Implementation of small molecule high throughput screens for fibrosis inhibition

开发新疗法和生物探针的一个重要实验方法涉及高 药物吞吐量和小分子筛选。在这种方法最常见的迭代中，基于细胞培养的 开发了检测系统，在此基础上筛选出数万种潜在的治疗剂。 潜在的治疗药物被应用到检测中，一些结果被“读出”，表明这种稀有的试剂 达到了预期的治疗效果。高通量筛分还没有系统地应用于 肌肉营养不良症。到目前为止进行的高通量筛查试图上调内源性促营养素 在细胞中弥补肌营养不良蛋白缺乏，筛选能够影响终止密码子通读的试剂，以及 鉴定能诱导靶向外显子跳跃的反义寡核苷酸。在这里，我们建议在这样的基础上扩大 应用系统方法筛选与细胞周期调控相关的高通量药物 纤维化，这是肌肉营养不良的一个重要方面。这项试点和可行性拨款使 使用中心内的所有三个核心。核心B被广泛用于促进提纯和扩大 小鼠肌肉来源的成纤维细胞，报告评估.流式细胞仪分析中的构建，HTS的培养和分析 以及高含量成像仪数据的解释。该项目还使用Core C中的小鼠来评估生理 以及FDA批准的潜在化合物的组织学效应。该项目使用核心D来执行基因组 对基因表达进行广泛的分析，协助数据分析和解释，并就 启动子选择。试点和可行性项目的目的是开发和实施一个蜂窝记者 适合高通量筛选的检测成纤维细胞活化的方法。我们建议鉴定一种基因 纯化的成纤维细胞在培养中的表达反应，代表成纤维细胞对 因此，可以开发细胞报告分析来检测成纤维细胞的激活情况。在这一目标中 我们计划识别关键基因，开发特异的荧光蛋白基因表达报告结构，并 将这些构建物导入永生化成纤维细胞系。我们预计这一技术在体外的应用 化验将导致检测到能够降低纤维细胞活性的分子。

An important experimental approach to the development of new therapies and biological probes involves high throughput drug and small molecule screening. In the most common iteration of this approach, a cell culturebased assay system is developed upon which tens of thousands of potential therapeutic agents are screened. Potential therapeutic agents are applied to the assays, with some resulting "read out" indicating the rare agent that has achieved the desired therapeutic effect. High throughput screens have not been systematically applied to the muscular dystrophies. The high throughput screens performed to date attempt to up-regulate endogenous utrophin in cells to compensate for dystrophin deficiency, screen for agents able to effect read-through of stop codons, and identify antisense oligonucleotides that induce targeted exon skipping. Here, we propose expanding on such approaches by applying a systematic approach to high throughput drug screening relevant to the modulation of fibrosis, which is an important aspect of muscular dystrophies. This Pilot and Feasibility grant makes substantial use of all three cores within the Center. Core B is used extensively to facilitate the purification and expansion of mouse muscle derived fibroblasts, assessment of reporter.construct in FACS analysis, plating for HTS, and analysis and interpretation of high content imager data. The project also uses mice within Core C to assess physiological and histological effects of potential FDA approved compounds. The project uses Core D to perform the genome wide analysis of gene expression and to assist in data analysis and interpretation, and to provide guidance on promoter selection. The Aim of the pilot and feasibility project is to develop and implement a cellular reporter assay to detect fibroblast activation suitable for high throughput screening. We propose to identify a gene expression response within purified fibroblasts in culture that represents a component of the fibroblast response to injury, and therefore allows the development of cellular reporter assays to detect fibroblast activation. In this aim we plan to identify key genes, develop specific fluorescent protein gene expression reporter constructs, and transfect these constructs into an immortalized fibroblast cell line. We anticipate that the use of this in vitro assay will lead to the detection of molecules capable of decreasing fibrocyte activation.