Type of Document Dissertation Author Stoops, Sydney Lear Author's Email Address Sydney.L.Stoops@Vanderbilt.edu URN etd-03262012-115610 Title Discovery, Optimization, and Understanding the Mechanism of Action of Small Molecules that Restore E-cadherin Expression Degree PhD Department Pharmacology Advisory Committee
Advisor Name Title Professor Brian Wadzinski Committee Chair Professor Albert Reynolds Committee Member Professor Craig Lindsley Committee Member Professor Lawrence Marnett Committee Member Professor Robert Coffey Committee Member Professor Stephen Fesik Committee Member Keywords
- Epithelial-Mesenchymal Transition
Date of Defense 2011-12-09 Availability unrestricted AbstractE-cadherin is a transmembrane protein that maintains intercellular contacts and cellular polarity in epithelial tissues. The down-regulation of E-cadherin is thought to aid in the induction of an epithelial-to-mesenchymal (EMT) transition resulting in an increased potential for invasion into surrounding tissues and entry into the bloodstream. Loss of E-cadherin has been observed in a variety of human tumors resulting from somatic mutations, chromosomal deletions, proteolytic cleavage of E-cadherin, and most commonly silencing of the CDH1 gene promoter.
A novel High-throughput screen was developed to identify small molecules that restored E-cadherin expression in the SW620 cell line followed by medicinal chemistry employing iterative analog library synthesis to better identify the structure-activity relationship. Preliminary optimization of the screening hit has shown it is possible to synthesize small molecules that have an improved ability to restore E-cadherin expression compared to the initial screening hits.
Recent endeavors have been taken to elucidate the mechanism of action of these small molecules to restore E-cadherin expression. Quantitative PCR analysis has shown that E-cadherin mRNA expression occurs after 3 hours of treatment with active analogs, suggesting the small molecules are altering transcription of the CDH1 gene. This was supported by experiments conducted using various plasmid constructs containing truncated segments of the E-cadherin promoter region and luciferase reporter. It was shown that active analogs had a significant increase in luciferase activity as compared to DMSO or an inactive analog, which were used as controls. More specifically, we were able to narrow the site of action the active analogs to a 200 bp fragment of the E-cadherin promoter region.
Elucidation of the mechanism of action will aid in identifying the novel molecular target. Such information would allow for further development of more efficacious and potent small molecules as well as further research to understand the importance of this interaction in the role of EMT and as a therapeutic target.
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