<p>Secondary metabolites are vital to human health and strategies to improve their production and detection are equally essential. The blue pigmented metabolite actinorhodin produced by <em>Streptomyces coelicolor</em>, a genus renowned for their diverse secondary metabolites, provides a unique opportunity to identify small molecules probes of secondary metabolism. Small molecules capable of altering secondary metabolism will have widespread application in the streptomycetes due to their ease of addition to any culture condition. Taking advantage of the phenotypic versatility of the <em>S. coelicolor</em> lifecycle, we extended our search for small molecule modulators further to include the entire developmental process. In addition to alterations in secondary metabolism, these processes include growth inhibition, precocious sporulation and alterations in aerial hyphae formation and sporulation. This work provides the foundation for studying <em>Streptomyces</em> by chemical manipulation. Those compounds which stimulate secondary metabolism were narrowed down to 19 ARCs (for antibiotic remodeling compounds). From these, a set of 4 structurally related molecules, the ARC2 series, was identified as weak inhibitors of fatty acid biosynthesis and most likely lead to alterations in secondary metabolism through shifting precursors from primary to secondary metabolism. Consistent with the conservation of fatty acid biosynthesis within bacteria, the effect of the ARC2 series extends in general to the actinomycetes. This provides a simple strategy to alter the secondary metabolic profiles of a diverse range of actinomycetes.</p> / Doctor of Philosophy (PhD)

Individuals with high levels of plasma triglycerides are at high risk to develop cardiovascular disease (CVD), currently one of the major causes of death worldwide. Recent epidemiological studies show that loss-of-function mutations in the APOC3 gene lower plasma triglyceride levels and reduce the incidence of coronary artery disease. The APOC3 gene encodes for apolipoprotein (APO) C3, known as an inhibitor of lipoprotein lipase (LPL) activity. Similarly, a common gain-of-function mutation in the LPL gene is associated with reduced risk for CVD. LPL is central for the metabolism of lipids in blood. The enzyme acts at the endothelial surface of the capillary bed where it hydrolyzes triglycerides in circulating triglyceride-rich lipoproteins (TRLs) and thereby allows uptake of fatty acids in adjacent tissues. LPL activity has to be rapidly modulated to adapt to the metabolic demands of different tissues. The current view is that LPL is constitutively expressed and that the rapid modulation of the enzymatic activity occurs by some different controller proteins. Angiopoietin-like protein 4 (ANGPTL4) is one of the main candidates for control of LPL activity. ANGPTL4 causes irreversible inactivation through dissociation of the active LPL dimer to inactive monomers. Other proteins that have effects on LPL activity are the APOCs which are surface components of the substrate TRLs. APOC2 is a well-known LPL co-factor, whereas APOC1 and APOC3 independently inhibit LPL activity. Given the important role of LPL for triglyceride homeostasis in blood, the aim of this thesis was to find small molecules that could increase LPL activity and serve as lead compounds in future drug discovery efforts. Another aim was to investigate the molecular mechanisms for how APOC1 and APOC3 inhibit LPL activity. Using a small molecule screening library we have identified small molecules that can protect LPL from inactivation by ANGPTL4 during incubations in vitro. Following a structure-activity relationship study we have synthesized lead compounds that more efficiently protect LPL from inactivation by ANGPTL4 in vitro and also have dramatic triglyceride-lowering properties in vivo. In a separate study we show that low concentrations of fatty acids possess the ability to prevent inactivation of LPL by ANGPTL4 under in vitro conditions. With regard to APOC1 and APOC3 we demonstrate that when bound to TRLs, these apolipoproteins prevent binding of LPL to the lipid/water interface. This results in decreased lipolysis and in an increased susceptibility of LPL to inactivation by ANGPTL4. We demonstrate that hydrophobic amino acid residues that are centrally located in the APOC3 molecule are critical for attachment of this protein to lipid emulsion particles and consequently for inhibition of LPL activity. In summary, this work has identified a lead compound that protects LPL from inactivation by ANGPTL4 in vitro and lowers triglycerides in vivo. In addition, we propose a molecular mechanism for inhibition of LPL activity by APOC1 and APOC3.

<p>Aminoglycoside antibiotics are commonly used to treat bacterial infections but are highly susceptible to chemical modification, leading to resistance. Chemical modification can be hindered through the use of small molecule inhibitors that target bacterial enzymes involved in resistance, most notably kinases. Current methods for the discovery of small molecule inhibitors of kinases and related “kinase-like” enzymes are limited in throughput and utilize slow, tedious, and expensive assays. This thesis is focused on the development of highly versatile and scaleable kinase and “kinase-like” screening platforms for the discovery of small molecule inhibitors of these drug targets. The work begins with the validation of a matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) platform utilizing phosphorylation of kanamycin, an aminoglycoside antibiotic, by aminoglycoside phosphotransferase 3ʹIIIa (APH 3ʹIIIa) as a model system. Using a product-to-substrate signal ratio as an internal standard, the assay was used to functionally screen over 200 compounds, combined into mixtures to enhance assay throughput. Moreover, the assay was used to determine inhibitory dissocation constants for newly discovered modulators. Throughput was further increased to a novel dual-kinase assay targeting a bacterial enzyme, APH 3ʹIIIa and a human kinase, protein kinase A (PKA), which was validated using the previous small molecule library. Alternative assay development platforms were also studied using imaging mass spectrometry of reaction microarrays and the fabrication of sol-gel derived bioaffinity chromatography columns. The MS-based kinase assays developed herein are highly amenable to high throughput screening, and have the potential to be extended to other important therapeutic targets.</p> / Doctor of Philosophy (PhD)