Discoveries of the past two decades have begun to demonstrate that the communities of microorganisms residing on and in the human body are able to influence the health of their human hosts through multiple mechanisms. One such pathway is through the production of bioactive metabolites which are taken up into the host’s circulation. While previous research suggests that human blood contains many such metabolites, to date, a relatively small number have been closely studied in highly targeted efforts. Here we develop and apply an untargeted liquid chromatography mass spectrometry (LC-MS) workflow for the discovery of microbial produced metabolites that are measured in human circulation and associated with human health. Chapter 1 of this dissertation is an introduction, providing a review of relevant literature, illustrating a need for an untargeted approach to microbial metabolite discovery, and discussing the challenges addressed in this work. Chapter 2 is a reprint of published work wherein we develop machine learning based approaches to LC-MS peak selection allowing for automated processing of high-throughput LC-MS data that is highly sensitive and highly accurate. Chapter 3 is a reprint of work submitted for publication wherein we apply a high-throughput LC-MS platform to the measurement of thousands of metabolites in hundreds of isolated human-associated bacterial strains in vitro as well as thousands of human blood plasma samples and identify thousands of microbially produced metabolites in circulation. Additionally, we highlight four circulating microbial metabolites that robustly associate with liver disease severity from pre-diagnosis through advanced fibrosis. Finally, Chapter 4 is a conclusion wherein we discuss the meaning of these findings in the context of other work at the intersection of health and the microbiome, while commenting on the limitations and opportunities for future extensions. This work demonstrates the utility of untargeted LC-MS for elucidating microbial small molecule products and motivates future efforts to characterize these chemical entities, particularly oxidized fatty acids, and their roles in human health.

Abstract

SYNTHESIS AND STRUCTURE-ACTIVITY RELATIONSHIPS STUDIES OF 4-((4-HYDROXY-3- METHOXYBENZYL)AMINO)BENZENESULFONAMIDE DERIVATIES AS POTENT AND SELECTIVE

Auric Sowell-Kantz

Human lipoxygenases (LOXs) are a family of iron-containing enzymes which

catalyze the oxidation of polyunsaturated fatty acids to provide the corresponding bioactive hydroxyeicosatetraenoic acid (HETE) metabolites. These eicosanoid signaling molecules are involved in a number of physiologic responses such as platelet aggregation, inflammation, and cell proliferation. Our group has taken a particular interest in platelet-type 12-(S)-LOX (12-LOX) because of its demonstrated role in skin diseases, diabetes, platelet hemostasis, thrombosis, and cancer. We previously reported the discovery of ML127, a potent and selective 12-LOX inhibitor which has proven to be a valuable tool compound for researchers in the field. However, the limited tolerance for structural modification and modest PK profile for this chemotype prompted us to continue our discovery efforts toward novel 12-LOX inhibitors. Herein, we report the identification and medicinal chemistry optimization of a 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide-based scaffold. Top compounds, exemplified by 35 (ML355) and 36, display nM potency against 12- LOX and excellent selectivity over related lipoxygenases and cyclooxygenases. In addition to possessing favorable ADME properties, 35 and 36 inhibit PAR-4 induced aggregation and calcium mobilization in human platelets, and reduce 12-HETE in mouse/human beta cells.