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

描述(申请人提供)：Wnt/Wingless(WG)途径是一种进化上保守的细胞信号途径，调节后生动物发育的许多方面。Wnt途径的失调与肝脏、结肠、乳房和皮肤的肿瘤发生有关。转录因子β-连环蛋白(？-cat)是Wnt途径最重要的效应因子之一。由于连环蛋白反应转录(CRT)与许多癌症的发生有关，因此它是开发调节β-CAT核活动的治疗药物的良好靶点。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)技术，在果蝇和人类细胞系中鉴定了新的和特异的CRT化合物调节剂。目的/假设：我们假设我们的初步筛选策略是针对CRT的，并且WNT途径的新的复合调节剂可以作为WNT相关疾病和发育模型的有效治疗试剂。具体目的：1)在细胞分析中确定(并提高)候选小分子阻断CRT诱导/依赖表型的特异性和有效性；2)确定在初级筛选中确定的候选小分子影响CRT并确定其蛋白质靶点的分子机制；3)在异种移植模型和WNT相关肿瘤的小鼠模型中测试铅抑制化合物阻断WNT/CRT依赖表型的能力/有效性。研究设计：初步筛选的目标是确定作用于Axin介导的降解复合体下游的核？-CAT活性的新抑制剂。为了确定候选小分子的作用机制，我们将利用免疫共沉淀、免疫定位和EMSA分析来测试它们改变？-CAT与已知蛋白质相互作用伙伴的相互作用的能力，或者它们改变？-CAT/TCF-转录复合体的DNA结合性质的能力。我们将利用酶联免疫吸附试验、下拉试验(与纯化的蛋白)以及表面等离子体共振分析来确定候选化合物与纯化的β-CAT或其他目标蛋白的直接结合。我们将验证来自我们的中试筛选的候选化合物在各种对Wnt有反应的哺乳动物和癌细胞系中的抑制作用，包括HEK293细胞、C57 mg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瘤建立和转移模型中的作用。我们还将结合电子对接模型进行SAR研究，以提高初级筛选中确定的新型化合物的功效/效力。随后，将使用已经优化的基于细胞的和体内的WNT/CRT活性测试来验证“改进的”候选化合物的有效性。 与公共健康相关：这项提案的总体目标是开发和实施创新的筛选技术，以识别Wnt/Wingless信号通路的新型复合调节剂。这项研究的结果将对开发治疗WNT诱导的致癌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.