Fluorescence sensing and glycosidase inhibition effect of multivalent glycosidase inhibitors based on Naphthalimide-deoxynojirimycin conjugates.

Synthetic glycoconjugates as chemical probes have been widely developed for the detection of glycosidase enzymes. However, the binding interactions between iminosugar derivatives and glycosidases were limited, especially for the binding interactions between multivalent glycosidase inhibitors and α-glycosidases. In this paper, three naphthalimide-DNJ conjugates were synthesized. Furthermore, the binding interactions and glycosidase inhibition effects of them were investigated. It was found that the strong binding interactions of multivalent glycosidase inhibitors with enzymes were related to the efficient inhibitory activity against glycosidase. Moreover, the lengths of the chain between DNJ moieties and the triazole ring for the naphthalimide-DNJ conjugates influenced the self-assembly properties, binding interactions and glycosidase inhibition activities with multisource glycosidases. Compound 13 with six carbons between the DNJ moiety and triazole ring showed the stronger binding interactions and better glycosidase inhibition activities against α-mannosidase (jack bean) and α-glucosidase (aspergillus niger). In addition, compound 13 showed an effective PBG inhibition effect in mice with 51.18 % decrease in blood glucose at 30 min. This result opens a way for detection of multivalent glycosidase inhibition effect by a fluorescent sensing method.

Synthesis, α-mannosidase inhibition studies and molecular modeling of 1,4-imino-á´ -lyxitols and their C-5-altered N-arylalkyl derivatives.

A synthesis of 1,4-imino-ᴅ-lyxitols and their N-arylalkyl derivatives altered at C-5 is reported. Their inhibitory activity and selectivity toward four GH38 α-mannosidases (two Golgi types: GMIIb from Drosophila melanogaster and AMAN-2 from Caenorhabditis elegans, and two lysosomal types: LManII from Drosophila melanogaster and JBMan from Canavalia ensiformis) were investigated. 6-Deoxy-DIM was found to be the most potent inhibitor of AMAN-2 (K i = 0.19 µM), whose amino acid sequence and 3D structure of the active site are almost identical to the human α-mannosidase II (GMII). Although 6-deoxy-DIM was 3.5 times more potent toward AMAN-2 than DIM, their selectivity profiles were almost the same. N-Arylalkylation of 6-deoxy-DIM resulted only in a partial improvement as the selectivity was enhanced at the expense of potency. Structural and physicochemical properties of the corresponding inhibitor:enzyme complexes were analyzed by molecular modeling.

Pre-exposure of voglibose exerts cerebroprotective effects through attenuating activation of the polyol pathway and inflammation.

Chronic hyperglycemia induces activation of the polyol-sorbitol pathway, which is a major contributor to microvascular complications like stroke. The current study was designed to elucidate the therapeutic role of α-glucose inhibitor in chronic hyperglycemia-induced impaired polyol pathway and associated micro-complications. Male albino-Wistar rats (200-250 g) were treated with voglibose 10 mg kg-1  day-1 /p.o. for 2 weeks before middle cerebral artery occlusion; 72 hr after surgery, neurological score was evaluated and blood was collected for the assessment of various serum biochemical parameters like CRP, CK-MB, LDH, lipid profile, and blood glucose levels. In the end, brain samples were excised for determination of brain infarct volume, brain hemisphere weight difference, Na+-K+ ATPase activity oxidative stress-related parameters, aldose reductase activity, and gene expression studies. Results from the present study indicate that pre-treatment with voglibose showed significant improvement in lipid parameters but did not impact glucose levels. Voglibose has shown a statistically significant (p < .05) reduction in neurological score and brain infarct volume, and the difference in brain hemisphere weight as compared to the disease control group. Voglibose significantly (p < .05) improve all biochemical parameters and reduced Na+-K+ ATPase and aldose reductase activity. Moreover, voglibose produced a significant reduction in oxidative stress and down-regulation of TNF-α and BCl-2 gene expression which reduces the risk of factors related to stroke. In conclusion, the pleiotropic effect of voglibose on cerebrovascular complications may be due to inhibition of aldose reductase or anti-inflammatory pathways.

Synthesis of N-benzyl substituted 1,4-imino-l-lyxitols with a basic functional group as selective inhibitors of Golgi α-mannosidase IIb.

Inhibition of the biosynthesis of complex N-glycans in the Golgi apparatus is one of alternative ways to suppress growth of tumor tissue. Eight N-benzyl substituted 1,4-imino-l-lyxitols with basic functional groups (amine, amidine, guanidine), hydroxyl and fluoro groups were prepared, optimized their syntheses and tested for their ability to inhibit several α-mannosides from the GH family 38 (GMIIb, LManII and JBMan) as models for human Golgi and lysosomal α-mannoside II. All compounds were found to be selective inhibitors of GMIIb. The most potent structure bearing guanidine group, inhibited GMIIb at the micromolar level (Ki = 19 ±â€¯2 µM) while no significant inhibition (>2 mM) of LManII and JBMan was observed. Based on molecular docking and pKa calculations this structure may form two salt bridges with aspartate dyad of the target enzyme improving its inhibitory potency compared with other N-benzyl substituted derivatives published in this and previous studies.

Two New Succinimide Derivatives Cladosporitins A and B from the Mangrove-derived Fungus Cladosporium sp. HNWSW-1.

Two new succinimide-containing derivatives, cladosporitins A (1) and B (2), were isolated from the fermentation cultures of the mangrove-derived fungus Cladosporium sp. HNWSW-1, along with a new pyrone, clapone (3), as well as the previously reported talaroconvolutin A (4) and anthraquinone (5). The structures of the isolated compounds were elucidated by 1D, 2D NMR, and HRMS spectral analysis. Compound 2 showed cytotoxicity against BEL-7042, K562 and SGC-7901 cell lines with IC50 values of 29.4 ± 0.35 µM, 25.6 ± 0.47 µM, and 41.7 ± 0.71 µM, respectively, whereas compound 4 exhibited cytotoxicity against Hela and BEL-7042 cell lines with IC50 values of 14.9 ± 0.21 µM and 26.7 ± 1.1 µM, respectively. In addition, compounds 4 and 5 displayed inhibitory activity against α-glycosidase, with IC50 values of 78.2 ± 2.1 µM and 49.3 ± 10.6 µM, respectively.

Strategy for designing selective α-l-rhamnosidase inhibitors: Synthesis and biological evaluation of l-DMDP cyclic isothioureas.

This study shows that the cyclization of l-DMDP thioureas to bicyclic l-DMDP isothioureas improved α-l-rhamnosidase inhibition which was further enhanced by increasing the length of the alkyl chain. The addition of a long alkyl chain, such as decyl or dodecyl, to the nitrogen led to the production of highly potent inhibitors of α-l-rhamnosidase; it also caused broad inhibition spectrum against ß-glucosidase and ß-galactosidase. In contrast, the corresponding N-benzyl-l-DMDP cyclic isothioureas display selective inhibition of α-l-rhamnosidase; 3',4'-dichlorobenzyl-l-DMDP cyclic isothiourea (3r) was found to display the most potent and selective inhibition of α-l-rhamnosidase, with IC50 value of 0.22µM, about 46-fold better than the positive control 5-epi-deoxyrhamnojirimycin (5-epi-DRJ; IC50=10µM) and occupied the active-site of this enzyme (Ki=0.11µM). Bicyclic isothioureas of ido-l-DMDP did not inhibit α-l-rhamnosidase. These new mimics of l-rhamnose may affect other enzymes associated with the biochemistry of rhamnose including enzymes involved in progression of tuberculosis.

Antidiabetic potential of a peptide isolated from an endophytic Aspergillus awamori.

AIM: To exploit the potential of endophytic fungi for pharmaceutically important antidiabetic alpha glycosidase inhibitors. METHODS AND RESULTS: Thirty six endophytic fungi were isolated from Acacia nilotica and screened for the production of alpha amylase and glucosidase inhibitors. Inhibitory activity against both alpha amylase (81%) and alpha glucosidase (80%) was exhibited in an isolate, identified to be Aspergillus awamori. Purification of the inhibitor was carried out on Sephadex LH-20 column and semi prep HPLC. The inhibitor was characterized to be proteinaceous in nature with an approximate molecular mass of 22 kDa. UHPLC amino acid analysis indicated the presence of amino acids serine, threonine, tyrosine and valine in the peptide. The purified inhibitor exhibited mixed type of inhibition against alpha amylase and alpha glucosidase with IC50 values of 3·75 and 5·625 µg ml(-1) respectively. The inhibitor was stable over a wide range of pH and temperature. Optimization of process parameters to increase the yield of the inhibitor was undertaken using one factor at a time approach as well as RSM statistical analysis. The interaction of dextrose and proteose peptone for the test organism was significant with first order effect of pH. Increase of 13% was obtained in the inhibitory activity after optimization of process parameters. Mutagenicity testing by Ames test revealed nonmutagenic nature of the peptide. CONCLUSION: Endophytic A. awamori is capable of producing a peptide with alpha glycosidase inhibitory activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibitor obtained in this study possesses dual (alpha glucosidase and alpha amylase) inhibitory activity, low IC50 values, is highly stable under extreme conditions of pH and temperature, and is nonmutagenic in nature. By virtue of its properties it can be commercially produced and exploited for better management of diabetes.

Insect trehalase: physiological significance and potential applications.

Trehalose, a non-reducing disaccharide, is widespread throughout the biological world. It is the major blood sugar in insects playing a crucial role as an instant source of energy and in dealing with abiotic stresses. The hydrolysis of trehalose is under the enzymatic control of trehalase. The enzyme trehalase is gaining interest in insect physiology as it regulates energy metabolism and glucose generation via trehalose catabolism. The two forms of insect trehalase namely, Tre-1 and Tre-2, are important in energy supply, growth, metamorphosis, stress recovery, chitin synthesis and insect flight. Insect trehalase has not been reviewed in depth and the information available is quite scattered. The present mini review discusses our recent understanding of the regulation, mechanism and biochemical characterization of insect trehalase with respect to its physiological role in vital life functions. We also highlight the molecular and biochemical properties of insect trehalase that makes it amenable to competitive inhibition by most glycosidase inhibitors. Due to its crucial role in carbon metabolism in insects, application of inhibitors against trehalose can form a promising area towards formulating strategies for insect pest control.

Concise synthesis of C-1-cyano-iminosugars via a new Staudinger/aza Wittig/Strecker multicomponent reaction strategy.

A new Staudinger/aza Wittig/Strecker multicomponent reaction sequence to C-1-cyano iminoalditols has been developed. When applied to 5-azidodeoxy-d-xylose and -d-glucose as substrates the method leads smoothly in good yield and with excellent stereoselectivity to respectively, 1,5-dideoxy-1,5-imino-d-idurono nitrile and 2,6-didesoxy-2,6-imino-d-glycero-d-ido-heptononitrile.

Solvent-controlled synthesis of bulky and polar-bulky galactonoamidines.

The goal of this study was the design and synthesis of bulky and polar-bulky galactonoamidines that have a potential to interact with both catalytic amino acids in the active site of human α-galactosidase. While a library of more than 25 compounds was previously synthesized following established protocols, the coupling of the selected amines with activated perbenzylated galactothionolactam yielded only small amounts for some of the perbenzylated targets. A computational approach disclosed relative energy differences of selected adducts and suggested a solvent change that then allowed a successful synthesis of the precursor compounds in 20-75%. Subsequent attempts to globally deprotect perbenzylated galactonoamidines by Pd catalyzed hydrogenation resulted in unwanted Pd coordination, incomplete debenzylation reactions, partial compound hydrolysis, and even complete decomposition. A lengthy protocol was elaborated to purify the targeted carbohydrate derivatives after modified debenzylation conditions.

A new picrotoxane-type sesquiterpene from Dendrobium nobile Lindl.

A picrotoxane-type sesquiterpene, dendroterpene E (1), together with five benzene derivatives (2-6), were isolated from the stems of Dendrobium nobile Lindl. Their structures were elucidated by spectroscopic analysis and X-ray diffraction analysis. Compound 1 was a new picrotoxane-type sesquiterpene with a C-9/C-1/O/C-11 oxetane ring, which was first encountered in this type of compounds. Compounds 1-3 exhibited inhibitory activities against α-glycosidase.

Metabolites From the Mangrove-Derived Fungus Cladosporium sp. HNWSW-1.

Two new benzoic acids, cladoslide A (1) and cladoslide B (2); one new ß-carboline derivative, cladospomine (3); and one new pyridin-2(1H)-one, cladoslide C (4), were isolated from the fermentation cultures of the mangrove-derived fungus Cladosporium sp. HNWSW-1, along with the previously reported N-acetyl-ß-oxotryptamine (5), (4S,5S,11R)-iso-cladospolide B (6), (4S,5S,11S)-iso-cladospolide B (7), and (4R,5S,11R)-iso-cladospolide B (8). Their structures were elucidated by spectroscopic analysis, Rh2(OCOCF3)4-induced ECD experiments, and Marfey's method. Compound 1 showed cytotoxicity against the K562 cell line with IC50 values of 13.10 ± 0.08 µM. Moreover, compounds 1 and 5 exhibited inhibitory activity against α-glycosidase with IC50 values of 0.32 ± 0.01 mM and 0.17 ± 0.01 mM, respectively.

Four New Picrotoxane-Type Sesquiterpenes From Dendrobium nobile Lindl.

Four picrotoxane-type sesquiterpenes, dendroterpene A-D (1-4), together with four known compounds (5-8), were isolated from the stems of Dendrobium nobile Lindl. Their structures were elucidated by spectroscopic analysis, X-ray diffraction analysis, analysis of the ECD data according to the Klyne's lactone sector rule, and quantum ECD calculation. Compounds 1 and 2 are two new picrotoxane-type sesquiterpenes with a new carbon skeleton containing a formamide group, which may be derived from the previously reported dendrobiumane B skeleton by the C(9)-C(11) carbon bond cleavage. Compounds 3, 5, 6, and 8 exhibited inhibitory activity against α-glycosidase. Compounds 5 and 6 were cytotoxic against SGC-7901, K562, A549, BEL-7402, and Hela cell lines.

Cassia grandis fruit extract reduces the blood glucose level in alloxan-induced diabetic rats.

INTRODUCTION: Cassia grandis Lf fruits are ethnobotanically used for digestive disorders, anemia, and for reducing blood glucose. However, there are no studies about the antidiabetic activity nor the oral toxicity of the plant fruit-extracts. This paper aims to evaluate the hypoglycemic effect of C. grandis fruits extract in vivo, and assess the acute oral toxicity, and sub-acute oral toxicity. The antioxidant activity and the α-glycosidase inhibitor effect were also evaluated. METHODS: The extract was obtained by maceration of the fruit pulp with 70% hydroalcoholic solution (1:2, m:v). The extractive solution was concentrated in a vacuum rotary evaporator, up to a drug: solvent ratio of 2:1 (g/ml). Soluble solids, relative density, refractive index, pH, total phenolics, and flavonoids were determined. A preliminary phytochemical screening was made, followed by the quantitation of volatiles by GC/MS. The acute and sub-acute oral toxicity was evaluated in Sprague Dawley rats, by using biochemical and hematological parameters. The radical scavenging activity (DPPH, ABTS) and α-glycosidase inhibitory effect were tested. The hypoglycemic effect was assessed in alloxan-induced diabetic rats. RESULTS: The extract of C. grandis contains alkaloids, coumarins, flavonoids, free amino acids, amines, phenols, tannins, reduced sugars, resins, saponins, steroids, and triterpenes, plus 38 volatile compounds, being linalool the most abundant (1,66%). The extract exhibited an LD50 > 2000 mg/kg, and after a continuous administration (1000 mg/kg, 28-days), the hematological and biochemical parameters were normal. The extract showed hypoglycemic effect, being the dose 200 mg/kg no statistically different from glibenclamide at 25 mg/kg. Good antioxidant activity and a potent α-glycosidase inhibitory effect were also observed. CONCLUSION: C. grandis extract is an excellent hypoglycemic and non-toxic plant product. The hypoglycemic mechanism could be associated with the antioxidant effect and with the α-glycosidase inhibition. Up to the best of our knowledge, this is the first report on the hypoglycemic effect in vivo of C. grandis fruits extract.

Glycoreplica Peptides.

"Glycoreplica peptides" are prepared using a phage display peptide library and monoclonal antibodies that recognize the carbohydrate epitopes of glycoconjugate antigens. The peptides obtained not only mimic the shapes of original glycoconjugate antigens but also have some of their functions. We herein describe how to identify the amino acid alignments of glycoreplica peptides using phage display selection against carbohydrate-binding proteins. Target-specific peptides and proteins may be selected from the large repertory of a peptide/protein library using phage display technology. Glycoreplica peptides have the potential to become alternatives to carbohydrate ligands such as mimotopes for vaccinations and carbohydrate-derived drugs for carbohydrate-related diseases.

C-5a-substituted validamine type glycosidase inhibitors.

A series of N-alkyl derivatives of the D-galactosidase inhibitor 1,4-di-epi-validamine featuring lipophilic substituents at position C-5a was prepared and screened for their glycosidase inhibitory properties. Products turned out selective for ß-galactosidases as well as ß-glucosidases.

A Morita-Baylis-Hillman based route to C-5a-chain-extended 4-epi-isofagomine type glycosidase inhibitors.

By Morita-Baylis-Hillman reaction of 2,3-O-isopropylidene-D-glyceraldehyde with α,ß-unsaturated carbonyl as well as hetero analogous carbonyl compounds such as acrylonitrile, suitable precursors of isofagomine and of 4-epi-isofagomine are available. Elaboration of the structures by amine introduction, followed by intramolecular ring closure and subsequent hydroboration of the double bond provides 4-epi-isofagomine derivatives featuring chain extensions at C-5a which are determined by the structures of the carbonyl compounds employed. As an example, the synthesis of C-(5aR)- and C-(5aS)-5a-C-pentyl-4-epi-isofagomines, powerful inhibitors of ß-galactosidases, is outlined. In line with reported data, the (C-5aR) epimer was found a highly potent experimental pharmacological chaperone for GM1-associated human lysosomal ß-galactosidase mutant R201C.

Influence of the configurational pattern of sp(2)-iminosugar pseudo N-, S-, O- and C-glycosides on their glycoside inhibitory and antitumor properties.

The synthesis of a complete series of cyclic carbamate-type sp(2)-iminosugar N-, S-, O- and C-octyl pseudoglycosides related to nojirimycin, mannojirimycin and galactonojirimycin, all having the α-pseudoanomeric configuration, is reported. The gem-diamine-type N-pseudoglycosides can be accessed directly from the corresponding reducing sp(2)-imisosugar precursors by reaction with octylamine in methanol, whereas per-O-acetyl or 1-fluoro derivatives were used as pseudoglycosyl donors for the preparation of S-pseudoglycosides or O- and C-pseudoglycosides, respectively. Evaluation of their inhibitory properties against a panel of glycosidases evidenced selectivity profiles that strongly depend on the configurational pattern and the nature of the glycosidic linkage. On the contrary, the antiproliferative activity determined against a panel of tumor cell lines was largely independent of the relative orientation of the hydroxyl groups in the sp(2)-iminosugar moiety. Indeed, sp(2)-iminosugar representatives exhibiting significant growth inhibition potencies were identified in all three configurationally different types of compounds studied, namely α-d-gluco, α-d-manno and α-d-galacto glycoside analogs. Interestingly, none of the compounds affected viability and mortality of normal cells at the used concentrations. Altogether, the results strongly suggest that the anticancer activity of amphiphilic sp(2)-iminosugar glycosides might be unrelated, or not solely related, to their glycosidase inhibitory activity.

Inhibitor versus chaperone behaviour of d-fagomine, DAB and LAB sp(2)-iminosugar conjugates against glycosidases: A structure-activity relationship study in Gaucher fibroblasts.

A library of sp(2)-iminosugar conjugates derived from the piperidine iminosugar d-fagomine and the enantiomeric pyrrolidine iminosugars DAB and LAB has been generated in only two steps involving direct coupling of the fully unprotected polyhydroxylated heterocycles with isothiocyanates, to give monocyclic thiourea adducts, and further intramolecular nucleophilic displacement of the δ-located primary hydroxyl group by the thiocarbonyl sulphur atom, affording bicyclic isothioureas. These transformations led to a dramatic shift in the inhibitory selectivity from α- to ß-glucosidases, with inhibition potencies that depended strongly on the nature of the aglycone-type moiety in the conjugates. Some of the new derivatives behaved as potent inhibitors of human ß-glucocerebrosidase (GCase), the lysosomal enzyme whose dysfunction is responsible for Gaucher disease. Moreover, GCase inhibition was 10-fold weaker at pH 5 as compared to pH 7, which is generally considered as a good property for pharmacological chaperones. Surprisingly, most of the compounds strongly inhibited GCase in wild type fibroblasts at rather low concentrations, showing an unfavourable chaperone/inhibitor balance on disease-associated GCase mutants in cellulo. A structure-activity relationship analysis points to the need for keeping a contiguous triol system in the glycone moiety of the conjugates to elicit a chaperone effect. In any case, the results reported here represent a proof of concept of the utmost importance of implementing diversity-oriented strategies for the identification and optimization of potent and specific glycosidase inhibitors and chaperones.

Conformationally-locked N-glycosides: exploiting long-range non-glycone interactions in the design of pharmacological chaperones for Gaucher disease.

Pyranoid-type glycomimetics having a cis-1,2-fused glucopyranose-2-alkylsulfanyl-1,3-oxazoline (Glc-PSO) structure exhibit an unprecedented specificity as inhibitors of mammalian ß-glucosidase. Notably, their inhibitory potency against human ß-glucocerebrosidase (GCase) was found to be strongly dependent on the nature of aglycone-type moieties attached at the sulfur atom. In the particular case of ω-substituted hexadecyl chains, an amazing influence of the terminal group was observed. A comparative study on a series of Glc-PSO derivatives suggests that hydrogen bond acceptor functionalities, e.g. fluoro or methyloxycarbonyl, significantly stabilize the Glc-PSO:GCase complex. The S-(16-fluorohexadecyl)-PSO glycomimetic turned out to be a more potent GCase competitive inhibitor than ambroxol, a non glycomimetic drug currently in pilot trials as a pharmacological chaperone for Gaucher disease. Moreover, the inhibition constant increased by one order of magnitude when shifting from neutral (pH 7) to acidic (pH 5) media, a favorable characteristic for a chaperone candidate. Indeed, the fluoro-PSO derivative also proved superior to ambroxol in mutant GCase activity enhancement assays in N370S/N370S Gaucher fibroblasts. The results presented here represent a proof of concept of the potential of exploiting long-range non-glycone interactions for the optimization of glycosidase inhibitors with chaperone activity.