| Each year, approximately 500,000 new cases of cervical cancer are diagnosed worldwide, and about 275,000 women die from this cancer. Virtually all cases of cervical cancer are attributable to infection with human papillomavirus (HPV). It is the goal of this dissertation to utilize our understanding of the mechanisms of HPV associated carcinogenesis to identify small molecules with the potential to serve as lead or candidate compounds in drug discovery against HPV and cervical cancer.; Two HPV oncogenes, E6 and E7, have been shown to be necessary and sufficient for cellular transformation. The best characterized function of E6 is the targeting of the p53 tumor suppressor protein for degradation. E6 mediates p53 degradation by interacting with the cellular E3 ubiquitin ligase E6AP (E6-associated protein) to form a complex that is able to bind and ubiquitinate p53. Molecular inhibition of E6 or E6AP function in HPV-positive cells stabilizes cellular p53 and induces cell cycle arrest and apoptosis, suggesting that inhibition of E6/E6AP activity may be a potential therapeutic approach in treating cervical cancer.; In this dissertation, I describe a high-throughput small molecule screen to identify chemical inhibitors of E6/E6AP-mediated p53 degradation using a novel biochemical p53 degradation assay amenable to scale-up. I first carefully established, validated, and optimized this novel p53 degradation system. I then scaled up the reagents necessary to utilize this system in a small molecule high-throughput screen. Once complete, I proceeded to conduct the screen, testing 57,200 compounds in total and identifying 528 total hits, for a "hit rate" of 0.91%. From these hits, I ultimately identified (1) five molecules capable of biochemically inhibiting p53 ubiquitination (2) seven molecules capable of inhibiting p53 degradation in HPV-positive cervical carcinoma cell lines, and (3) a potential target and mechanism of action of one of the small molecules. Although I was not able to find any molecules that specifically inhibit E6-mediated p53 degradation in cells, I did identify one molecule, molecule R, which was potent in inhibiting general protein degradation within cells. My preliminary experiments suggest that R may have inhibitory effects on proteasome activity.; In summary, I've established and validated a high-throughput small molecule screening approach for the identification of compounds capable of inhibiting E6-mediated p53 degradation. It is my hope that the work from this dissertation will contribute new tools and useful compounds that will ultimately aid the academic and pharmaceutical fields in their endeavor to develop small molecule drugs against HPV and cervical cancer. |
