The number of people living with diabetes worldwide is continually increasing. The majority of these people will eventually die of cardiovascular disease, the major underlying cause of which is atherosclerosis. Despite the efforts of many researchers, gaps in our knowledge still exist regarding the molecular mechanism(s) linking the two conditions. Current data suggests that the hexosamine biosynthetic pathway (HBP) may have a role in the development of hyperglycemia-accelerated atherosclerosis. About 2-3% of glucose entering a cell is diverted into this pathway where it is modified through a series of reactions to yield the end product, UDP-N-acetylglucosamine (UDP-GlcNAc); a substrate for both N- and O-linked glycosylation of various molecules. N-linked glycosylation occurs in the endoplasmic reticulum (ER) and is an important process in the maintenance of ER homeostasis. We hypothesized that a dysregulation in the HBP can ultimately trigger ER stress – an event associated with the development of atherosclerosis. We have established a method that allows us to monitor levels of UDP-GlcNAc both in cultured cells and mouse tissues through high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Using this technique, we’ve shown that both glucosamine supplementation and overexpression of the rate limiting enzyme of the HBP, GFAT, in cultured cells results in elevated UDP-GlcNAc levels. Furthermore, glucosamine was shown to trigger ER stress. In contrast, three GFAT inhibitors that were previously identified in a high throughput screen were shown to decrease UDP-GlcNAc levels and one inhibitor, dehydroiso-β-lapachone, appears to prevent ER stress induction. Finally, we use complementary methods to show that the HBP is augmented in the livers of hyperglycemic mice. This process may play a role in the accelerated development of atherosclerosis. Together, these results provide further insight into the role of the HBP in diabetic atherosclerosis and the established methods provide a platform for the further investigation of this mechanism. / Thesis / Master of Science (MSc)

<p>Human L-glutamine:D-fructose-6-phosphate amidotransferase 1 (hGFAT1) is the first and rate-limiting enzyme of the hexosamine biosynthesis pathway (HBP) and is a potential target to help prevent secondary complications of type II diabetes. GFAT catalyzes the irreversible reaction between L-glutamine and D-fructose-6-phosphate to produce L-glutamate and D-glucosamine-6-phosphate. hGFAT1 is not commercially available and is difficult to obtain from natural sources. Thus, a recombinant method to generate and purify the enzyme was developed and is discussed herein.</p> <p>There are only a handful of known inhibitors available to study the enzyme and the majority of these are toxic, non-specific, or substrate analogs. A high-throughput screening campaign was undertaken in pursuit of novel hGFAT1 inhibitors. The bioactive subset of the Canadian Compound Collection was assayed in duplicate for GFAT inhibitory activity using a modified version of the Morgan-Elson assay. Out of the 3950 bioactives, 9 were identified as lead compounds. All of the compounds identified from the bioactive collection are novel GFAT inhibitors.</p> <p>A structure-activity relationship (SAR) analysis was performed on the lead compounds. Derivatives of the leads were also purchased or synthesized for inhibitory testing. Four distinct classes of compounds were identified as GFAT inhibitors: isoquinolines, aminothiazoles, pyridinones and quinones. The most potent lead compound elucidated from the SAR was dehydroiso-β-lapachone (IC₅₀ 1.5±0.5 µM). The mode of inhibition of dehydroiso-β-lapachone was determined to be non-competitive for both binding domains of recombinant hGFAT1.</p> <p>To validate the lead compounds as inhibitors of native hGFAT1 and to determine their cell permeability, a cell based assay was developed. HepG2 cell cultures were treated with an inhibitor and HBP metabolism was determined by measuring the levels of the end-product uridine diphosphate <em>N</em>-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc was separated and detected by UPLC-ESI-TOF-MS and metabolite levels were normalized to cell concentration. The leads, alloxan, lapachol and amrinone all displayed hGFAT1 inhibition in cell culture.</p> / Doctor of Philosophy (PhD)