High-Content Screening (HCS) is a form of high throughput screening (HTS) that couples automated microscopy with high throughput image analysis to quantify biological effects. Thus, it can be thought of as applied biology bridging basic bench research to drug discovery and clinical medicine. The first aim of this proposal is to develop a HCS strategy to identify small molecules that stimulate the differentiation of murine embryonic stem cells (mESC) toward the cardiac lineage. Screening campaigns are difficult and expensive, and success can be undermined by variation or errors in the underlying biology, robotics, instrumentation and data analysis. Some of these problems can result in systematic errors resistant to correction by established methods. Thus, the second aim of this proposal focuses on development and testing of a novel correction method for redressing systematic errors. The third aim is to implement the HCS campaign and analyze the most interesting hits through confirmatory and secondary screens, medicinal chemistry optimization, and then mechanism of action and target identification. Two libraries were screened: 1) ∼14,000 small synthetic compounds screened at 2 doses and added 1 molecule per sample well, and 2) a chemical mixture library where each sample well is tested against thousands of structurally similar molecules at equimolor concentration. Two molecules from the individual compound screen and one compound from the mixture library screen were selected for structure activity relationship (SAR) analyses. For each compound class, the data define an SAR for cardiomyogenesis from ESCs. Studies focused on the biological mechanism of action studies have revealed two molecules bias germ layer commitment toward early endoderm at the expense of mesoderm. Biotin-tethered probes used to affinity capture small molecule protein targets together with biological assays have suggested metabolic stress is a modulator of mESC specification of the 3 germ layers.
|Advisor:||Mercola, Mark, Cheresh, David|
|Commitee:||Chen, Ju, Cheresh, David, Evans, Sylvia, Mercola, Mark, Subramaniam, Shankar|
|School:||University of California, San Diego|
|School Location:||United States -- California|
|Source:||DAI-B 73/07(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Pharmacology, Developmental biology|
|Keywords:||Automation, Cardiopoiesis, Screening errors, Stem cells, Systematic errors|
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