeted Screen for Novel Chemical Modulators of Wnt/Beta-Cat Signaling Pathway.

描述（由申请人提供）：Wnt/无翅（wg）通路是一种进化上保守的细胞信号传导通路，调节后生动物发育的许多方面。Wnt通路的失调与肝脏、结肠、乳腺和皮肤的肿瘤发生有关。Wnt途径最重要的效应子之一是由转录因子？连环蛋白（？-猫）。由于连环蛋白应答转录（CRT）与许多癌症的发生有关，因此它成为开发可调节？最近，我们采用了一种创新的基于RNAi的靶向化学遗传高通量筛选（HTS）来鉴定果蝇和人类细胞系中CRT的新型和特异性化合物调节剂。目的/假设：我们假设我们的初步筛选策略特异性靶向CRT，并且Wnt途径的新型化合物调节剂可以在Wnt相关疾病和发育模型中充当有效的治疗试剂。具体目标：1）确定（并改善）候选小分子在基于细胞的测定中阻断CRT诱导的/依赖性表型的特异性以及功效; 2）确定在初级筛选中鉴定的候选小分子影响CRT的分子机制并鉴定其蛋白质靶标; 3）测试先导抑制化合物在异种移植模型以及Wnt相关癌症的小鼠模型中阻断Wnt/CRT依赖性表型的能力/功效。研究设计：初步筛选的目的是鉴定新的核受体抑制剂。cat活性，其作用于Axin介导的降解复合物的下游。为了确定候选小分子的机制，我们将测试它们改变？猫的相互作用与其已知的蛋白质相互作用的合作伙伴，或他们的能力，改变DNA结合特性的？使用免疫共沉淀、免疫定位和EMSA测定来检测cat/TCF-转录复合物。我们将利用ELISA和下拉测定（纯化蛋白），以及表面等离子体共振测定来确定候选化合物与纯化？cat或其他靶蛋白。我们将在多种Wnt响应性哺乳动物和癌细胞系中验证来自我们的中试筛选的候选化合物的抑制作用，所述细胞系包括HEK 293细胞、C57 mg小鼠乳腺上皮细胞、MCF 7人乳腺癌细胞系和HCT 116 & HT 29结肠癌细胞系。我们将评估这些化合物在体内阻断小鼠异种移植物中Wnt/CRT诱导的肿瘤建立和转移模型的作用。我们还将结合计算机对接模型进行SAR研究，以提高在初步筛选中鉴定的新型化合物的疗效/效价。“改进的”候选化合物随后将使用已经优化的Wnt/CRT活性的基于细胞的和体内测定法进行经验验证。 公共卫生相关性：该提案的总体目标是开发和实施创新的筛选技术，用于鉴定Wnt/Wingless信号通路的新型化合物调节剂。这项研究的结果将对开发用于治疗Wnt诱导的致癌作用的靶向疗法产生广泛的影响。

DESCRIPTION (provided by applicant): The Wnt/wingless (wg) pathway is an evolutionarily conserved cell-signaling pathway that regulates many aspects of metazoan development. Dysregulation of the Wnt pathway has been associated with tumorigenesis of the liver, colon, breast and skin. One of the most important effectors of the Wnt pathway is encoded by the transcription factor, ?-catenin (?-cat). Since Catenin Responsive Transcription (CRT) has been implicated in the genesis of many cancers, it makes a good target for developing therapeutics that could modulate the nuclear activity of ?-cat. Recently, we employed an innovative RNAi-based targeted chemical genetic high-throughput-screen (HTS) to identify novel and specific compound modulators of CRT in Drosophila and human cell lines. Objective/Hypothesis: We hypothesize that our primary screening strategy specifically targets CRT and that the novel compound modulators of the Wnt pathway could serve as effective therapeutic reagents in Wnt- relevant disease and developmental models. Specific Aims: 1) Determine (and improve) the specificity as well as efficacy of candidate small molecules in blocking CRT-induced/dependent phenotypes in cell-based assays; 2) Determine the molecular mechanisms by which candidate small molecules identified in the primary screen impact CRT and identify their protein targets; 3) Test the ability/efficacy of lead inhibitory compounds in blocking Wnt/CRT-dependent phenotypes in xenograft models, as well as in mouse models of Wnt-relevant cancers. Study design: The goal of the primary screen was to identify novel inhibitors of nuclear ?-cat activity that act downstream of the Axin-mediated degradation complex. In order to determine the mechanism of candidate small molecules, we will test their ability to alter ?-cat's interaction with its known protein interaction partners, or their ability to alter the DNA binding properties of ?-cat/TCF-transcriptional complex using co- immunoprecipitation, immunolocalization, and EMSA assays. We will utilize ELISA, and pull-down assays (with purified proteins), together with Surface Plasmon Resonance assays to determine direct binding of candidate compounds to purified ?-cat or other target proteins. We will validate the inhibitory effect of candidate compounds from our pilot screen in a variety of Wnt-responsive mammalian and cancer cell lines, including, HEK293 cells, C57mg mouse mammary epithelial cells, MCF7 human breast adenocarcinoma cell line, and the HCT116 & HT29 colon cancer cell lines. We will assess the effect of these compounds in blocking Wnt/CRT-induced tumor establishment and metastasis models in mouse xenografts, in vivo. We will also perform SAR studies coupled with in silico docking models to improve the efficacy/potency of the novel class of compounds identified in the primary screen. The "improved" candidate compounds will subsequently be validated empirically using the already optimized cell-based and in vivo assays for Wnt/CRT activity. PUBLIC HEALTH RELEVANCE: The overall goal of this proposal is to develop and implement innovative screening technologies for the identification of novel compound modulators of the Wnt/Wingless signaling pathway. Results from this study will have a broad impact in the development of targeted therapeutics for the treatment of Wnt-induced carcinogenesis.