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

Wnt/Beta-Cat 信号通路新型化学调节剂的靶向筛选。

基本信息

批准号：
8462926
负责人：
Ramanuj Dasgupta
金额：
$ 32.96万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-23 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8462926
关键词：
Adenocarcinoma Cell Adherens Junction Affect Affinity Chromatography Animal Model Binding Biological Assay Breast Breast Adenocarcinoma Cancer Patient Cancer cell line Cancerous Cell Line Cell Nucleus Cell Proliferation Cells Cessation of life Chemicals Clinical Co-Immunoprecipitations Colon Complex Computer Simulation Coupled DNA Binding Data Development Differentiation and Growth Disease Dissection Docking Drosophila genus E-Cadherin EMSA Enhancers Enzyme-Linked Immunosorbent Assay Epithelial Cells Exhibits Family Family Dasypodidae Felis catus Gene Targeting Genetic Screening Genetic Transcription Goals HCT116 Cells HT29 Cells Human Human Cell Line Intercellular Junctions Kinetics Large Intestine Carcinoma Lead Ligands Liver MCF7 cell Malignant Neoplasms Malignant neoplasm of ovary Mammalian Cell Mammary gland Mass Spectrum Analysis Maximum Tolerated Dose Mediating Modeling Molecular Morphology Mus Nature Neoplasm Metastasis Nuclear Pathway interactions Phenotype Primary carcinoma of the liver cells Property Proteins RNA Interference Reagent Regulation Research Design Signal Pathway Signal Transduction Skin Specificity Structure-Activity Relationship Surface Plasmon Resonance TCF7L2 gene Technology Testing Therapeutic Tissues Uterine Cancer Xenograft Model Xenograft procedure base carcinogenesis cell motility chemical genetics colon cancer cell line combat design high throughput screening improved in vivo inhibitor/antagonist innovation insight melanoma mouse model novel protein expression screening small molecule therapeutic target tool transcription factor tumor tumor growth tumor xenograft tumorigenesis

项目摘要

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.

描述（由申请人提供）：Wnt/无翼（WG）途径是一种进化保守的细胞信号途径，可调节后生动物发育的许多方面。 Wnt途径的失调与肝脏，结肠，乳腺和皮肤的肿瘤发生有关。 Wnt途径的最重要效应子之一是由转录因子？-Catenin（？-CAT）编码的。由于Catenin反应式转录（CRT）与许多癌症的起源有关，因此它是开发可以调节核活性的疗法的良好靶标。最近，我们采用了创新的基于RNAi的靶向化学遗传高通量屏幕（HTS）来识别果蝇和人类细胞系中CRT的新颖和特定复合调节剂。客观/假设：我们假设我们的主要筛查策略专门针对CRT，而Wnt途径的新型复合调节剂可以用作WNT相关疾病和发育模型中的有效治疗试剂。具体目的：1）确定（和改善）候选小分子在阻断基于细胞测定中CRT诱导/依赖的表型方面的特异性和功效； 2）确定候选小分子在主要筛选中识别出CRT并鉴定其蛋白质靶标的分子机制； 3）测试铅抑制性化合物在封闭异种移植模型中阻断Wnt/CRT依赖性表型以及Wnt相关癌的小鼠模型中的能力/功效。研究设计：主要筛选的目的是鉴定核CAT活性的新型抑制剂，该核能活性在轴介导的降解络合物下游作用。为了确定候选小分子的机制，我们将测试它们改变其已知蛋白质相互作用伙伴的相互作用的能力，或使用使用共沉淀，免疫定位，免疫定位和EMSA分析来改变？-CAT/TCF-转录复合物的DNA结合特性的能力。我们将利用ELISA和下拉测定法（带有纯化的蛋白质），以及表面等离子体共振测定法，以确定候选化合物与纯化的？-CAT或其他靶蛋白的直接结合。我们将在各种WNT响应性哺乳动物和癌细胞系中验证候选化合物对候选化合物的抑制作用，包括HEK293细胞，C57MG小鼠乳腺上皮细胞，MCF7人类乳腺癌细胞和HCT116＆HT29结肠癌细胞系MCF7人乳腺癌细胞。我们将评估这些化合物在体内的小鼠异种移植物中阻断Wnt/CRT诱导的转移模型的作用。我们还将进行SAR研究，并在计算机对接模型中进行结合，以提高主要筛选中鉴定出的新型化合物的功效/效力。随后，使用已经优化的基于细胞的细胞和体内分析进行WNT/CRT活性，将对“改进的”候选化合物进行经验验证。