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

描述（由申请人提供）：Wnt/wingless （wg）通路是一种进化上保守的细胞信号通路，调节后生动物发育的许多方面。Wnt通路的失调与肝脏、结肠、乳房和皮肤的肿瘤发生有关。Wnt通路最重要的效应物之一是由转录因子编码的。连环蛋白(?猫)。由于Catenin反应性转录（CRT）与许多癌症的发生有关，因此它是开发可调节-cat核活性的治疗方法的良好靶点。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新的特异性CRT化合物调节剂。目的/假设：我们假设我们的初步筛选策略专门针对CRT，并且Wnt通路的新型化合物调节剂可以作为Wnt相关疾病和发育模型的有效治疗试剂。具体目标：1)确定（并提高）候选小分子在基于细胞的检测中阻断crt诱导/依赖表型的特异性和有效性；2)确定primary screen中确定的候选小分子影响CRT的分子机制，并确定其蛋白靶点；3)在异种移植物模型以及Wnt相关癌症小鼠模型中，测试铅抑制化合物阻断Wnt/ crt依赖表型的能力/功效。研究设计：初步筛选的目的是鉴定新的核？-cat活性，作用于轴蛋白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？-cat与其已知蛋白质相互作用伙伴的相互作用，或者它们改变DNA结合特性的能力？-cat/ tcf转录复合物使用共免疫沉淀，免疫定位和EMSA测定。我们将利用ELISA、下拉试验（纯化蛋白）和表面等离子体共振试验来确定候选化合物与纯化蛋白的直接结合。-cat或其他靶蛋白。我们将验证中试筛选的候选化合物在多种wnt反应性哺乳动物和癌细胞系中的抑制作用，包括HEK293细胞、C57mg小鼠乳腺上皮细胞、MCF7人乳腺腺癌细胞系以及HCT116和HT29结肠癌细胞系。我们将在体内评估这些化合物在阻断Wnt/ crt诱导的小鼠异种移植物肿瘤建立和转移模型中的作用。我们还将结合硅对接模型进行SAR研究，以提高在初级筛选中鉴定的新型化合物的功效/效力。“改进”的候选化合物随后将使用已经优化的基于细胞和体内的Wnt/CRT活性试验进行经验验证。

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.