Browsing by Subject "Indoles"
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Item Approach to interstitial lung disease(2022-09-30) Adams, TraciItem Chemistry to Biology and Back Again: Small Molecule Regulation of HIF Transcription Factors and the Development of a Platform for the Discovery of New Biocatalyzed Organic Transformations(2013-04-08) Rogers, Jamie Lee 1985-; Tambar, Uttam; MacMillan, John; Ready, Joseph M.; Gardner, Kevin H.This work is divided into two parts. The first is the description of the regulation of the hypoxic response pathway via small molecule inhibitors. The hypoxia response pathway is a way in which cells sense and regulate oxygen levels in cells. Specifically, when oxygen levels in the cells are low, a family of transcription factors known as hypoxia inducible factors (HIFs) is up regulated. Importantly, the hypoxic response pathway is often mis–regulated in cancer. As a result, the regulation of this pathway offers a promising target for cancer treatment. Previous work on HIF identified a large internal cavity, which provided an opportunity for allosteric binding and regulation of HIF. After identification of a small molecule inhibitor of HIF through a high–throughput screening campaign, an SAR analysis was performed on the lead molecule. This led to a greater understanding of the structural requirements to strong binding with HIF. In addition, a sterol natural product was identified through the screen that also inhibits HIF. This molecule led to the search for the endogenous ligand of the HIF transcription factor and has developed a better understanding of the natural regulation of the hypoxic response pathway. The second part of this work describes the development of a new discovery platform for the identification of new, biocatalyzed organic transformations. Biocatalyzed organic transformations have been used by organic chemists for decades as these reactions offer many benefits to the synthetic chemist. For example, reactions catalyzed by biological enzymes tend to be very stereo- and regiospecific. In addition, biocatalyzed transformation can occur on unfunctionalized organic substrates. However, research into new biocatalyzed reactions has been limited due to the challenges in searching for these new reactions. We have developed a discovery platform designed to screen a vast library of bacteria for new reactivity by introducing 13C labeled substrates to cultures. This platform is illustrated with the discovery of a tunable indole oxidation reaction.Item Interplay Between Tryptophan Metabolites and the Virulence Factors of Enteric Pathogens(2020-08-01T05:00:00.000Z) Kumar, Aman; Winter, Sebastian E.; Sperandio, Vanessa; Conrad, Nicholas; Tu, BenjaminThe human gut consists of a complex milieu of several small molecules that helps in shaping its overall chemistry and biogeography. Trillions of bacteria, collectively known as the gut microbiota, colonize this landscape and occupy a specific niche. Small molecules derived from diet, gut microbial metabolism, and host metabolic activity have an impact on dictating the underlying gut microbiome composition. Gut bacterial populations effectively sense these molecular signatures and modulate gene expression to colonize this niche. An invading intestinal pathogen, to effectively colonize the gut, must sense and respond to the molecular signatures in the gut, which leads to effective colonization and infection. Diet is the principal source of energy by the intake of three main nutrients: carbohydrates, proteins, and fats. Tryptophan is an essential amino acid taken from the diet and plays an important role in protein biosynthesis. Tryptophan is also metabolized to different small molecules and its metabolic products are known to be present in high concentrations in the body. Upon ingestion, tryptophan is readily available in the luminal environment of the gut. Abundant levels of tryptophan are also absorbed by intestinal epithelial cells and is made available in different cellular compartments for normal physiological processes. Tryptophan present in the gut lumen can be further metabolized to numerous small molecules by the action of the gut microbial metabolic activity. Indole is one of the most abundant tryptophan-derived metabolites present in the gut and is absorbed by the host epithelial cells. The host can also metabolize tryptophan to various small molecules including serotonin that is made available in the gut lumen upon release from the host cells. Bacteria in the gut sense these bacterial and host-derived small molecules to colonize and maintain their niche. Similarly, an invading food-borne pathogen such as Enterohemorrhagic Escherichia coli (EHEC), which causes gastroenteritis by primarily colonizing the human colon, sense these small molecules and respond in a way that is conducive for its colonization and virulence gene expression. The role of these highly abundant tryptophan-derived small molecules in dictating the infectivity of an enteric pathogen remains unknown. Because the concentrations of these small molecules naturally present in the body are in the range of the drug concentrations that are used to treat certain diseases, it is possible to repurpose the information gained from these studies to treat intestinal infections. In the present study, we focused on two highly abundant tryptophan-derived small molecules present in the gut. Indole is derived from bacterial metabolism while serotonin is present via the action of host metabolism of tryptophan. Indole and serotonin are structurally similar and therefore may have similar effects against a pathogen in vivo. Indeed, we identified that both indole and serotonin decrease the virulence of the human pathogen Enterohemorrhagic E. coli (EHEC) and the mouse pathogen Citrobacter rodentium. We used multiple strategies including genetic manipulation, pharmacological inhibitors, and knock-out murine models to show that both indole and serotonin are inhibitory signals for virulence gene expression in EHEC and C. rodentium. We further investigated the mechanism used by these pathogens to sense these signals. We identified the first bacterial receptor for both indole and serotonin, and showed that these signals are sensed by a bacterial membrane bound histidine kinase CpxA. Upon sensing indole or serotonin, CpxA dephosphorylates itself and the transcription factor CpxR. In its phosphorylated state, CpxR directly activates expression of the virulence genes, its dephosphorylation prevents its action, decreasing expression of these genes. Together, our studies highlight the importance of sensing small molecules and understanding the gut biogeography by invading pathogens to successfully colonize the gut.