Implementation of small molecule high throughput screens for fibrosis inhibition

实施小分子高通量筛选以抑制纤维化

• 批准号: 7680826
• 负责人: STANLEY F. NELSON
• 金额: $ 5.93万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7680826
• 关键词: Antisense Oligonucleotides; Biological Assay; Biological Response Modifier Therapy; Cell Culture Techniques; Cell Line; Cells; Characteristics; Classification; Data; Data Analyses; Detection; Development; Dystrophin; Exons; FDA approved; Fibroblasts; Fibrosis; Gene Expression; Genes; Grant; Injury; Lead; Microarray Analysis; Mus; Muscle; Muscle function; Muscular Dystrophies; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Physiological; Preclinical Drug Evaluation; Proteins; Reading; Reporter; Screening procedure; System; Terminator Codon; Testing; Therapeutic Agents; Therapeutic Effect; Utrophin; base; boys; cellular development; genome wide association study; genome-wide analysis; high throughput screening; in vitro Assay; promoter; response; response to injury; small molecule; success

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.

开发新疗法和生物探针的重要实验方法涉及高 吞吐药和小分子筛选。在这种方法的最常见迭代中，基于细胞培养 开发了测定系统，筛选了数万个潜在的治疗剂。 潜在的治疗剂被应用于测定中，其中一些“读取”表明罕见的药物 已经达到了所需的治疗效果。高吞吐量屏幕尚未系统地应用于 肌肉营养不良。迄今为止执行的高吞吐量屏幕尝试上调内源性乌托芬 在补偿肌营养不良蛋白缺乏症的细胞中，筛选能够实现终止密码子的读取物的筛选，并且 确定诱导靶向外显子跳动的反义寡核苷酸。在这里，我们建议扩大这种扩展 通过将系统的方法应用于与调制相关的高通量药物筛查中的方法 纤维化，这是肌肉营养不良的重要方面。这项飞行员和可行性赠款可实现 在中心内使用所有三个核心。 Core B广泛用于促进纯化和扩展 小鼠肌肉衍生的成纤维细胞，评估报告中的facs分析中的构造，用于HTS的电镀和分析 以及高素质成像器数据的解释。该项目还使用Core C内的小鼠评估生理 潜在FDA批准化合物的组织学影响。该项目使用核心D执行基因组 广泛分析基因表达并协助数据分析和解释，并为 启动子选择。飞行员和可行性项目的目的是开发和实施蜂窝记者 测定可检测适用于高吞吐量筛选的成纤维细胞激活。我们建议识别一个基因 培养中纯化成纤维细胞中的表达反应，代表成纤维细胞反应的成分 损伤，因此允许开发细胞报告基因测定法检测成纤维细胞激活。在这个目标中 我们计划鉴定关键基因，开发特定的荧光蛋白基因表达报告基因构建体，并 将这些构建体转染到永生的成纤维细胞系中。我们预计会在体外使用这种使用 测定将导致检测能够降低纤维细胞活化的分子。