Mechanistic Studies of the Transcription Inhibitor Triptolide and its Selective Targeting to Cancer Cells under Hypoxia

Small molecules (< 900 daltons or g/mol) are essential tools in biology, chemistry and medicine. The versatility of small molecules to modulate or monitor biological processes through modulation of protein activity, macromolecular concentration and protein-protein interactions are unrivaled by other techniques. However, the specificity of inhibition by small molecules is dependent on a number of factors, including its structure, potency, concentrations and features of its target/s. The rarity of specific inhibitors for proteins and anticipated mechanism-based toxicities of potent bioactive compounds pose challenges for their use as targeted therapy in the clinic. The goal of my PhD thesis was to identify new and potent inhibitors of cancer cell proliferation with prodrug and selective targeting properties. Moreover, this thesis also focuses on mechanistic studies of prodrug products (i.e. released parent compound) and the impact of tissue microenvironment on newly identified inhibitors for future design and synthesis of transcription-based therapeutics. Established antiproliferative agents have been conjugated to glucose in efforts to target cancer cells as seen with paclitaxel, ifosfamide and platinum. We decided to focus on glucose-conjugated triptolides (glutriptolides) given the specificity and potent antiproliferative activity of triptolide in addition to promising in vivo anti- tumor activity of a recently identified glutriptolide analogue. Triptolide is a potent inhibitor of cell proliferation in all cell lines tested through specific inhibition of the XPB subunit of the general transcription factor TFIIH, which regulates eukaryotic transcription initiation. This thesis attempts to advance our understanding of approaches for selective targeting of toxic agents like triptolide to cancer cells over cells from normal tissue. The first contribution of this thesis is the identification of a more potent glutriptolide analogue with a prodrug feature, selective targeting of cancer over normal cells and improved stability in human serum. The second contribution of this thesis is the discovery of increased glutriptolide potency against cancer cells under hypoxia in contrast to existing anti-cancer drugs. Finally, we disclose the identification of E2 and E3 ubiquitylating enzymes that mediate the degradation of the catalytic subunit of RNA polymerase II induced by triptolide