Structural studies of alpha-amylase inhibition

The increasing prevalence of type 2 diabetes has stimulated research aimed at discovering new drugs to treat the condition. Human Pancreatic α-Amylase (HPA) is responsible for cleaving starch into the smaller oligosaccharides that then become the substrates for the intestinal disaccharidases that hydrolyze them into glucose. Inhibition of HPA activity correlates to a reduction in postprandial blood glucose levels. This thesis uses x-ray crystallography to characterize the binding interactions between alpha-amylases and several newly discovered inhibitors that have potential therapeutic value, as well as one compound, voglibose, currently in clinical use. Voglibose is an excellent inhibitor of the intestinal disaccharidases, but a poor inhibitor of α-amylase. The HPA-voglibose complex is the first structure of voglibose with any α-glucosidase, and reveals a surprising binding mode for this inhibitor. Montbretin A is a glycosylated acyl flavonol that inhibits HPA with a Ki of 8 nM. This inhibitor exhibits a unique binding mode that allows it to fill the volume of the active site cleft near the enzyme’s three catalytic carboxylates. Two montbretin A fragments, myricetin and ethyl caffeate, also inhibit HPA with Ki values of 100 μM and 1.3 mM, and the crystal structures of their complexes with HPA were also solved. These two inhibitors impede enzyme activity through opposite mechanisms: myricetin blocks the active site, aided by increased order in an active site loop, and ethyl caffeate increases disorder in four loops that form part of the substrate binding cleft. The HPA-ethyl caffeate complex structure is also the first of HPA bound to a noncompetitive inhibitor. Amystatin is a 44 amino acid peptide that exhibits nanomolar inhibition of HPA. The crystal structure of the porcine pancreatic α-amylase-amystatin complex, reveals that this peptide represents a brand new class of HPA inhibitor.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat