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

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

Lack of Bax Prevents Influenza A Virus-Induced Apoptosis and Causes Diminished Viral Replication ▿

The ectopic overexpression of Bcl-2 restricts both influenza A virus-induced apoptosis and influenza A virus replication in MDCK cells, thus suggesting a role for Bcl-2 family members during infection. Here we report that influenza A virus cannot establish an apoptotic response without functional Bax, a downstream target of Bcl-2, and that both Bax and Bak are directly involved in influenza A virus replication and virus-induced cell death. Bak is substantially downregulated during influenza A virus infection in MDCK cells, and the knockout of Bak in mouse embryonic fibroblasts yields a dramatic rise in the rate of apoptotic death and a corresponding increase in levels of virus replication, suggesting that Bak suppresses both apoptosis and the replication of virus and that the virus suppresses Bak. Bax, however, is activated and translocates from the cytosol to the mitochondria; this activation is required for the efficient induction of apoptosis and virus replication. The knockout of Bax in mouse embryonic fibroblasts blocks the induction of apoptosis, restricts the infection-mediated activation of executioner caspases, and inhibits virus propagation. Bax knockout cells still die but by an alternative death pathway displaying characteristics of autophagy, similarly to our previous observation that influenza A virus infection in the presence of a pancaspase inhibitor leads to an increase in levels of autophagy. The knockout of Bax causes a retention of influenza A virus NP within the nucleus. We conclude that the cell and virus struggle to control apoptosis and autophagy, as appropriately timed apoptosis is important for the replication of influenza A virus

mTOR/p70S6K signaling distinguishes routine, maintenance-level autophagy from autophagic cell death during influenza A infection

AbstractAutophagy, a stress response activated in influenza A virus infection helps the cell avoid apoptosis. However, in the absence of apoptosis infected cells undergo vastly expanded autophagy and nevertheless die in the presence of necrostatin but not of autophagy inhibitors. Combinations of inhibitors indicate that the controls of protective and lethal autophagy are different. Infection that triggers apoptosis also triggers canonical autophagy signaling exhibiting transient PI3K and mTORC1 activity. In terminal autophagy phospho-mTOR(Ser2448) is suppressed while mTORC1, PI3K and mTORC2 activities increase. mTORC1 substrate p70S6K becomes highly phosphorylated while its activity, now regulated by mTORC2, is required for LC3-II formation. Inhibition of mTORC2/p70S6K, unlike that of PI3K/mTORC1, blocks expanded autophagy in the absence of apoptosis but not moderate autophagy. Inhibitors of expanded autophagy limit virus reproduction. Thus expanded, lethal autophagy is activated by a signaling mechanism different from autophagy that helps cells survive toxic or stressful episodes

Triptolide: reflections on two decades of research and prospects for the future

This review highlights advances in material sourcing, molecular mechanisms, clinical progress and new drug design strategies for triptolide from a Chinese medicinal herb, along with some prospects for the future course of development of triptolide.</p