Implementation of small molecule high throughput screens for fibrosis inhibition

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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.