Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemistry

First Advisor

Ronen Marmorstein

Abstract

Human papillomavirus (HPV) is the most common sexually transmitted pathogen, and is associated with almost all cervical cancers, about 20 percent of head and neck cancers and an array of other cancers. Current HPV vaccines offer preventative care, however, long-term benefits are unknown and the vaccines are not effective for therapy. High risk forms of HPV mediate cell transformation via two viral oncoproteins, E6 and E7, which lead to cell cycle disruption and cancer. E7 deregulates the cell cycle and abrogates other pathways mediated by the retinoblastoma protein, pRb. pRb is essential for regulating many cellular activities through its binding and inhibition of E2F transcription activators, and pRb inactivation leads to many cancers. pRb activity can be perturbed by viral oncoproteins, including HPV, that contain an LxCxE motif. E6 mediates cell transformation, in part, by forming a complex with the cellular E3 ligase E6-Association Protein (E6AP) to target p53 for degradation.

To tackle these problems, we performed two high throughput solution screens of ~88,000 compounds to search for (1) compounds that inhibit the ability of HPV-E7 to disrupt pRb/E2F complexes and (2) small molecule inhibitors of the E6/E6AP interaction. The HPV-E7 screen led to the identification of thiadiazolidinedione compounds that bind to pRb with mid-high nanomolar affinity, are competitive with the binding of viral oncoproteins containing an LxCxE motif and are selectively cytotoxic in HPV positive cells alone and in mice. The HPV-E6 screen resulted in 30 inhibitors with in vitro IC50 values in the low-micromolar to mid-nanomolar range. Six of these compounds were shown to associate with HPV-E6, block p53 degradation and promote apoptosis in high risk HPV positive cells. These E6 and E7 inhibitors provide promising scaffolds for the development of therapies to treat HPV-mediated pathologies.

An in silico screen was also done to identify small molecules that bind directly to E7 and prevent its interaction with E2F. This resulted in two compounds that prevented E7-mediated displacement of E2F from pRb, however, the IC50 values were very high, and not pursued further. The interaction between E7 and another cellular target, p300, was characterized by various biochemical and biophysical techniques. The interaction seemed very weak, different from the adenovirus E1A interaction with p300. This suggested that the E1A/p300 interaction would be easier to characterize.

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