Adult forms of glycogenosis type II. A defect in an early stage of acid alpha-glucosidase realization

The activity of acid alpha-glucosidase in cultured fibroblasts from adult patients with the lysosomal storage disease glycogenosis type II is only 10% of normal. A normal activity per molecule is found for the mature as well as for the precursor form of acid alpha-glucosidase in adult mutant fibroblasts. Excessive lysosomal breakdown of mature enzyme purified from mutant fibroblasts and taken up by acceptor cells does not occur. However, the NH4Cl-stimulated secretion of a precursor form of acid alpha-glucosidase by adult mutant fibroblasts is markedly reduced. The results are indicative of a defect during the production of acid alpha-glucosidase.     

A new resorufin-based α-glucosidase assay for high-throughput screening

Mutations in α-glucosidase cause accumulation of glycogen in lysosomes, resulting in Pompe disease, a lysosomal storage disorder. Small molecule chaperones that bind to enzyme proteins and correct the misfolding and mistrafficking of mutant proteins have emerged as a new therapeutic approach for the lysosomal storage disorders. In addition, α-glucosidase is a therapeutic target for type II diabetes, and α-glucosidase inhibitors have been used in the clinic as alternative treatments for this disease. We have developed a new fluorogenic substrate for the α-glucosidase enzyme assay, resorufin α-d-glucopyranoside. The enzyme reaction product of this new substrate emits at a peak of 590 nm, reducing the interference from fluorescent compounds seen with the existing fluorogenic substrate, 4-methylumbelliferyl-α-d-glucopyranoside. Also, the enzyme kinetic assay can be carried out continuously without the addition of stop solution due to the lower pK_a of the product of this substrate. Therefore, this new fluorogenic substrate is a useful tool for the α-glucosidase enzyme assay and will facilitate compound screening for the development of new therapies for Pompe disease.     

Two potent competitive inhibitors discriminating alpha-glucosidase family I from family II

The inhibition kinetics for isoacarbose (a pseudotetrasaccharide, IsoAca) and acarviosine-glucose (pseudotrisaccharide, AcvGlc), both of which are derivatives of acarbose, were investigated with various types of alpha-glucosidases obtained from microorganisms, plants, and insects. IsoAca and AcvGlc, competitive inhibitors, allowed classification of alpha-glucosidases into two groups. Enzymes of the first group were strongly inhibited by AcvGlc and weakly by IsoAca, in which the K(i) values of AcvGlc (0.35-3.0 microM) were 21- to 440-fold smaller than those of IsoAca. However, the second group of enzymes showed similar K(i) values, ranging from 1.6 to 8.0 microM for both compounds. This classification for alpha-glucosidases is in total agreement with that based on the similarity of their amino acid sequences (family I and family II). This indicated that the alpha-glucosidase families I and II could be clearly distinguished based on their inhibition kinetic data for IsoAca and AcvGlc. The two groups of alpha-glucosidases seemed to recognize distinctively the extra reducing-terminal glucose unit in IsoAca.     

A simple and efficient synthesis of novel inhibitors of alpha-glucosidase based on benzimidazole skeleton and molecular docking studies

A novel series of benzimidazole derivatives were prepared starting from o-phenylenediamine and 4-nitro-o-phenylenediamine with iminoester hydrochlorides. Acidic proton in benzimidazole was exchanged with ethyl bromoacetate, then ethyl ester group was transformed into hydrazide group. Cyclization using CS₂/KOH leads to the corresponding 1,3,4-oxadiazole derivative, which was treated with phenyl isothiocyanate resulted in carbothioamide group, respectively. As the target compounds, triazole derivative was obtained under basic condition and thiadiazole derivative was obtained under acidic condition from cyclization of carbothioamide group. Most reactions were conducted using both the microwave and conventional methods to compare yields and reaction times. All compounds obtained in this study were investigated for α-glucosidase inhibitor activity. Compounds 6a, 8a, 4b, 5b, 6b and 7b were potent inhibitors with IC₅₀ values ranging from 10.49 to 158.2 μM. This has described a new class of α-glucosidase inhibitors. Molecular docking studies were done for all compounds to identify important binding modes responsible for inhibition activity of α-glucosidase.     

A novel alpha-glucosidase inhibitor from pine bark

Inhibitors of carbohydrate-hydrolysing enzymes play an important role for the treatment of diabetes. To our knowledge, a number of inhibitors such as, 1-deoxynojirimycin, acarbose and voglibose have been identified as a result of screening of secondary metabolites up to now. In this note, we report the inhibitory effect on carbohydrate hydrolysis of ethanol extracts from more than 1400 species of plants with the aim of identifying a potential antihyperglycemic drug. Pinus densiflora bark extracts showed the highest inhibition activity against several carbohydrate-hydrolysing enzymes.     

Altering the substrate chain-length specificity of an alpha-glucosidase

Dextran glucosidases show high sequence identity (50%) to Bacillus sp. SAM1606 alpha-glucosidase, which is more specific for short-chain substrates. Sequence comparison of these enzymes as well as molecular modeling studies predicted that the extension of loop 4 of the (beta/alpha)(8)-barrel fold may be responsible for the narrower specificity of SAM1606 alpha-glucosidase with respect to substrate chain length. Indeed, deletion mutants of SAM1606 alpha-glucosidase that lack this extension showed higher relative activities toward dextran and long-chain isomaltooligosaccharides. Kinetic and thermodynamic analyses of oligosaccharide hydrolysis catalyzed by SAM1606 alpha-glucosidase and its deletion mutants suggested that the loss of such extension(s) in loop 4 should energetically destabilize the Michaelis complexes with long-chain substrates to result in smaller differences between the activation free energies for the enzymatic hydrolyses of isomaltoheptaose and isomaltose than those observed for the wild-type enzyme. This is the reason that dextran glucosidase, whose loop 4 is shorter in length, shows broader substrate chain-length specificity than does SAM1606 alpha-glucosidase.     

Immortalization of murine muscle cells from lysosomal α-glucosidase deficient mice: A new tool to study pathophysiology and assess therapeutic strategies for Pompe disease

Glycogen storage disease type II (GSDII) is an autosomal recessive disorder caused by defects in the acid α-glucosidase (GAA) gene leading to lysosomal glycogen accumulation, mainly in cardiac and muscle tissues. In order to facilitate biological investigation on this disease and to avoid time-consuming direct cell isolation and culture, we have established murine myogenic GSDII cell lines. Lentiviral/retroviral expression of SV40 T antigen, Bmi-1 or cyclin-dependent kinase 4 (CDK4) genes was used to induce the immortalization of primary satellite cells from GSDII mice. The resulting immortalized myoblasts exhibit phenotypic characteristics of their parental cells, including profound GAA deficiency, glycogen accumulation and the ability to fully differentiate into myotubes when placed in proper culture conditions. These cell lines will constitute a powerful tool for both basic and applied studies focused on a better understanding of the pathophysiological mechanisms involved in GSDII and for assessing putative therapeutic strategies.     

Design,synthesis and biological evaluation of novel coumarin thiazole derivatives as α-glucosidase inhibitors

A new series of coumarin thiazole derivatives 7a-7t were synthesized, characterized by ¹H NMR, ¹³C NMR and element analysis, evaluated for their α-glucosidase inhibitory activity. The majority of the screened compounds displayed potent inhibitory activities with IC₅₀ values in the range of 6.24 ± 0.07–81.69 ± 0.39 μM, when compared to the standard acarbose (IC₅₀ = 43.26 ± 0.19 μM). Structure–activity relationship (SAR) studies suggest that the pattern of substitution in the phenyl ring is closely related to the biological activity of this class of compounds. Among all the tested molecules, compound 7e (IC₅₀ = 6.24 ± 0.07 μM) was found to be the most active compound in the library of coumarin thiazole derivatives. Enzyme kinetic studies showed that compound 7e is a non-competitive inhibitor with a K_i of 6.86 μM. Furthermore, the binding interactions of compound 7e with the active site of α-glucosidase were confirmed through molecular docking. This study has identified a new class of potent α-glucosidase inhibitors for further investigation.     

Canine glycogen storage disease type II a biochemical study of an acid α-glucosidase-deficient Lapland dog

A biochemical study was performed in a Lapland dog suspected of glycogen storage disease type II (acid α-glucosidase deficiency, Pompe's disease). Glycogen content was substantially elevated in heart and skeletal muscle but not in the liver. Severly reduced activities of acid α-glucosidase (EC 3.2.1.20) were found in heart, skeletal muscle, liver and cultured tongue fibroblasts. The deficiency was located in the glycoprotein fraction, which supported its lysosomal origin. The electrophorogram showed after acid incubation that the affected dog was missing the activity band, while after neutral incubation the pattern was similar to control. The obtained biochemical data are compared with the known data of the human pathology.     

Purification and characterization of an alpha-glucosidase from germinating millet seeds

An α-glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) was isolated from germinating millet (Panicum miliaceum L.) seeds by a procedure that included ammonium sulfate fractionation, chromatography on CM-cellulofine/Fractogel EMD SO₃, Sephacryl S-200 HR and TSK gel Phenyl-5 PW, and preparative isoelectric focusing. The enzyme was homogenous by SDS-PAGE. The molecular weight of the enzyme was estimated to be 86,000 based on its mobility in SDS-PAGE and 80,000 based on gel filtration with TSKgel super SW 3000, which showed that it was composed of a single unit. The isoelectric point of the enzyme was 8.3. The enzyme readily hydrolyzed maltose, malto-oligosaccharides, and α-1,4-glucan, but hydrolyzed polysaccharides more rapidly than maltose. The K_m value decreased with an increase in the molecular weight of the substrate. The value for maltoheptaose was about 4-fold lower than that for maltose. The enzyme preferably hydrolyzed amylopectin in starch, but also readily hydrolyzed nigerose, which has an α-1,3-glucosidic linkage and exists as an abnormal linkage in the structure of starch. In particular, the enzyme readily hydrolyzed millet starch from germinating seeds that had been degraded to some extent.     

Purification and biochemical characterisation of a membrane-bound alpha-glucosidase from the parasite Entamoeba histolytica

An alpha-glucosidase was solubilised from a mixed membrane fraction of Entamoeba histolytica and purified to homogeneity by a two-step procedure consisting of ion exchange chromatography in a Mono Q column and affinity chromatography in concanavalin A-sepharose. Although the enzyme failed to bind the lectin, this step rendered a homogenous and more stable enzyme preparation that resolved into a single polypeptide of 55 kDa after SDS-PAGE. As measured with 4-methylumbelliferyl-alpha-D-glucopyranoside (MUalphaGlc) as substrate, glycosidase activity was optimum at pH 6.5 with different buffers and at 45 degrees C. Although the enzyme preferentially hydrolysed nigerose (alpha1,3-linked), it also cleaved kojibiose (alpha1,2-linked), which was the second preferred substrate, and to a lesser extent maltose (alpha1,4), trehalose (alpha1,1) and isomaltose (alpha1,6). Activity on alpha1,3- and alpha1,2-linked disaccharides was strongly inhibited by the glycoprotein processing inhibitors 1-deoxynojirimycin and castanospermine but was unaffected by australine. Glucose and particularly 3-deoxy-D-glucose and 6-deoxy-D-glucose were strong inhibitors of activity, whereas 2-deoxy-D-glucose and other monosaccharides had no effect. Enzyme activity on MUalphaGlc was very sensitive to inhibition by diethylpyrocarbonate suggesting a critical role of histidine residues in enzyme catalysis. Other amino acid modifying reagents such as N-ethylmaleimide and N-(3-dimethylaminopropyl)-N'ethylcarbodiimide showed a moderate effect or none at all, respectively. Results are discussed in terms of the possible involvement of this glycosidase in N-glycan processing.     

Synthesis,biological evaluation and molecular docking study of N-arylbenzo[d]oxazol-2-amines as potential α-glucosidase inhibitors

A novel series of N-arylbenzo[d]oxazol-2-amines (4a–4m) were synthesized and evaluated for their α-glucosidase inhibitory activity. Compounds 4f–4i, 4k and 4m displayed potent inhibitory activity against α-glucosidase with IC₅₀ values in the range of 32.49 ± 0.17–120.24 ± 0.51 μM as compared to the standard drug acarbose. Among all tested compounds, compound 4g having 4-phenoxy substitution at the phenyl ring was found to be the most active inhibitor of α-glucosidase with an IC₅₀ value of 32.49 ± 0.17 μM. Analysis of the kinetics of enzyme inhibition indicated that compound 4g is a noncompetitive inhibitor of α-glucosidase with a K_i value of 31.33 μM. Binding interaction of compound 4g with α-glucosidase was explored by molecular docking simulation.     

Comparative evaluation of D-glucosyl thiouronium, glucosylthio heterocycles, Daonil, and insulin as inhibitors for hepatic glycosidases

Comparison of the in vivo and in vitro effects of S-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)thiuronium bromide (1), 2-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosylthio-1,3,4-thiadiazolin-5-thione (2), and 2-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosylthio)-1,3-benzoxazole (3), as well as the antidiabetics Daonil and insulin on glycosidase enzymes has been investigated. Compound 1 inhibited both alpha- and beta-glucosidases, but the inhibition was more potent with the beta-enzyme. Compound 2 was found to be a weaker inhibitor of these enzymes, while compound 3 showed a slight apparent activation.     

Inhibition of alpha-glucosidase by oleanolic acid and its synthetic derivatives

Oleanolic acid (1) and five synthetic derivatives (2-6) were tested spectrophotometrically for inhibition of urease, beta-lactamase, acetyl cholinesterase and alpha-glucosidase. All products showed a positive response only against alpha-glucosidase but not against the other enzymes; IC(50) calculations showed that the dihydroxy-olide derivative (4) was the most potent among all tested samples.     

Enzyme markers and isolation of the microvillar and perimicrovillar membranes of Dysdercus peruvianus (hemiptera: pyrrhocoridae) midgut cells

The midgut of Dysdercus peruvianus is divided into four sections (V₁-V₄). All the cells have microvilli ensheathed by a lipoprotein membrane (perimicrovillar membrane) extending toward the lumen as narrow tubes with dead ends. Subcellular fractionation of V₁ and V₂ tissue in isotonic and hypotonic conditions showed that -glucosidase is associated with membranous structures larger than those associated with β-glucosidase. The /β-glucosidase activity ratio is 34 ± 4 in V₁ tissue and 170 ± 10 in membranes recovered from the V₁ luminal contents. These membranes are resolved in sucrose gradients into low density (1.087 ± 0.001 g/cm³) -glucosidase-carrying membranes (/β-glucosidase activity ratio of 330±30) and high density (1.132 ± 0.002g/cm³) β-glucosidase-carrying-membranes. Low-density membranes have 1090 ± 60 μg lipid/mg protein and apparently are not contaminated by high-density ones (electron micrographs). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that membranes recovered from V₁ luminal contents are composed mainly of a-glucosidase-rich membranes. The data suggest that -glucosidase-rich membranes are perimicrovillar membranes which may be partly lost into luminal contents on dissection, with densities and lipid/protein ratios similar to that of myelin sheaths, in accordance with previous freeze-fracture data. β-Glucosidase-rich membranes are probably microvillar membranes with densities increased by the presence of associated portasomes.     

A new diantheramide and a new cyclic peptide from the seeds of Vaccaria hispanica

A new diantheramide, 4,4′-dihydroxy-2′-methoxydianthramide (1), and a new cyclic peptide, named segelin I (2) were isolated from the seeds of Vaccaria hispanica. Their structures were elucidated by detailed spectroscopic analysis and chemical methods. Compounds 1 and 2 were revealed to show significantly in vitro α-glucosidase inhibitory activity with IC₅₀ values of 0.080 ± 0.002 mM and 0.28 ± 0.002 mM, respectively, which were more potent than the reference compound acarbose (IC₅₀ 0.410 ± 0.001 mM).     

Quinazoline-1-deoxynojirimycin hybrids as high active dual inhibitors of EGFR and α-glucosidase

A series of novel quinazoline-1-deoxynojirimycin hybrids were designed, synthesized and evaluated for their inhibitory activities against two drug target enzymes, epidermal growth factor receptor (EGFR) tyrosine kinase and α-glucosidase. Some synthesized compounds exhibited significantly inhibitory activities against the tested enzymes. Comparing with reference compounds gefitinib and lapatinib, compounds 7d, 8d, 9b and 9d showed higher inhibitory activities against EGFR (IC₅₀: 1.79–10.71 nM). Meanwhile the inhibitory activities of 7d, 8d and 9c against α-glucosidase (IC₅₀ = 0.14, 0.09 and 0.25 µM, respectively) were obvious higher than that of miglitol (IC₅₀ = 2.43 µM), a clinical using α-glucosidase inhibitor. Interestingly, compound 9d as a dual inhibitor showed high inhibitory activity to EGFR^wt tyrosine kinase (IC₅₀ = 1.79 nM), also to α-glucosidase (IC₅₀ = 0.39 µM). The work could be very useful starting point for developing a new series of enzyme inhibitors targeting EGFR and/or α-glucosidase.     

Triterpenoids from the seedpods of Holarrhena curtisii King and Gamble

Two new hydroperoxy pentacyclic triterpenoids, 3β-hydroxy-11α-hydroperoxyolean-12-en-28-oic acid (1) and 3β-hydroxy-11α-hydroperoxyursan-12-en-28-oic acid (2), together with nine known triterpenoids, squalene (3), β-amyrin acetate (4), α-amyrin acetate (5), lupeol acetate (6), lupeol (7), lanosta-7,24-dien-3β-ol (8), cycloeucalenol (9), oleanolic acid (11) and ursolic acid (12), a known phytosterol, 24-methylenepollinastanol (10), and two known flavanols, (–)-catechin (13) and (–)-gallocatechin (14), were isolated from the methanolic extract of the fresh seedpods of Holarrhena curtisii. Their structures were determined by spectroscopic analysis (one and two dimensional nuclear magnetic resonance, high resolution electrospray ionization mass spectrometry and attenuated total reflectance-Fourier transform infrared spectroscopy). All compounds (except squalene) were evaluated for their in vitro α-glucosidase inhibitory activity. Compounds 1, 2, 11 and 12, which had a pentacyclic triterpenoid acid skeleton, showed a strong in vitro α-glucosidase inhibitory activity compared to that of the standard control, acarbose.     

Induction and preliminary characterization of intracellular β-glucosidases from a cellulolytic Streptomyces strain

Abstract The cellulolytic actinomycete Streptomyces sp. QM-B814 posasses an intracellular β-glucosidase system which is induced by cellobiose and carboxymethylcellulose. Maximal β-glucosidase activity was attained 8–10 h after inducer addition to exponential phase growing cultures. The induction is depressed in the presence of glucose. The system is composed of two electrophoretically different β-glucosidase forms showing relative molecular masses of about 60 and 35 kDa, and p I values in the range 4.2–4.5. Both β-glucosidases are synthesized de novo. The enzymes share substrate preference and are both inhibited by δ-gluconolactone and p -chloromercuribenzoate. The induction pattern and glucose inhibition are similar for both enzymes.