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Exploring novel human pancreatic alpha-amylase inhibitors : a departure from carbohydrate-based therapeutics

University of British Columbia

Human pancreatic α-amylase (HPA) catalyzes a key step in the degradation of ingested starch. Accordingly HPA activity has been positively correlated to post-prandial blood glucose levels and has been identified as a viable target for inhibition and the development of therapeutics towards the treatment of diabetes and obesity. This work directs the hunt away from traditional saccharide-based inhibitors, which represent all carbohydrate metabolism therapeutics currently in use, to novel inhibitors with improved selectivity and potency. In Chapter 2, the synthesis of flavonol-based HPA inhibitors based upon the structure of Montbretin A, a complex flavonol glycoside, is explored. Through the synthesis of a library of Montbretin A analogues we were able to identify an inhibitor of HPA with a KI of 44 nM that formed new interactions within the amylase active site. Chapter 3 details work on the peptide-based HPA inhibitor helianthamide previously isolated from the Caribbean Sea anemone Stichodactyla helianthus. Recombinant expression of helianthamide as a fusion peptide was achieved in Escherichia coli and Pichia pastoris. Kinetic analysis indicated that recombinant helianthamide is one of the most potent HPA inhibitors known to date, with a KI of 0.01 nM. Structural analysis of the recombinant material indicated that it contained three disulfide bonds in a 1-5, 2-4, 3-6 pattern. Site-directed mutagenesis of helianthamide indicated that disruption of disulfide bonds led to a large decrease in potency, while alanine variants of residues forming polar contacts with HPA’s active site residues led to smaller decreases in potency, indicating that the intact tertiary structure of helianthamide is necessary for blockage of the amylase active site. Small peptides were synthesized based on the sequence of helianthamide, but most showed modest or no inhibition of HPA.

Text

2019-03-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/64848

Genome Science and Technology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/