Screening and Selection of New Antagonists of the RING-Mediated Hdm2/Hdmx Interaction
Final rept. 1 Mar 2010-28 Apr 2013
UNIVERSITY OF SOUTHERN CALIFORNIA LOS ANGELES
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Prostate cancer poses a major public health problem in the United States and worldwide. It has the highest incidence and is the second most common cause of cancer deaths in North American men resulting in over 30,000 deaths per annum. Consequently, there is an urgent need to develop novel therapeutic approaches. We propose to use a novel cyclotide-based molecular scaffold for generating molecular libraries that will be screened and selected in vivo to identify antagonists of the RING-mediated Hdm2Hdmx interaction. Our innovative approach will use cell-based E. coli libraries in which individual bacteria express a different cyclotide. This comprises a new single cell-single compound approach to identify protein-protein binding antagonists. These compounds will be subjected to a two-step screen, the first involving a high throughput FRET-based FACS screen in bacteria, and the second a bioluminescence complementation assay in cancer cells. Together, these assays will identify cyclotides that disrupt Hdm2-Hdmx interactions, activate p53, and elicit p53-dependent cytotoxicity in prostate cancer cells. During the second year of this project we have accomplished the following 1 Developed a more efficient method for the biosynthesis of cyclotides in E. coli cells 2 We have validated our FRET-based reporter to sort cell populations 3 Developed and characterized a novel cyclotide with p53 activating properties.
- Medicine and Medical Research