In Situ Extension as an Approach for Identifying Novel α-Amylase Inhibitors

Abstract

A new approach for the discovery and subsequent structural elucidation of oligosaccharide-based inhibitors of ␣-amylases based upon autoglucosylation of known ␣-glucosidase inhibitors is presented. This concept, highly analogous to what is hypothesized to occur with acarbose, is demonstrated with the known ␣-glucosidase inhibitor, D-gluconohydroximino-1,5-lactam. This was transformed from an inhibitor of human pancreatic ␣-amylase with a K i value of 18 mM to a trisaccharide analogue with a K i value of 25 M. The three-dimensional structure of this complex was determined by x-ray crystallography and represents the first such structure determined with this class of inhibitors in any ␣-glycosidase. This approach to the discovery and structural analysis of amylase inhibitors should be generally applicable to other endoglucosidases and readily adaptable to a high throughput format.

Order and Disorder: Differential Structural Impacts of Myricetin and Ethyl Caffeate on Human Amylase, an Antidiabetic Target

“…This glucose is then, in turn, absorbed into the blood stream. [17][18][19][20] These types of conventional inhibitors make use of the extended substrate binding cleft of HPA, which contains five high affinity binding subsites (-3 -+2) for glucose and in which the three active site residues Asp197, Glu233 and Asp300 reside. The presence of high blood glucose levels and the glucose spiking phenomenon after meals, can in itself lead to serious medical complications such as progressive worsening of diabetic symptoms and the introduction of comorbidities such as cardio-vascular disease, kidney dysfunction, neuropathy and vision loss.…”

“…The potential therapeutic application of flavonoids for diabetic patients would therefore be in firstly reducing the amount of glucose derived from food and thereby blood glucose levels, and secondly to reduce the spiking of glucose concentrations in the blood stream following a meal. 17,18 In contrast, myricetin and related flavonols, with their planar conjugated ring structures, represent very different inhibitor binding motifs from those based on a polymeric glucose framework. 4-8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Indeed, elevated post-prandial glucose levels have been shown to be an accurate predictor of cardio-vascular disease associated with diabetes, even more so than conventional markers such as fasting glucose or glycated hemoglobin.…”

“…3,9 HPA is an endoglycosidase whose structure and function has been extensively studied. 17,18 Detailed kinetic and structural studies have shown that Asp197 acts as the catalytic nucleophile, while Glu233 functions as the acid/base catalyst. 16 The high-resolution structure of HPA shows it to comprise three domains, with the active site located to one end of a TIM barrel and associated with an extended substrate binding cleft, consistent with its ability to bind polymeric starch molecules.…”

“…16 The high-resolution structure of HPA shows it to comprise three domains, with the active site located to one end of a TIM barrel and associated with an extended substrate binding cleft, consistent with its ability to bind polymeric starch molecules. 17,18,20 Prominent amongst alternative classes of inhibitors being studied as prospects having greater specificity and affinity for HPA, are derivatives of flavonols [21][22][23][24][25][26][27][28][29][30] and caffeic acids 31-33 that occur naturally. Cleavage is accomplished via a classical double displacement mechanism, with net retention of anomeric configuration.…”

First Principles Insight into the α-Glucan Structures of Starch: Their Synthesis, Conformation, and Hydration

“…301,302 An approach to obtain such inhibitors has been by use of CGTase coupling either a maltoside or a maltotrioside with a lactam. 303,304 …”