Inhibitors Of Glycoside Hydrolases Research Articles

Allylic Carbocyclic Inhibitors Covalently Bind Glycoside Hydrolases.

Allylic cyclitols were investigated as covalent inhibitors of glycoside hydrolases by chemical, enzymatic, proteomic, and computational methods. This approach was inspired by the C7 cyclitol natural product streptol glucoside, which features a potential carbohydrate leaving group in the 4-position (carbohydrate numbering). To test this hypothesis, carbocyclic inhibitors with leaving groups in the 4- and 6- positions were prepared. The results of enzyme kinetics analyses demonstrated that dinitrophenyl ethers covalently inhibit α-glucosidases of the GH13 family without reactivation. The labeled enzyme was studied by proteomics, and the active site residue Asp214 was identified as modified. Additionally, computational studies, including enzyme homology modeling and density functional theory (DFT) calculations, further delineate the electronic and structural requirements for activity. This study demonstrates that previously unexplored 4- and 6-positions can be exploited for successful inhibitor design.

Glycoside hydrolase stabilization of transition state charge: new directions for inhibitor design.

Carbasugars are structural mimics of naturally occurring carbohydrates that can interact with and inhibit enzymes involved in carbohydrate processing. In particular, carbasugars have attracted attention as inhibitors of glycoside hydrolases (GHs) and as therapeutic leads in several disease areas. However, it is unclear how the carbasugars are recognized and processed by GHs. Here, we report the synthesis of three carbasugar isotopologues and provide a detailed transition state (TS) analysis for the formation of the initial GH-carbasugar covalent intermediate, as well as for hydrolysis of this intermediate, using a combination of experimentally measured kinetic isotope effects and hybrid QM/MM calculations. We find that the α-galactosidase from Thermotoga maritima effectively stabilizes TS charge development on a remote C5-allylic center acting in concert with the reacting carbasugar, and catalysis proceeds via an exploded, or loose, SN2 transition state with no discrete enzyme-bound cationic intermediate. We conclude that, in complement to what we know about the TS structures of enzyme-natural substrate complexes, knowledge of the TS structures of enzymes reacting with non-natural carbasugar substrates shows that GHs can stabilize a wider range of positively charged TS structures than previously thought. Furthermore, this enhanced understanding will enable the design of new carbasugar GH transition state analogues to be used as, for example, chemical biology tools and pharmaceutical lead compounds.

Revealing the mechanism for covalent inhibition of glycoside hydrolases by carbasugars at an atomic level

Mechanism-based glycoside hydrolase inhibitors are carbohydrate analogs that mimic the natural substrate’s structure. Their covalent bond formation with the glycoside hydrolase makes these compounds excellent tools for chemical biology and potential drug candidates. Here we report the synthesis of cyclohexene-based α-galactopyranoside mimics and the kinetic and structural characterization of their inhibitory activity toward an α-galactosidase from Thermotoga maritima (TmGalA). By solving the structures of several enzyme-bound species during mechanism-based covalent inhibition of TmGalA, we show that the Michaelis complexes for intact inhibitor and product have half-chair (2H3) conformations for the cyclohexene fragment, while the covalently linked intermediate adopts a flattened half-chair (2H3) conformation. Hybrid QM/MM calculations confirm the structural and electronic properties of the enzyme-bound species and provide insight into key interactions in the enzyme-active site. These insights should stimulate the design of mechanism-based glycoside hydrolase inhibitors with tailored chemical properties.

Polyhydroxylated Quinolizidine Iminosugars as Nanomolar Selective Inhibitors of α-Glucosidases.

Polyhydroxylated quinolizidines bearing a hydroxymethyl group at the ring junction were synthesized from a readily available l-sorbose-derived ketonitrone. Evaluated as glycoside hydrolase inhibitors, these quinolizidines revealed to be potent and selective α-glucosidase inhibitors. Quinolizidine 9a is the first quinolizidine-scaffolded iminosugar exhibiting nanomolar inhibition of a glycoenzyme.

First Glycoside Hydrolase Family 2 Enzymes from Thermus antranikianii and Thermus brockianus with β-Glucosidase Activity.

Two glycoside hydrolase encoding genes (tagh2 and tbgh2) were identified from different Thermus species using functional screening. Based on amino acid similarities, the enzymes were predicted to belong to glycoside hydrolase (GH) family 2. Surprisingly, both enzymes (TaGH2 and TbGH2) showed twofold higher activities for the hydrolysis of nitrophenol-linked β-D-glucopyranoside than of -galactopyranoside. Specific activities of 3,966 U/mg for TaGH2 and 660 U/mg for TbGH2 were observed. In accordance, Km values for both enzymes were significantly lower when β-D-glucopyranoside was used as substrate. Furthermore, TaGH2 was able to hydrolyze cellobiose. TaGH2 and TbGH2 exhibited highest activity at 95 and 90°C at pH 6.5. Both enzymes were extremely thermostable and showed thermal activation up to 250% relative activity at temperatures of 50 and 60°C. Especially, TaGH2 displayed high tolerance toward numerous metal ions (Cu2+, Co2+, Zn2+), which are known as glycoside hydrolase inhibitors. In this study, the first thermoactive GH family 2 enzymes with β-glucosidase activity have been identified and characterized. The hydrolysis of cellobiose is a unique property of TaGH2 when compared to other enzymes of GH family 2. Our work contributes to a broader knowledge of substrate specificities in GH family 2.

Structure of γ-conglutin: insight into the quaternary structure of 7S basic globulins from legumes.

γ-Conglutin from lupin seeds is an unusual 7S basic globulin protein. It is capable of reducing glycaemia in mammals, but the structural basis of this activity is not known. γ-Conglutin shares a high level of structural homology with glycoside hydrolase inhibitor proteins, although it lacks any kind of inhibitory activity against plant cell-wall degradation enzymes. In addition, γ-conglutin displays a less pronounced structural similarity to pepsin-like aspartic proteases, but it is proteolytically dysfunctional. Only one structural study of a legume 7S basic globulin, that isolated from soybean, has been reported to date. The quaternary assembly of soybean 7S basic globulin (Bg7S) is arranged as a cruciform-shaped tetramer comprised of two superposed dimers. Here, the crystal structure of γ-conglutin isolated from Lupinus angustifolius seeds (LangC) is presented. The polypeptide chain of LangC is post-translationally cleaved into α and β subunits but retains its covalent integrity owing to a disulfide bridge. The protomers of LangC undergo an intricate quaternary assembly, resulting in a ring-like hexamer with noncrystallographic D3 symmetry. The twofold-related dimers are similar to those in Bg7S but their assembly is different as a consequence of mutations in a β-strand that is involved in intermolecular β-sheet formation in γ-conglutin. Structural elucidation of γ-conglutin will help to explain its physiological role, especially in the evolutionary context, and will guide further research into the hypoglycaemic activity of this protein in humans, with potential consequences for novel antidiabetic therapies.

Enzymatic Synthesis of Acarviosyl-maltooligosaccharides Using Disproportionating Enzyme 1

Acarbose is a pseudo-tetrasaccharide and one of the most effective inhibitors of glycoside hydrolases. Its derivatives, acarviosyl-maltooligosaccharides, which have longer maltooligosaccharide parts than the maltose unit of acarbose, were synthesized using a disproportionating enzyme partially purified from adzuki cotyledons. The enzyme was identified as a typical type-1 disproportionating enzyme (DPE1) by primary structure analysis. It produced six compounds from 100 mM acarbose and 7.5% (w/v) of maltotetraose-rich syrup. The masses of the six products were confirmed to accord with acarviosyl-maltooligosaccharides with the degrees of polymerization = 5-10 (AC5-AC10) by electrospray ionization mass spectrometry. (1)H and (13)C NMR spectra indicated that AC5-AC10 were α-acarviosyl-(1→4)-maltooligosaccharide, which have maltotriose-maltooctaose respectively in the maltooligosaccharide part. A predominance of AC7 in the products at the early stage of the reaction indicated that DPE1 catalyzes the transfer of the acarviosyl-glucose moiety from acarbose to the acceptors. ACn can be useful tools as new inhibitors of glycoside hydrolases.

Structural Basis for Inhibition of Xyloglucan-specific Endo-β-1,4-glucanase (XEG) by XEG-Protein Inhibitor

Microorganisms such as plant pathogens secrete glycoside hydrolases (GHs) to digest the polysaccharide chains of plant cell walls. The degradation of cell walls by these enzymes is a crucial step for nutrition and invasion. To protect the cell wall from these enzymes, plants secrete glycoside hydrolase inhibitor proteins (GHIPs). Xyloglucan-specific endo-β-1,4-glucanase (XEG), a member of GH family 12 (GH12), could be a great threat to many plants because xyloglucan is a major component of the cell wall in most plants. Understanding the inhibition mechanism of XEG by GHIP is therefore of great importance in the field of plant defense, but to date the mechanism and specificity of GHIPs remain unclear. We have determined the crystal structure of XEG in complex with extracellular dermal glycoprotein (EDGP), a carrot GHIP that inhibits XEG. The structure reveals that the conserved arginines of EDGP intrude into the active site of XEG and interact with the catalytic glutamates of the enzyme. We have also determined the crystal structure of the XEG-xyloglucan complex. These structures show that EDGP closely mimics the XEG-xyloglucan interaction. Although EDGP shares structural similarity to a wheat GHIP (Triticum aestivum xylanase inhibitor-IA (TAXI-IA)) that inhibits GH11 family xylanases, the arrangement of GH and GHIP in the XEG-EDGP complex is distinct from that in the xylanase-TAXI-IA complex. Our findings imply that plants have evolved structures of GHIPs to inhibit different GH family members that attack their cell walls.

Interactions between Glycoside Hydrolase Family 94 Cellobiose Phosphorylase and Glucosidase Inhibitors

Azasugars are known as potent inhibitors of glycoside hydrolases. In this study, we examined the inhibition of Cellvibrio gilvus cellobiose phosphorylase (CBP) by four azasugars (isofagomine, 1-deoxynojirimycin, castanospermine and calystegine B2) and a non-azasugar (glucono-1,5-lactone). Isofagomine strongly inhibited CBP, whereas 1-deoxynojirimycin, castanospermine, and glucono-1,5-lactone exhibited moderate or weak inhibition. Calystegine B2 did not inhibit CBP. Kinetic analysis in the presence of sulfate indicated that it is an extremely weak competitive inhibitor against phosphate. Moreover, crystal structures of CBP complexed with isofagomine or 1-deoxynojirimycin were determined, revealing molecular recognition of the glucosidase inhibitors by the phosphorolytic enzyme. These inhibitors are bound at subsite −1 and form several hydrogen bonds with the protein and anion (phosphate or sulfate). The strong inhibition by isofagomine is probably due to an electrostatic interaction between its endocyclic amino group and phosphate.

Stereoselective Synthesis of 3‐Deoxy‐4‐S‐(1→4)‐Thiodisaccharides and Their Inhibitory Activities Towards β‐Glycoside Hydrolases

AbstractThe sulfur linkage of β‐(1→4)‐thiodisaccharides was constructed with excellent diastereoselectivity by Michael addition of 2,3,4,6‐tetra‐O‐acetyl‐1‐thio‐β‐D‐galactose (2) or its β‐D‐glucose isomer (3) to sugar‐derived (2S, 6S)‐6‐acetoxymethyl‐2‐(2‐propyloxy)‐2H‐pyran‐3(6H)‐one (1). These reactions led to the per‐O‐acetyl glycosides of 3‐deoxy‐4‐S‐glycopyranosyl‐4‐thiohexopyranosid‐2‐ulose (4 and 5, respectively). Similar conjugated addition to the enone 1 of the isothiouronium salts 6 or 7, precursors in the synthesis of 2 or 3, also afforded the thiodisaccharides 4 or 5, respectively, with exclusive formation of the isomer that has an R configuration for the C‐4 stereocenter of the reducing‐end. The carbonyl function of 4 and 5 was reduced, and the resulting products were O‐deacetylated to give the free 4‐S(1→4)‐thiodisaccharides 10, 11, 14, and 15, which have a deoxy functionality adjacent to the thio group. These compounds were tested as inhibitors of glycoside hydrolases. Thus 11, the 3‐deoxy‐4‐thiomimetic of Galβ(1→4)Gal, proved to be a competitive inhibitor of the β‐galactosidase from E. coli (Ki = 0.16 mM). (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

Structural analysis of a newly identified class of plant protective microbial glycoside hydrolase inhibitors

Structural analysis of a newly identified class of plant protective microbial glycoside hydrolase inhibitors

Molecular identification of wheat endoxylanase inhibitor TAXI-I , member of a new class of plant proteins

T riticum a estivum endo x ylanase i nhibitors (TAXIs) are wheat proteins that inhibit family 11 endoxylanases commonly used in different (bio)technological processes. Here, we report on the identification of the TAXI-I gene which encodes a mature protein of 381 amino acids with a calculated molecular mass of 38.8 kDa. When expressed in Escherichia coli, the recombinant protein had the specificity and inhibitory activity of natural TAXI-I, providing conclusive evidence that the isolated gene encodes an endoxylanase inhibitor. Bioinformatical analysis indicated that no conserved domains nor motifs common to other known proteins are present. Sequence analysis revealed similarity with a glycoprotein of carrot and with gene families in Arabidopsis thaliana and rice, all with unknown functions. Our data indicate that TAXI-I belongs to a newly identified class of plant proteins for which a molecular function as glycoside hydrolase inhibitor can now be suggested.

The synthesis of a carbohydrate-like dihydrooxazine and tetrahydrooxazine as putative inhibitors of glycoside hydrolases: A direct synthesis of isofagomine

The treatment of benzyl 2,3-O-isopropylidene-β-L-xylopyranoside with N-hydroxyphthalimide under Mitsunobu conditions, followed by protecting-group interchange, gave benzyl 4-O-[(tert-butoxycarbonyl)amino]-2,3- O-isopropylidene-α-D-arabinoside. Mild acid hydrolysis and catalytic hydrogenolysis afforded 4-O-[(tert-butoxycarbonyl)amino]-D-arabinose that, upon heating in water, gave the dihydrooxazine [(4R,5S,6R)-5,6-dihydro-4,5-dihydroxy-6-hydroxymethyl-4H-1,2-oxazine] as a crystalline solid. A single-crystal structure determination of this solid showed it to exist in the 5H6 conformation. Reduction of the dihydrooxazine gave the tetrahydrooxazine [(4R,5S,6R)-4,5-dihydroxy-6-hydroxymethyl-3,4,5,6-tetrahydro-2H-1,2-oxazine]. The dihydrooxazine was an effective inhibitor of two β-glucosidases (Ki = 27 and 35 µM). Benzyl 2,3-O-isopropylidene-β-L-xylopyranoside, via the derived imidazylate, was converted into a nitrile that, upon reduction and protecting-group manipulations, gave benzyl 4-C-aminomethyl-4-deoxy-α-D-arabinoside. Treatment of this amine with hydrogen and palladium-on-carbon gave isofagomine.Key words: dihydrooxazine, tetrahydrooxazine, isofagomine, iminosugars, glycosidase inhibitors.

Mixed-Linkage Cellooligosaccharides: A New Class of Glycoside Hydrolase Inhibitors

Mixed-Linkage Cellooligosaccharides: A New Class of Glycoside Hydrolase Inhibitors

Mixed-linkage cellooligosaccharides: a new class of glycoside hydrolase inhibitors.

A new class of inhibitors for beta-D-glycoside hydrolases, in which a single alpha-(1-->4)-glycosidic bond is incorporated into an otherwise all-beta-(1-->4)-linked oligosaccharide, is described. Such mixed beta/alpha-linkage cellooligosaccharides are not transition-state mimics, but instead are capable of utilising binding energy from numerous subsites, spanning either side of the catalytic centre, without the need for substrate distortion. This binding is significant; a mixed alpha/beta-D-tetrasaccharide acts competitively on a number of cellulases, displaying inhibition constants in the range of 40-300 microM. Using the Bacillus agaradhaerens enzyme Cel5A as a model system, one such mixed beta/alpha-cellooligosaccharide, methyl 4(II),4(III)-dithio-alpha-cellobiosyl-(1-->4)-beta-cellobioside, displays a K(i) value of 100 microM, an inhibition at least 150 times better than is observed with an equivalent all-beta-linked compound. The three-dimensional structure of B. agaradhaerens Cel5A in complex with methyl 4(II),4(III)-dithio-alpha-cellobiosyl-(1-->4)-beta-cellobioside has been determined at 1.8 A resolution. This confirms the expected mode of binding in which the ligand, with all four pyranosides in the (4)C(1) chair conformation, occupies the -3, -2 and +1 subsites whilst evading the catalytic (-1) subsite. Such "by-pass" compounds offer great scope for the development of a new class of beta-D-glycoside hydrolase inhibitors.

ChemInform Abstract: Cyclic Guanidinium Ions as Analogues of Glycosyl Cations are Competitive and Noncompetitive Inhibitors of Glycoside Hydrolases.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Stable Dose-Related Partial Inhibition of Sucrase Activity Can Be Produced by a Glycoside Hydrolase Inhibitor

Stable Dose-Related Partial Inhibition of Sucrase Activity Can Be Produced by a Glycoside Hydrolase Inhibitor

The Influence of a New Glycoside Hydrolase Inhibitor on Blood Glucose and Serum Insulin Concentration in Non-Insulin Treated Diabetics

The Influence of a New Glycoside Hydrolase Inhibitor on Blood Glucose and Serum Insulin Concentration in Non-Insulin Treated Diabetics

A GLYCOSIDE-HYDROLASE INHIBITOR IN TREATMENT OF DUMPING SYNDROME

BAY g 5421, a glycoside-hydrolase inhibitor, produced symptomatic improvement in ten patients with the dumping syndrome. 100 mg BAY g 5421, given before a 50 g sucrose meal, produced pronounced attenuation of both hyperglycæmic and hypoglycæmic phases of plasma glucose levels; and it greatly reduced the rise in plasma levels of gastric inhibitory polypeptide and insulin. Gastric emptying, studied simultaneously by an isotopic method, showed little difference between tests, suggesting that the improvement achieved was not mediated by slowing gastric emptying.

家族性高脂質血症IV型に関する2, 3の検討

Plasma lipids and lipoproteins, glucose tolerance and insulin secretory function were examined in 81 relatives of 12 index cases with type IV hyperlipidemia, hyperinsulinemia and fatty liver as well as in 90 nonrelatives, including the spouses, as controls. Hyperinsulinemia mostly coupled with type IV or type IIb hyperlipidemia and abnormal liver function suggesting fatty liver was shown to exist in 37% of the relatives. Thus, hyperinsulinemia might be the basic trait of this form of familial hyperlipidemia, although the mode of transmission remains to be elucidated.The electronmicroscopic morphometry was performed on the biopsied liver specimens from the hyperinsulinemic and hyperlipidemic patients. In the hepatocyte, the membrane-bound ribosomes in rough endoplasmic reticulum, which are known to produce secretory proteins, were significantly increased in the patients. Number of VLDL particles in the hepatocyte Golgi complexes was also increased significantly. A highly significant positive correlation was found between the density of the membrane-bound ribosomes and the number of VLDL per hepatocyte, suggesting that the production and secretion of VLDL in the hyperinsulinemic patients might be accelerated in relation to the increased VLDL-apoprotein synthesis. Therefore, hyperinsulinemia could be the causative factor of this form of type IV hyperlipidemia.A glycoside-hydrolase inhibitor (Bay g 5421-Bayer AG, West Germany) was applied for treatment of hyperinsulinemia combined with hyperlipidemia. After administration of this drug, the absorption of dietary starch was shown to be delayed and the postprandial insulin elevation was markedly decreased. The administration of Bay g 5421 in the daily dosage of 300mg for 4 weeks produced no change in body weight but significant reduction in plasma lipids in the hyperinsulinemic hyperlipidemic patients.