Iminosugar-Dihydroazulenes as Mutant L444P Glucocerebrosidase Enhancers.

A remarkable enhancer of human glucocerebrosidase enzyme (GCase) was identified among a set of dihydroazulene-tagged iminosugars. An unprecedented 3.9-fold increase in GCase activity was detected on fibroblasts bearing the homozygous L444P mutation, which is frequently associated with neuronopathic Gaucher forms, and which commonly results refractory to chaperone-induced refolding.

Tacrine-sugar mimetic conjugates as enhanced cholinesterase inhibitors.

We have used the Cu(i)-catalyzed azide-alkyne Huisgen cycloaddition reaction to obtain two families of bivalent heterodimers where tacrine is connected to an azasugar or iminosugar, respectively, via linkers of variable length. The heterodimers were investigated as cholinesterase inhibitors and it was found that their activity increased with the length of the linker. Two of the heterodimers were significantly stronger acetylcholinesterase inhibitors than the monomeric tacrine. Molecular modelling indicated that the longer heterodimers fitted better into the active gorge of acetylcholinesterase than the shorter counterparts and the former provided more efficient simultaneous interaction with the tryptophan residues in the catalytic anionic binding site (CAS) and the peripheral anionic binding site (PAS).

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.

Structural Basis of Outstanding Multivalent Effects in Jack Bean α-Mannosidase Inhibition.

Multivalent design of glycosidase inhibitors is a promising strategy for the treatment of diseases involving enzymatic hydrolysis of glycosidic bonds in carbohydrates. An essential prerequisite for successful applications is the atomic-level understanding of how outstanding binding enhancement occurs with multivalent inhibitors. Herein we report the first high-resolution crystal structures of the Jack bean α-mannosidase (JBα-man) in apo and inhibited states. The three-dimensional structure of JBα-man in complex with the multimeric cyclopeptoid-based inhibitor displaying the largest binding enhancements reported so far provides decisive insight into the molecular mechanisms underlying multivalent effects in glycosidase inhibition.

Mechanistic Insight into the Binding of Multivalent Pyrrolidines to α-Mannosidases.

Novel pyrrolidine-based multivalent iminosugars, synthesized by a CuAAC approach, have shown remarkable multivalent effects towards jack bean α-mannosidase and a Golgi α-mannosidase from Drosophila melanogaster, as well as a good selectivity with respect to a lysosomal α-mannosidase, which is important for anticancer applications. STD NMR and molecular modeling studies supported a multivalent mechanism with specific interactions of the bioactive iminosugars with Jack bean α-mannosidase. TEM studies suggested a binding mode that involves the formation of aggregates, which result from the intermolecular cross-linked network of interactions between the multivalent inhibitors and two or more dimers of JBMan heterodimeric subunits.

Iminosugar-based ceramide mimicry for the design of new CERT START domain ligands.

The enigmatical dichotomy between the two CERT/GPBP protein isoforms, their vast panel of biological implications and the scarcity of known antagonist series call for new ligand chemotypes identification. We report the design of iminosugar-based ceramide mimics for the development of new START domain ligands potentially targeting either protein isoforms. Strategic choice of (i) an iminoxylitol core structure and (ii) the positioning of two dodecyl residues led to an extent of protein binding comparable to that of the natural cargo lipid ceramide or the archetypical inhibitor HPA-12. Molecular docking study evidenced a possible mode of protein binding fully coherent with the one observed in crystalline co-structures of known ligands. The present study thus paves the way for cellular CERT inhibition studies en route to the development of pharmacological tools aiming at deciphering the respective function and therapeutic potential of the two CERT/GPBP protein isoforms.

The multivalent effect in glycosidase inhibition: probing the influence of valency, peripheral ligand structure, and topology with cyclodextrin-based iminosugar click clusters.

In view of recent reports of a strong multivalent effect in glycosidase inhibition, a library of ß-CD-based multivalent iminosugars has been efficiently synthesized by way of Cu(I) -catalyzed azide-alkyne cycloaddition (CuAAC). In combination with the first application of isothermal titration calorimetry (ITC) experiments to the study of multivalent iminosugar-enzyme interactions, the inhibition properties of these click clusters were evaluated on a panel of glycosidases. The structural parameters that were varied include valency, peripheral ligand structure, and topology. The inhibition results obtained with the iminosugar clusters further highlight the importance of multivalency in the inhibition of α-mannosidase. Generally, the evaluated multivalent iminosugars displayed comparable thermodynamic signatures of binding towards α-mannosidase (Jack bean): that is, large negative enthalpies of complexation coupled with small entropies of either sign. In addition, the enthalpy-entropy compensation observed in all tested cases may be attributed to a common mechanism of dissociation for the enzyme-multivalent iminosugar interactions. The measured binding stoichiometries indicated that each iminosugar cluster interacts with no more than one protein molecule.

Enzyme inhibition by iminosugars: analysis and insight into the glycosidase-iminosugar dependency of pH.

Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme-inhibitor complexes with three (EH3I), two (EH2I), one (EHI), or no protons (EI), are possible. In the present work an analysis method is presented that from pH-inhibition data allows one to distinguish between the different complexes and determine which protonation state is preferred. It is also possible to determine the pH-independent binding constants of the inhibitor. Analysis of pH data for imino- and azasugar inhibition of ß-glucosidases revealed that basic glycosidase inhibitors bind as the monoprotonated (EHI) complex. Three neutral inhibitors were also studied and two of these were also bound exclusively as the EHI complex while a third bound both as a EHI and a EH2I complex.

Rapid modifications of N-substitution in iminosugars: development of new ß-glucocerebrosidase inhibitors and pharmacological chaperones for Gaucher disease.

The rapid discovery of ß-glucocerebrosidase (GCase) inhibitors and pharmacological chaperones for Gaucher disease is described. The N-aminobutyl DNJ-based iminosugar was synthesized and conjugating with a variety of carboxylic acids to generate a N-diversely substituted iminosugar-based library. Several members of this library were found to be nanomolar-range inhibitors of GCase; the inhibition constant Ki of the most potent was found to be 71nM. Although these new molecules showed reasonable chaperoning activity (1.5- to 1.9-fold) in the N370S fibroblast of Gaucher patient-derived cell line, this was accompanies by a concomitant decrease in the cellular α-glucosidase activity, which might limit their further therapeutic potential. Next, newly developed N-substituents were assembled with pyrrolidine-based scaffolds to generate new molecules for further evaluation. The new 2,5-dideoxy-2,5-imino-d-mannitol (DMDP)-based iminosugar 22 was found to exhibit a satisfactory chaperoning activity to enhance GCase activity by 2.2-fold in Gaucher N370S cell line, without impairment of cellular α-glucosidase activity.

Skeletal rearrangement of seven-membered iminosugars: synthesis of (-)-adenophorine, (-)-1-epi-adenophorine and derivatives and evaluation as glycosidase inhibitors.

The mirror image of natural product (+)-adenophorine along with its 1-epi-, 1-homo-analogs and other derivatives have been synthesized and evaluated as glycosidase inhibitors. The synthetic strategy is based on the skeletal rearrangement of tetrahydroxylated C-alkyl azepanes obtained via a Staudinger/azaWittig/alkylation sequence starting from a sugar-derived azidolactol. Several organometallic species have been investigated for the alkylation step including organomagnesium, organolithium, organozinc, organoaluminum and organocerium reagents. While diallylzinc proved to be the most efficient to introduce an allyl substituent, disappointing results were obtained with the other organometallic species leading either to lower yields or no reaction. Enzymatic assays indicate that (-)-adenophorine is a moderate α-l-fucosidase inhibitor.

α-Glucosidase-inhibitory iminosugars from the leaves of Suregada glomerulata.

A water extract of the leaves of Suregada glomerulata (Euphorbiaceae) was found to inhibit rat small intestinal α-glucosidase. An examination of the extract afforded 20 iminosugars including one pyrrolidine and 19 piperidines. The structures of the 10 new compounds (11-20) were determined by NMR, and MS spectroscopic data analyses, and chemical correlations. The novelty of the identified compounds mainly stems from the loss of a hydroxy at C-4 and the presence of an 8-hydroxyoctyl side chain. Nine N-alkyl derivatives including N-methyl (1a, 8a, and 13a), N-butyl (1b, 2b, and 9b) and N,N-dimethyl (1c, 2c, and 9c) were synthesized. The compounds were tested for rat small intestinal α-glucosidase inhibitory activity. In total, 15 compounds, including compounds 11, 12, 15, and 19 and the three derivatives 8a, 9b, and 13a, showed inhibitory activity with IC50 values less than 40µM. In vivo results showed that total alkaloids of S. glomerulata (10mg/kg) and four major iminosugars 1, 2, 3, and 9 (10mg/kg) can lower the postprandial blood glucose level after sucrose and starch load in healthy male ICR mice.

Molecular mechanism for stabilization of a mutant α-galactosidase A involving M51I amino acid substitution by imino sugars.

Small molecules including imino sugars are expected to act as chaperones for a mutant α-galactosidase A (GLA), which will be useful for pharmacological chaperone therapy for Fabry disease. However, there is little detailed information about the molecular mechanism. We paid attention to an M51I mutant GLA which had been reported to strongly react to an imino sugar. The predicted structural change caused by this amino acid substitution is very small and located on the surface of the molecule. We produced the mutant enzyme in yeast, and determined its enzymological characteristics. The enzymological parameter values are almost the same as those of the wild-type GLA, although the mutant enzyme is unstable not only under neutral pH conditions but also under acidic ones. Then, we directly examined the effect of imino sugars including 1-deoxygalactonojirimycin and galactostatin bisulfite on the purified mutant enzyme. The imino sugars apparently improved the stability of the mutant enzyme under both neutral and acidic pH conditions. The results of surface plasmon resonance biosensor assaying suggested that the imino sugars retained their binding activity as to the mutant enzyme under both neutral and acidic pH conditions. This information will facilitate improvement of pharmacological chaperone therapy for Fabry disease.

Biochemical and structural study on a S529V mutant acid α-glucosidase responsive to pharmacological chaperones.

Recently, pharmacological chaperone therapy for Pompe disease with small molecules such as imino sugars has attracted interest. But mutant acid α-glucosidase (GAA) species responsive to imino sugars are limited. To elucidate the characteristics of a mutant GAA responsive to imino sugars, we performed biochemical and structural analyses. Among cultured fibroblast cell lines derived from Japanese Pompe patients, only one carrying p.S529V/p.S619R amino acid substitutions responded to 1-deoxynojirimycin (DNJ), and an expression study revealed that DNJ, N-butyl-deoxynojirimycin and nojirimycin-1-sulfonic acid increased the enzyme activity of the S529V mutant GAA expressed in Chinese hamster ovary cells. The results of western blotting analysis suggested that these imino sugars facilitated the intracellular transportation of the mutant GAA and stabilized it. Among these imino sugars, DNJ exhibited the strongest action on the mutant GAA. Structural analysis revealed that DNJ almost completely occupied the active site pocket, and interacted with amino acid residues comprising it through van der Waals contacts and hydrogen bonds. This information will be useful for improvement of pharmacological chaperone therapy for Pompe disease.

N-Substituted Valiolamine Derivatives as Potent Inhibitors of Endoplasmic Reticulum α-Glucosidases I and II with Antiviral Activity.

Most enveloped viruses rely on the host cell endoplasmic reticulum (ER) quality control (QC) machinery for proper folding of glycoproteins. The key ER α-glucosidases (α-Glu) I and II of the ERQC machinery are attractive targets for developing broad-spectrum antivirals. Iminosugars based on deoxynojirimycin have been extensively studied as ER α-glucosidase inhibitors; however, other glycomimetic compounds are less established. Accordingly, we synthesized a series of N-substituted derivatives of valiolamine, the iminosugar scaffold of type 2 diabetes drug voglibose. To understand the basis for up to 100,000-fold improved inhibitory potency, we determined high-resolution crystal structures of mouse ER α-GluII in complex with valiolamine and 10 derivatives. The structures revealed extensive interactions with all four α-GluII subsites. We further showed that N-substituted valiolamines were active against dengue virus and SARS-CoV-2 in vitro. This study introduces valiolamine-based inhibitors of the ERQC machinery as candidates for developing potential broad-spectrum therapeutics against the existing and emerging viruses.

Synthesis, computational study and glycosidase inhibitory activity of polyhydroxylated conidine alkaloids--a bicyclic iminosugar.

New bicyclic conidine iminosugars 1d and 1e were synthesized from D-glucose. Thus, D-glucose was converted to sugar beta-amino acids 3a and 3b in good yields. Individual treatment of 3a/3b with the Mukaiyama reagent afforded sugar beta-lactams 4a/4b that on reduction with LiAlH(4)/AlCl(3) gave azetidines 5a/5b with a sugar appendage. Reductive aminocyclization of sugar azetidines 5a/5b afforded the corresponding conidine iminosugars 1d/1e. Based on the (1)H NMR and DFT calculation studies the conformation of 1d was assigned as half chair A2 and that of 1e as a boat B2. The glycosidase inhibitory activities of 1d and 1e such as alpha-mannosidase, alpha-glucosidase and alpha-galactosidase were studied. The alpha-amylase activity was compared with acarbose. Compound 1d was found to be a moderate inhibitor of glycosidases while 1e was noticed to be a good inhibitor of alpha-mannosidase and a moderate inhibitor of other glycosidases. These results were substantiated by molecular docking studies using WHAT IF software and the AUTODOCK 3.0 program.

Nanomolar affinity, iminosugar-based chemical probes for specific labeling of lysosomal glucocerebrosidase.

Three different photoprobes were synthesized to label beta-glucosidases; one probe was based on glucose, two probes on the iminosugar deoxynojirimycin. The affinity of the probes for three different beta-glucosidases was determined. Furthermore, their labeling efficiencies, binding specificities through competition with deoxynojirimycin, and binding specificities in the presence of cell lysate, were evaluated. Especially one showed very high affinity towards non-lysosomal glucoceramidase (IC(50)=20 nM).

Glycomimetics at the mirror: medicinal chemistry of L-iminosugars.

Inhibition of carbohydrate processing enzymes is a topic of great interest, as these enzymes are involved in a plethora of key biochemical events, such as digestion, lysosomal catabolism of glycoconjugates and post-translational glycoprotein processing. Among the most potent inhibitors of such enzymes, iminosugars have emerged as versatile tools for medicinal chemists, especially those in quest for new therapeutic agents. Supply of iminosugars from natural sources or by chemical synthesis has provided excellent targets for medical intervention, ranging from antidiabetics and antivirals to inhibitors of genetic disorders. Although a huge body of literature has been reported around iminosugars, most data have focused on D-series iminosugars, whereas relatively little attention has been devoted to the corresponding L-enantiomers, due to their supposed lack of biological activity profile, as well as their scarce availability from natural sources. Notwithstanding, recent insights into the molecular details of enzyme-inhibitor interactions have led to a reassessment of L-iminosugars for pharmaceutical purposes. On one hand, they have been used as tools for intensive SAR (structure-activity-relationship) studies, in order to gain new information on the enzymatic inhibition mechanisms. Likewise, early reports on biological activity of L-iminosugars have led to reconsider their therapeutic skills. This review focuses on the most significant discoveries regarding medicinal chemistry of L-iminosugars. The important role L-iminosugars play in unravelling the inhibition mechanisms of specific enzymes is herein recognized; moreover, the high potential of this class of inhibitors as novel drug candidates is under discussion.

Synthesis and biological characterisation of novel N-alkyl-deoxynojirimycin alpha-glucosidase inhibitors.

The N-alkylated deoxynojirimycin compound, N-(6'-(4''-azido-2''-nitrophenylamino)hexyl)-1-deoxynojirimycin (6) was synthesised as a potential photoaffinity probe for endoplasmic reticulum (ER) alpha-glucosidases I and II. Surprisingly this compound was a highly potent inhibitor of alpha-glucosidase I (IC(50), 17 nM) in an in vitro assay and proved equally effective at inhibiting cellular ER glucosidases, as determined by a free oligosaccharide (FOS) analysis. A modest library of compounds was synthesised to obtain structure-activity information by variation of the N-alkyl chain length and modifications to the azido-nitrophenyl group. All of these compounds failed to improve on the efficacy of compound 6, but most showed greater enzyme inhibitory potency than N-butyl-deoxynojirimycin (NB-DNJ), a pharmacological agent that has been evaluated for the treatment of several viruses for which infectivity is dependent on host cell glycosylation.

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 beta-glucosidase (acid beta-Glu, beta-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 beta-Glu. Recently, bicyclic nojirimycin (NJ) analogues with structure of sp2 iminosugars were found to behave as very selective, competitive inhibitors of the lysosomal beta-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 beta-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 beta-Glu mutations, especially for neuronopathic forms of Gaucher disease.

Glycosphingolipid depletion in PC12 cells using iminosugars protects neuronal membranes from anti-ganglioside antibody mediated injury.

Autoimmune neuropathies are frequently associated with pathogenic anti-ganglioside antibodies targeting ganglioside-rich neuronal and glial membranes. The extent of injury is determined by the concentration of membrane ganglioside and thus reduction might be expected to attenuate disease. In this study, we suppressed ganglioside biosynthesis in PC12 cells with the glucosylceramide synthase inhibitor, N-butyldeoxynojirimycin and observed reduced plasma membrane antibody binding and a major neuroprotective effect in complement-mediated lysis assays. These data demonstrate that iminosugar inhibitors, currently used to treat type 1 Gaucher disease, are also of potential value for depleting antigen and thereby suppressing tissue injury in anti-ganglioside antibody-associated neuropathy.