Hurben, Alexander2023-09-192023-09-192023-04https://hdl.handle.net/11299/257127University of Minnesota Ph.D. dissertation. April 2023. Major: Medicinal Chemistry. Advisor: Natalia Tretyakova. 1 computer file (PDF); xii, 476 pages.Human health is impacted by molecular level events. Within this context, DNA encodes for cellular instructions and proteins execute these genetic protocols to ensure the cell’s components are functioning properly. Thus, the underlying biochemistry within the cell is tightly regulated to ensure vitality. However, when these processes become compromised or damaged, there is increased susceptibility towards developing cancer and neurodegenerative diseases. Often, small chemical changes to our DNA and proteins can be the culprits of such dysregulation. These seemingly minuscule modifications can impair the cell’s proper functions, which can lead to cell death or uncontrolled growth, and ultimately manifest as degenerative disease or cancer. Electrophilic small molecules can be responsible for chemically altering cellular machinery. Additionally, enzymes can make chemical changes such as post-translational modifications on proteins and epigenetic DNA modifications, which can have pronounced effects on cell function and can be equally damaging if not properly regulated. Understanding how molecular events influence health is of paramount importance in designing therapeutics that effectively prevent and treat disease, as well as discovering biomarkers which enable early detection. Despite immense research efforts from the scientific community, there is much remaining to learn about these microscopic processes. This is due to their inherent complexity and lack of technologies to study them. This thesis aims to contribute to addressing this problem by developing new chemical tools which advance our knowledge of the molecular mechanisms that drive disease. This work is composed of seven chapters which explore reactive dopamine metabolites linked to Parkinson’s disease, tools to study the biological implications of elevated intracellular methylglyoxal concentrations, and the development of small molecule epigenetic modulators to regulate aberrant DNA methylation. Chapters I, II, and III of this thesis explores how dysregulated dopamine may contribute to Parkinson’s disease initiation. Chapter I commences with a review of dopamine metabolism and the subsequent generation of reactive dopamine derived metabolites in neurons. This is followed by an overview of protein damaged induced by these metabolites and a review of chemical tools and techniques implemented to study dysregulated dopamine in various experimental systems. Next, Chapter II describes our efforts in designing and implementing a dopamine derived chemical probe to profile dopamine modified proteins which found that dopamine metabolites disrupt protein-folding pathways critical for maintaining healthy neurons. We also detail our development of photoactivatable dopamine probes in Chapter III. Collectively, this work improves the understanding of dopamine protein modification and by extension, molecular events that may contribute to Parkinson’s, which may inform future Parkinson’s therapeutic development. Chapter IV and Chapter V of this thesis focuses on the reactive metabolite methylglyoxal. Methylglyoxal is a sugar-derived metabolite produced naturally in all cells. This reactive compound forms adducts with DNA and proteins, thereby altering their function and influencing cell signalling. Consequently, methylglyoxal protein adducts are implicated in numerous diseases such as cancer, neurodegeneration, diabetes, and cardiovascular disease. In many of these diseases, the cellular processes that break down methylglyoxal become compromised, leading to elevated levels of this reactive molecule within cells. Existing chemical tools to investigate methylglyoxal biology are limited, leading to an incomplete understanding of its physiological and disease-causing roles. Here, we disclose a chemical tool that confers light-mediated release of a methylglyoxal probe within cell models. We use this chemical to identity of the resulting protein adducts. This work enables studying protein adducts induced by methylglyoxal in a controlled fashion to illuminate how this reactive compound impacts various disease states. We also detail our efforts in profiling proteins which undergo covalent DNA crosslinking in the presence of methylglyoxal in Chapter V. This effort is the first study to identify this type of methylglyoxal adduct at a proteome wide scale, which provides a list of candidate methylglyoxal derived DNA-protein cross to investigate in future work. Collectively, these efforts further our understanding of basic methylglyoxal biology and its role in disease progression. Finally, Chapters VI and VII of this thesis describe our efforts towards developing small molecule epigenetic modulators. Regulating gene expression is critical for keeping cells healthy. Over- or under-expressed genes can lead to cancer and other diseases. Accordingly, cells have many methods to control when specific genes are turned on or off in order to function properly; DNA methylation being an example. There are many proteins which control the addition and removal of DNA methyl marks across the genome to ensure appropriate gene expression. Mutations in, or dysfunction of, these proteins can initiate certain cancers. One essential group of proteins involved in removing DNA methylation marks is ten-eleven translocation (TET) methylcytosine dioxygenases (TET). Given TET’s central role in cancer development, theses protein represent a potential drug target. However, there is a paucity of small molecules which selectively inhibit their function without affecting other cellular processes. Thus, there is a need to develop potent and selective compounds which block TET-meditated DNA demethylation. Within Chapter VI, we show our efforts towards the development of novel small molecule TET inhibitors which led us to uncover that copper contamination is responsible for the activity of a reported TET inhibitor. In Chapter VII, we present work on a novel TET inhibitor scaffold which features a bifunctional cofactor-substrate mimetic design. This work has the potential to generate new anticancer therapeutics and improve our understanding of how TET and DNA methylation is linked to cancer development. Ultimately, the work in this thesis provides a novel suite of chemical tools for studying dopamine dysregulation, methylglyoxal metabolism, and TET function. These tools provide insights into cellular damage caused by dopamine and methylglyoxal adducts as well as probes for altering DNA methylation status. Such tools are critical for mapping molecular mechanisms that drive disease.enChemical probeDNA methylationDopamineMethylglyoxalProtein adductsTETThesis or Dissertation