The optical antipode of anhydromyriocin, the (gamma)-lactone derived from the antibiotic myriocin, was synthesized from L-arabinose. The identity of myriocin and thermozymocidin was confirmed, and their relative and absolute stereochemistries were established. In contrast to its natural enantiomer, (-)-ent-anhydromyriocin showed but little antifungal activity.

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Sphingolipids have been implicated as potential atherogenic lipids. Inhibition of hepatic serine palmitoyl transferase (SPT) reduces plasma sphingomyelin (SM) levels leading to a reduction of atherosclerosis in apolipoprotein-E gene knockout (apoE-/-) mice. In my thesis I have investigated the possibility that the reduced atherosclerosis resulting from SPT inhibition is associated with decreases in plasma glycosphingolipids (GSL). Furthermore, I examined whether SPT inhibition can lead to regression of atherosclerotic lesions. This thesis shows the SPT inhibitor myriocin inhibits atherosclerosis in apoE-/- mice fed a high fat diet. Lesion inhibition was most pronounced at the aortic arch and distal sites of the thoracic and abdominal aorta. There was also a trend towards a reduction in lesion area at the aortic root. Myriocin treatment resulted in significant reductions in both plasma SM and GSL concentrations. Moreover, it was demonstrated that myriocin significantly inhibited the progression of established atherosclerosis. Although the inhibition of lesion progression was observed mainly in the distal regions of the aorta, regression of lesion size was not detected. In addition, this thesis demonstrated for the first time that selective inhibition of GSL synthesis by the GSL synthesis inhibitor, d-threo-1- ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidino-propanol (EtDO-P4) had no significant impact on lesion area in apoE-deficient mice. Thus, despite the previously observed positive correlations between plasma and aortic GSL concentrations and the development of atherosclerosis, this thesis indicates that inhibition of GSL synthesis does not inhibit atherosclerosis in vivo. In other studies we assessed the possibility that myriocin may also be acting to increase hepatic apoA-I production via the inhibition of ERK phosphorylation. To address this, HepG2 cells and primary mouse hepatocytes were treated with myriocin. This significantly increased apoA-I mRNA, and protein levels. It also increased apoA-I secrection, and decreased ERK phosphorylation. These in vitro data indicate that ERK phosphorylation plays a role in modulating apoA-I expression, and that myriocin???s mechanism of action is linked to this pathway. Overall, this thesis has expanded the current literature regarding the role of sphingolipid synthesis inhibition and atherosclerosis.

Sphingolipids and ceramides are essential components of cellular membranes and important signalling molecules. Because of a growing appreciation for their diverse biological roles, understanding of the biosynthesis and regulation of sphingolipids has recently become a key goal in drug discovery. Serine palmitoyltransferase (SPT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyses the condensation between L-serine and a long-chain acyl thioester such as palmitoyl-CoA (C16-CoA). This first step in sphingolipid biosynthesis is conserved in all organisms studied to date, from microbes to man. The fungal natural product myriocin is a potent inhibitor of SPT; however, the molecular details of inhibition are not fully understood. Myriocin contains a long alkyl chain and a polar head group thus it displays features of both SPT substrates. Therefore, the prevailing hypothesis is that inhibition of SPT occurs because myriocin acts as a mimic of a key transition state of the catalytic mechanism. Through a combination of UV-vis spectroscopy, mass spectrometry, x-ray crystallography and enzyme inhibition assays it has been possible to study the interaction between S. paucimobilis SPT and myriocin. I have shown that myriocin initially forms an inhibitory PLP:myriocin aldimine complex in the active site that displays a Ki of 967 nM. Interestingly, this complex is susceptible to unexpected, slow enzymatic degradation. The mechanism for myriocin breakdown has been elucidated as a retro-aldol type reaction, which results in cleavage of the C2-C3 bond producing a C18 aldehyde. This aldehyde is then capable of covalently modifying the active site lysine265, forming a second (suicide) inhibitory complex and rendering the enzyme catalytically inactive. Substitution of the active site lysine produced SPT K265A, an inactive enzyme that did not catalyse the breakdown of the PLP:myriocin complex. However, the determination of the crystal structure of the SPT K265A:PLP-myriocin complex revealed that the myriocin had undergone decarboxylation. Nevertheless, this SPT:PLP:decarboxymyriocin structure revealed details about myriocin’s mechanism of inhibition for the first time. The novel mechanism of myriocin degradation has implications on the structure activity relationship (SAR) and design of drugs targeted towards SPT, the role of feedback regulation by long chain aldehydes and further expands the range of reactions catalysed by this important enzyme. As well as inhibition studies the structure of bacterial SPT was also examined by preparing an N-terminally truncated S. paucimobilis SPT. This version, shortened by 21 amino acids, was ~5-fold slower than the wild-type enzyme and suggests that the N-terminus may play a role in catalysis. Additional work has been undertaken to study an unusual membrane-bound viral SPT, composed of two naturally fused open reading frames (SPT2-SPT1) with the proposed SPT2 domain at the N-terminus and the SPT1 domain at the C-terminus. To study soluble mimics of this interesting fusion I prepared a bacterial S. paucimobilis SPT fused wild-type and mutant construct and isolated a fused SPT2-SPT1 with what appears to be single PLPbinding site.

While cigarette smoking is a common-knowledge way to stay lean, it has long been known as a risk factor for diabetes and obesity. Here we establish that smoking causes fat gain and metabolic disruption in mice, effects which are exacerbated by a high-fat, high-sugar diet. We found that smoke exposure increases levels of ceramide—the lipid responsible for diet-induced insulin resistance—and that blocking ceramide production with the pharmacological inhibitor myriocin restored insulin sensitivity, stopped weight gain, and rescued mitochondrial respiration in vivo and in vitro.We also sought to assess the impact of the RAGE ligand HMGB1 on skeletal muscle metabolism. We found that respiration between vehicle and HMGB1-injected red gastrocnemius was comparable. In myotubes, adding myriocin treatment to the HMGB1 cells increased respiration above HMGB1 treatment alone. HMGB1 increased oxidative stress in cultured myotubes and increased the transcript levels of Spt2, the enzyme responsible for the rate-limiting step in ceramide synthesis, although transcript levels of markers of mitochondrial fission and fusion leave us unsure of HMGB1's impact on mitochondrial dynamics. HMGB1, even at an exceptionally low dose over only 2 weeks, did cause significant impairment in glucose and insulin tolerance tests. Considering HMGB1's accessibility as a therapeutic target, its involvement in metabolic disruption is worth pursuing further.