Abstract
Glycoside hydrolases (GH) are a large family of hydrolytic enzymes found in all domains of life. As such, they control a plethora of normal and pathogenic biological functions. Thus, understanding selective inhibition of GH enzymes at the atomic level can lead to the identification of new classes of therapeutics. In these studies, we identified a 4-⍺-glucoside of valienamine (8) as an inhibitor of Streptomyces coelicolor (Sco) GlgE1-V279S which belongs to the GH13 Carbohydrate Active EnZyme family. The results obtained from the dose–response experiments show that 8 at a concentration of 1000 µM reduced the enzyme activity of Sco GlgE1-V279S by 65%. The synthetic route to 8 and a closely related 4-⍺-glucoside of validamine (7) was achieved starting from readily available D-maltose. A key step in the synthesis was a chelation-controlled addition of vinylmagnesium bromide to a maltose-derived enone intermediate. X-ray structures of both 7 and 8 in complex with Sco GlgE1-V279S were solved to resolutions of 1.75 and 1.83 Å, respectively. Structural analysis revealed the valienamine derivative 8 binds the enzyme in an E2 conformation for the cyclohexene fragment. Also, the cyclohexene fragment shows a new hydrogen-bonding contact from the pseudo-diaxial C(3)–OH to the catalytic nucleophile Asp 394 at the enzyme active site. Asp 394, in fact, forms a bidentate interaction with both the C(3)–OH and C(7)-OH of the inhibitor. In contrast, compound 7 disrupts the catalytic sidechain interaction network of Sco GlgE1-V279S via steric interactions resulting in a conformation change in Asp 394. These findings will have implications for the design other aminocarbasugar-based GH13-inhibitors and will be useful for identifying more potent and selective inhibitors.
Highlights
Glycoside hydrolase (GH) enzymes are hydrolases involved in a large array of biological p henomena[1,2,3]
Aminocarbasugars related to validamine (2a) and valienamine (6)[29,30] show inhibitory activity against α-Glycoside hydrolases (GH) such as α-amylase, rice α-glucosidase and yeast α-glucosidase31,32. IC50 values for compounds 2–6 against α-amylase are in the range of 1 0−2 mol/L and against yeast α-glucosidase is between 1 0−5–10−4 mol/L31
Before synthesizing the target pseudodisaccharides, we synthesized valienamine (6) from commercially available D-(+)-glucose (9) to obtain spectroscopic diastereoselectivity metrics for the intermediates afforded by the stereoselective addition of vinylmagnesium bromide to ketone 15, Scheme 2
Summary
Glycoside hydrolase (GH) enzymes are hydrolases involved in a large array of biological p henomena[1,2,3]. 1-Aminocarbasugars are thought to mimic charge development in the TS by incorporating a basic nitrogen atom in place of C(1)–O14 One example of this compound class is Acarbose (1), an inhibitor of the GH13 enzyme human pancreatic α-amylase and a treatment for type-II diabetes mellitus (T2D), Fig. 115,16. Kinetic isotope effect studies have suggested a 2,5B TS could be at play for a yeast α-glucosidase[24] Taking these conformational itineraries into consideration in the inhibitor design is expected to enhance inhibitor-enzyme interactions. Voglibose (5), a derivative of 2a, has found use as an α-glucosidase inhibitor and treatment for T2D32 Based on these reports, we proposed the 4-⍺-glucoside of validamine (7) and 4-⍺-glucoside of valienamine (8) would be potential inhibitors of α-GHs that can accommodate an α-configured − 2 site sugar, for example, Sco GlgE1V279S, Fig. 1. That has been mutated to be 100% identical to the M1P binding site of Mtb GlgE, and affords much higher quality X-ray diffraction d ata[34]
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