Using Chemoproteomic Platforms to Discover Therapeutic and Toxicological Mechanisms

Dysregulation of cancer cell metabolism contributes to abnormal cell growth, the biological endpoint of cancer. There have been numerous affected oncogenes and metabolic pathways recorded in cancer, and how they contribute to cancer pathogenesis and malignancy is of great interest; various pharmacological manipulations take advantage of these metabolic abnormalities, and many targeted therapies that have arisen from this research. However, despite the many therapies currently on market or in clinical trials, much work still needs to be done./nMany of these cancer therapeutics include natural products and various small molecules that act through covalent mechanisms. In fact, a large number of pharmaceuticals, as well as endogenous metabolites and environmental chemicals, act through covalent interactions with proteins. Cancer, as well as other diseases such as Alzheimer’s disease and obesity, are often subject to drugs that irreversibly bind and inhibit their respective protein targets. Endogenous reactive metabolites and environmental chemical exposure, in turn, can also work through covalent interactions with proteins within the body to cause disease. Therefore, understanding what mechanisms can cause disease, and what therapeutic mechanisms can treat disease, are of great importance./nChemoproteomic technologies have arisen as a powerful strategy that enable the assessment of proteome-wide interactions of these irreversible agents directly in complex biological systems. Using these chemoproteomic strategies has afforded scientists a more thorough understanding of specific protein interactions of irreversibly-acting pharmaceuticals, endogenous metabolites, and environmental electrophiles to reveal novel pharmacological, biological, and toxicological mechanisms. Through these same platforms, researchers have also been able to identify therapeutically active small-molecules and the mechanisms of action underlying these hit compounds./nIn this dissertation, I present a thorough review of our current understanding of metabolic pathways and therapies in cancer. I also discuss several of the most-utilized chemoproteomic strategies that have facilitated our understanding of specific protein interactions of irreversibly-acting pharmaceuticals, endogenous metabolites, and environmental electrophiles to reveal novel pharmacological, biological, and toxicological mechanisms. Next, I demonstrate the utility and diversity of chemoproteomic platforms in two separate studies. First, I use two cysteine-reactive covalent ligand libraries to identify hit compounds that impair cell survival and proliferation in non-small cell lung carcinoma, and an activity-based chemoproteomic analysis to identify the protein target of my hit compounds. Finally, I discuss an activity-based proteomics method used to reveal the off-targets of the widely used herbicide, acetochlor, in vivo in mice. Overall, the work reported in this dissertation demonstrates how chemoproteomic platforms can be used to identify direct protein targets of small-molecule covalent ligands and environmental chemicals, and uncover their downstream pathophysiological and biochemical effects.

Advisor: Nomura, Daniel K