M. Isabel García-Moreno

Chaperone activity of bicyclic nojirimycin analogues for Gaucher mutations in comparison with N-(n-nonyl)deoxynojirimycin.

Gaucher disease (GD), the most prevalent lysosomal storage disorder, is caused by mutations of lysosomal β‐glucosidase (acid β‐Glu, β‐glucocerebrosidase); these mutations result in protein misfolding. Some inhibitors of this enzyme, such as the iminosugar glucomimetic N‐(n‐nonyl)‐1‐deoxynojirimycin (NN‐DNJ), are known to bind to the active site and stabilize the proper folding for the catalytic form, acting as “chemical chaperones” that facilitate transport and maturation of acid β‐Glu. Recently, bicyclic nojirimycin (NJ) analogues with structure of sp2 iminosugars were found to behave as very selective, competitive inhibitors of the lysosomal β‐Glu. We have now evaluated the glycosidase inhibitory profile of a series of six compounds within this family, namely 5‐N,6‐O‐(N′‐octyliminomethylidene‐NJ (NOI‐NJ), the 6‐thio and 6‐amino‐6‐deoxy derivatives (6S‐NOI‐NJ and 6N‐NOI‐NJ) and the corresponding galactonojirimycin (GNJ) counterparts (NOI‐GNJ, 6S‐NOI‐GNJ and 6N‐NOI‐GNJ), against commercial as well as lysosomal glycosidases. The chaperone effects of four selected candidates (NOI‐NJ, 6S‐NOI‐NJ, 6N‐NOI‐NJ, and 6S‐NOI‐GNJ) were further evaluated in GD fibroblasts with various acid β‐Glu mutations. The compounds showed enzyme enhancement on human fibroblasts with N188S, G202R, F213I or N370S mutations. The chaperone effects of the sp2 iminosugar were generally stronger than those observed for NN‐DNJ; this suggests that these compounds are promising candidates for clinical treatment of GD patients with a broad range of β‐Glu mutations, especially for neuronopathic forms of Gaucher disease.

A Fluorescent sp2-Iminosugar With Pharmacological Chaperone Activity for Gaucher Disease: Synthesis and Intracellular Distribution Studies

Gaucher disease (GD) is the most prevalent lysosomal‐storage disorder, it is caused by mutations of acid β‐glucosidase (β‐glucocerebrosidase; β‐Glu). Recently, we found that bicyclic nojirimycin (NJ) derivatives of the sp2‐iminosugar type, including the 6‐thio‐N′‐octyl‐(5N,6S)‐octyliminomethylidene derivative (6S‐NOI‐NJ), behaved as very selective competitive inhibitors of the lysosomal β‐Glu and exhibited remarkable chaperone activities for several GD mutations. To obtain information about the cellular uptake pathway and intracellular distribution of this family of chaperones, we have synthesized a fluorescent analogue that maintains the fused piperidine–thiazolidine bicyclic skeleton and incorporates a dansyl group in the N′‐substituent, namely 6‐thio‐(5N,6S)‐[4‐(N′‐dansylamino)butyliminomethylidene]nojirimycin (6S‐NDI‐NJ). This structural modification does not significantly modify the biological activity of the glycomimetic as a chemical chaperone. Our study showed that 6S‐NDI‐NJ is mainly located in lysosome‐related organelles in both normal and GD fibroblasts, and the fluorescent intensity of 6S‐NDI‐NJ in the lysosome is related to the β‐Glu concentration level. 6S‐NDI‐NJ also can enter cultured neuronal cells and act as a chaperone. Competitive inhibition studies of 6S‐NDI‐NJ uptake in fibroblasts showed that high concentrations of D‐glucose have no effect on chaperone internalization, suggesting that it enters the cells through glucose‐transporter‐independent mechanisms.

6-Amino-6-deoxy-5,6-di-N-(N′-octyliminomethylidene)nojirimycin: Synthesis, Biological Evaluation, and Crystal Structure in Complex with Acid β-Glucosidase

6‐Amino‐6‐deoxy‐5,6‐di‐N‐(N′‐octyliminomethylidene)nojirimycin, a reducing analogue of N‐nonyl‐1‐deoxynojirimycin, proved to be a potent and very selective inhibitor of β‐glucosidases, including human acid β‐glucosidase. Structural studies of the enzyme–inhibitor complex showed a binding mode in which the anomeric hydroxy group is accommodated in the “wrong” α configuration.

Castanospermine–trehazolin hybrids: a new family of glycomimetics with tuneable glycosidase inhibitory propertiesElectronic supplementary data (ESI) available: full characterization data for the new compounds 7–9, 11, 14–19. See http://www.rsc.org/suppdata/cc/b2/b200162d/

Bicyclic azasugar glycomimetics related to castanospermine and trehazolin have been prepared from sugar carbodiimides via aminooxazoline intermediates; preliminary enzyme inhibition tests showed a marked dependence of the selectivity and potency towards alpha and beta-glucosidases upon the nature of the exocyclic substituent.

Molecular Basis for β-Glucosidase Inhibition by Ring-Modified Calystegine Analogues

Calystegines, polyhydroxy nortropane alkaloids first isolated from the extracts of Calystegia sepium, represent the most recently discovered members of the azasugar and iminosugar glycomimetic families. 3] Similar to the well-studied polyhydroxypiperidine, indolizidine, pyrrolidine and pyrrolizidine-type iminocyclitols, calystegines exhibit potent glycoside hydrolase (glycosidase) inhibitory properties. Given the broad range of biological events in which glycosidases are involved, inhibitors of these enzymes have potential in therapies that are targeted at, for example, cancer, viral infections, tuberculosis, diabetes and glycosphingolipid storage disorders. The molecular basis for glycosidase inhibition by the calystegines remain poorly understood. Recent structural and mechanistic studies on a b-glucosidase from Thermotoga maritima, TmGH1, which belongs to family 1, clan GH-A, in the CAZy classification, showed unambiguously that calystegine B2 (1; Scheme 1), one of the most powerful representatives, binds at

Synthesis of thiohydantoin-castanospermine glycomimetics as glycosidase inhibitors.

The preparation of bicyclic carbohydrate mimics related to (+)-castanospermine incorporating a thiohydantoin moiety is reported. The synthetic approach is compatible with molecular diversity-oriented strategies and involves alpha-azidoesters, built at the C-5/C-6 segment in gluco- or galactofuranose scaffolds, as the key precursors. Reduction to the corresponding alpha-amino ester and in situ coupling with isothiocyanates afford thioureidoester intermediates that undergo spontaneous cyclization to the corresponding hydantoins, beta-elimination, and furanose --> indolizidine rearrangement in a tandem manner. Biological evaluation of the new sp(2)-iminosugar-type glycomimetics evidenced a strong influence of the nature of the substituents at the nitrogen or oxygen atoms on the glycosidase inhibitory properties.

One-step synthesis of non-anomeric sugar isothiocyanates from sugar azides.

Tandem Staudinger-aza-Wittig reaction of primary azidodeoxy sugars with triphenylphosphine-carbon disulfide affords the corresponding primary deoxyisothiocyanato sugars in high yield. No products arising from O --> N acyl migration or formation of dimeric carbodiimides were observed. Interestingly, a polymer-supported triarylphosphine can advantageously replace triphenylphosphine, thus limiting the purification step to a simple filtration process. The reaction also allows the preparation of 5-deoxy-5-isothiocyanato sugars, a hitherto unknown class of compounds, from the corresponding azide precursors. Secondary sugar azides bearing the azido group at an endocyclic carbon atom afforded much lower isothiocyanation yields under these reaction conditions.

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 β-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 β-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.

Polyhydroxylated N-(thio)carbamoyl piperidines: nojirimycin-type glycomimetics with controlled anomeric configuration

Abstract N -(Thio)carbamoyl d - xylo -nojirimycin derivatives have been prepared by intramolecular rearrangement of sugar thiourea precursors under basic conditions. The stereochemistry at the aminoketal stereocentre is under stereoelectronic control, with the diastereomer having the pseudoanomeric group in axial orientation being obtained in all cases.

The Impact of Heteromultivalency in Lectin Recognition and Glycosidase Inhibition: An Integrated Mechanistic Study

The vision of multivalency as a strategy limited to achieve affinity enhancements between a protein receptor and its putative sugar ligand (glycotope) has proven too simplistic. On the one hand, binding of a glycotope in a dense glycocalix-like construct to a lectin partner has been shown to be sensitive to the presence of a third sugar entity (heterocluster effect). On the other hand, several carbohydrate processing enzymes (glycosidases and glycosyltransferases) have been found to be also responsive to multivalent presentations of binding partners (multivalent enzyme inhibition), a phenomenon first discovered for iminosugar-type inhibitory species (inhitopes) and recently demonstrated for multivalent carbohydrate constructs. By assessing a series of homo- and heteroclusters combining α-d-glucopyranosyl-related glycotopes and inhitopes, it was shown that multivalency and heteromultivalency govern both kinds of events, allowing for activation, deactivation or enhancement of specific recognition phenomena towards a spectrum of lectin and glycosidase partners in a multimodal manner. This unified scenario originates from the ability of (hetero)multivalent architectures to trigger glycosidase binding modes that are reminiscent of those harnessed by lectins, which should be considered when profiling the biological activity of multivalent architectures.