High Throughput Molecular Screening for Compounds(RMI)

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DESCRIPTION (provided by applicant): The potent and pleiotropic cytokine, transforming growth factor beta1 (Tgfb1), is involved in a broad range of biological processes. Excessive Tgfbl activity causes fibrosis in the lungs, kidney and liver. Intracellular signaling by Tgfb1 is mediated largely through a class of molecules known as Smad proteins. The Smad proteins, Smad2 or Smad3, are phosphorylated by the Tgfb1 receptor complex, translocate to the nucleus and initiate nuclear signaling. Interference with SmadS-mediated Tgfb1 signaling prevents the development of tissue fibrosis in many organs. The the ability to modulate this pathway has obvious therapeutic replications. To facilitate screen of these compounds, we have developed fibroblasts that stably express fireflv luciferase selectively, sensitively, and quantitatively in response to Tgfb1 stimulation. We propose to optimize this system for high-throughput compound screening by way of 4 quarterly milestones. First, we will establish the robustness and reliability of the assay in a 384 well format; its tolerance of the effects of dimethyl sulfoxide (DMSO), a common organic solvent; optimal plating and culture conditions; and optimize assay timing and throughput. Secondly, we will develop a counterscreen using a measure of cell proliferation and viability to assure the absence of toxicity and to independently verify the biological efficacy of compounds by measuring their ability to influence the known mitogenic activity of Tgfb1 for fibroblasts. We will also screen a small but chemically diverse library, the 960 compound GenPlus library from MicroSource, in duplicate. Assay performance and counter-screening characteristics will be optimized based on this screen. Thirdly, we will perform a larger screen using a 20,000 compound diversity library (the Maybridge Screening collection) owned by the Yale Chemical Genomics Screening facility. Based on the number and quality of hits from this 20,000 compound screen, performed as singlet measures, we will choose approximately 1000 compounds for a second round of testing in duplicate. The top 100 compounds identified and confirmed through duplicate testing and counter-screening, will be further characterized pharmacologically. Fourth, we will begin development of a strategy for assessing the biological mechanism of these top 100 candidates by using a high-throughput fluorescence microscopy method to study the effect of the top 100 agonist/antagonist hits on phospho-Smad2/3/4 nuclear translocation using the In Cell Analyzer 1000. These studies are critical to develop robust and reliable assays to identify biologically active molecules from diverse collections that regulate the activity of important cellular processes. The current public availability of reagents of this sort is quite limited due to high potential commercial importance in the pharmaceutical industry.