Glucosyl epi‐cyclophellitol allows mechanism‐based inactivation and structural analysis of human pancreatic α‐amylase

Abstract

As part of a search for selective, mechanism-based covalent inhibitors of human pancreatic a-amylase we describe the chemoenzymatic synthesis of the disaccharide analog a-glucosyl epi-cyclophellitol, demonstrate its stoichiometric reaction with human pancreatic a-amylase and evaluate the time dependence of its inhibition. X-ray crystallographic analysis of the covalent derivative so formed confirms its reaction at the active site with formation of a covalent bond to the catalytic nucleophile D197. The structure illuminates the interactions with the active site and confirms OH4' on the nonreducing end sugar as a good site for attachment of fluorescent tags in generating probes for localization and quantitation of amylase in vivo.Keywords: activity-based probes; amylase; conduritol epoxide; GH13 structure; glycosyl enzyme; mechanism-based inhibition Digestion of starch in mammals starts with endocleavage of its a-1,4-glucosidic bonds by salivary and pancreatic a-amylases. This liberates oligosaccharides that are hydrolyzed further by a-glucosidases located on the gut wall, thereby delivering glucose to the bloodstream. Control of blood glucose levels can be achieved clinically by a-glucosidase inhibitors (AGI) such as acarbose and miglitol, which inhibit the gut wall enzymes [1,2]. However unpleasant side effects, in part associated with passage of the oligosaccharides to the lower gut, can limit patient compliance. An alternative strategy, which our laboratory has been exploring, is to employ specific inhibitors of the a-amylases that allow dietary oligosaccharides such as sucrose to be processed normally. This approach should minimize side effects while still reducing blood glucose levels. To that end, we have taken two approaches. High-throughput screening of natural product extracts is one approach, and has led us to the potent (K i = 8.1 nM) human pancreatic a-amylase (HPA) inhibitor montbretin A, which controls blood glucose in diabetic animals, and for which we have determined three-dimensional structures of its complex with HPA [3,4].The other approach has been through design of mechanism-based inhibitors that form covalent adducts with the HPA active site. While 2-deoxy-2-Abbreviations CP, cyclophellitol; ECP, epi-cyclophellitol; G-ECP, glucosyl epi-cyclophellitol; HPA, human pancreatic a-amylase; LaMalP, Lactobacillus acidophilus NCFM maltose phosphorylase.

The Synthesis of Cyclophellitol‐Aziridine and Its Configurational and Functional Isomers

“…To date, ABPs have been reported that enable selective profiling of retaining glycosidases such as β‐glucosidases, α‐glucosidases, α‐galactosidases[29b] and α‐fucosidases, which are designed based on compound 2 bearing N‐substituents featuring a fluorophore or biotin as a reporter entity. Cyclophellitol‐aziridine ( 2 ) has proven to be a superior scaffold compared to 2 (or 5)‐deoxy‐5‐fluoroglycosides for in vitro and in situ ABPP of retaining β‐glucosidase activities and adaptation of the configuration and substitution pattern will likely yield selective ABPs for retaining glycosidase families evolved to recognize and hydrolyze the underlying configurational carbohydrates – next to monosaccharides (exoglycosidases) likely also oligosaccharides (endoglycosidases). To fulfil this promise, though, synthetic methodology needs to be expanded to enable easy access to an array of configurational and functional cyclophellitol/cyclophellitol‐aziridine analogues.…”

Conformational Itinerary of Sucrose During Hydrolysis by Retaining Amylosucrase

“…Concerning the glycosyl-enzyme covalent intermediate (GEI) of the reaction, structural analyses of GH13 amylosucrase show that the -1 sugar bears a 4 C 1 conformation (Jensen et al, 2004). In the case of α-amylase, the GEI trapped using sugar analogs exhibits a similar 4 C 1 conformation (Zhang et al, 2009; Caner et al, 2016). QM/MM calculations of human pancreatic α-amylase captured the relevant role of the catalytic residues during the hydrolysis mechanism (Pinto et al, 2015), but the conformational itinerary of the -1 sugar was not investigated.…”

Effect of phenolic compounds from the rind of Punica granatum on the activity of three metabolism‐related enzymes

“…The amino acids residues of the active site pocket for 5EMY and allosteric sites for 4GQQ were retrieved from Refs. 53 and 21,54, respectively.…”