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一个重要实验方法涉及高 高通量药物和小分子筛选。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，在此基础上筛选了数以万计的潜在治疗剂。 潜在的治疗药物被应用于检测，一些结果“读出”表明稀有药物 已达到预期的治疗效果。高通量筛选尚未系统地应用于 肌肉营养不良症。迄今为止进行的高通量筛选试图上调内源性肌营养不良蛋白 在细胞中补偿肌营养不良蛋白的缺陷，筛选能够影响终止密码子通读的试剂，以及 鉴定诱导靶向外显子跳跃的反义寡核苷酸。在此，我们建议扩展此类 通过应用系统方法进行与调节相关的高通量药物筛选 纤维化，这是肌营养不良症的一个重要方面。这项试点和可行性拨款使 使用中心内的所有三个核心。 Core B 广泛用于促进纯化和扩增 小鼠肌肉来源的成纤维细胞、FACS 分析中的报告基因评估、HTS 电镀和分析 和高内涵成像仪数据的解释。该项目还使用 Core C 内的小鼠来评估生理学 以及 FDA 批准的潜在化合物的组织学影响。该项目使用Core D进行基因组分析 广泛分析基因表达并协助数据分析和解释，并提供指导 启动子选择。试点和可行性项目的目的是开发和实施细胞报告器 检测适合高通量筛选的成纤维细胞活化的测定。我们建议鉴定一个基因 培养物中纯化的成纤维细胞内的表达反应，代表成纤维细胞反应的一个组成部分 损伤，因此允许开发细胞报告测定法来检测成纤维细胞活化。为了这个目标 我们计划识别关键基因，开发特定的荧光蛋白基因表达报告构建体，以及 将这些构建体转染至永生化成纤维细胞系中。我们预计该技术在体外的应用 测定将导致能够减少纤维细胞活化的分子的检测。