Utilizing Chemoproteomic Platforms to Elucidate Toxicological Mechanisms

A large number of pharmaceuticals, endogenous metabolites, and environmental chemicals act through covalent mechanisms with protein targets. Yet, their specific interactions with the proteome still remain poorly defined for most of these reactive chemicals. Deciphering direct protein targets of reactive small-molecules is critical in understanding their biological action, off-target effects, and potential toxicological liabilities, as well as for the development of safer and more selective chemical agents. Chemoproteomic technologies have arisen as a powerful strategy that enables the assessment of proteome-wide interactions of these irreversible agents directly in complex biological systems. In Chapter one, we review several 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./nThe usefulness of chemoproteomic platforms in assessing the toxicity of environmental chemicals is further demonstrated in Chapter two; here, we utilize the chemoproteomic strategy termed Isotopic tandem orthogonal proteolysis-enabled activity-based protein profiling (isoTOP-ABPP) in identifying the direct protein binding, possible targets, mechanism of toxicity and target selectivity of the herbicide glyphosate. Glyphosate, the active ingredient in the commercial herbicide formulation Roundup®, is one of the most widely used pesticides in agriculture and home garden use. Whether glyphosate causes any mammalian toxicity remains highly controversial. While many studies have associated glyphosate with numerous adverse health effects, the mechanisms underlying glyphosate toxicity in mammals remain poorly understood. In chapter two, we used activity-based protein profiling to map the reactivity of glyphosate metabolites in vivo in mice. We show that glyphosate at high doses can be metabolized in vivo to reactive metabolites such as glyoxylate and react with several cysteines across many different protein targets in mouse liver. We show that glyoxylate inhibits several fatty acid oxidation enzymes and high-dose glyphosate treatment in mice increases the levels of several lipid metabolites, including triglycerides and cholesteryl esters, likely resulting from diversion of fatty acids away from oxidation and towards other lipid pathways. Our study underscores the utility of using chemoproteomic platforms to identify novel toxicological mechanisms of environmental chemicals such as glyphosate./nUnderstanding the many ways in which environmental chemicals, such as pesticides, or pharmaceuticals compounds interact with complex biological systems is absolutely necessary in determining the mechanism of toxicity, secondary targets, off-targets, selectivity, and therefore potential for toxicity of exogenous compounds. In this dissertation we demonstrate the versatility of chemoproteomics technologies, such as IsoTOP-ABPP, to aid in elucidating the direct targets and mechanism of action of environmental chemicals, such as the herbicide glyphosate. Furthermore, it is an additional aim of this research to demonstrate the potential of chemoproteomic platforms to be useful as part of the covalent compound discovery and development process towards developing safer chemicals.

Advisor: Nomura, Daniel