(5S)-5-Benzylswainsonines as potent and selective inhibitors of Golgi α-mannosidase II: synthesis, enzyme evaluation and molecular modelling.

Development of novel anti-cancer therapeutics based on Golgi α-mannosidase II (GMII) inhibition is considerably impeded by an undesired co-inhibition of lysosomal α-mannosidase leading to severe side-effects. In this contribution, we describe a fully stereoselective synthesis of (5S)-5-[4-(halo)benzyl]swainsonines as highly potent and selective inhibitors of GMII. The synthesis starts from a previously reported aldehyde readily available from l-ribose, and the key features include an intramolecular reductive amination with substrate-controlled stereoselectivity and a late-stage derivatisation of the benzyl group via ipso-substitution. These novel swainsonine analogues were found to be nanomolar inhibitors of the Golgi-type α-mannosidase AMAN-2 (Ki = 23-75 nM) with excellent selectivity (selectivity index = 205-870) over the lysosomal-type Jack bean α-mannosidase. Finally, molecular docking and pKa calculations were performed to provide more insight into the structure of the inhibitor:enzyme complexes, and a pair interaction energy analysis (FMO-PIEDA) was carried out to rationalise the observed potency and selectivity of the inhibitors.

Carbohydrate polymer degradation derivatives as possible natural mannanase inhibitors.

The role of mannanases is diverse and they are used in many industrial applications, in animal feed, in the food industry and in healthcare. They are also applied in biomass processing, because they play an important role in the breakdown of hemicellulose. Among the mannanase inhibitors, heavy metal ions and general enzyme inhibitors are mainly mentioned. Unfortunately, almost no data are available on carbohydrate-based natural inhibitors of mannanases. According to the literature, carbohydrates do not play an important role in the inhibition of mannanases, so neither do oligosaccharides. This is in contrast to the action and inhibition of other O-glycosyl hydrolases. My hypothesis is that mannanases, like other polysaccharide-degrading enzymes, work in the same way and can be inhibited by oligosaccharides. Evidence from docking and modeling results supports and makes probable the hypothesis that oligosaccharides can inhibit the activity of mannanases, similar to the inhibition of other O-glycosyl hydrolases. Among natural carbohydrate oligomers, several potential mannanase inhibitors have been identified and characterized. In addition to expensive research, it is very important to use research based on cheaper modeling to explore the processes. The results obtained are novel and forward-looking, enabling in-depth and targeted research to be carried out.

Golgi a-mannosidase II mediates the formation of vascular smooth muscle foam cells under inflammatory stress.

INTRODUCTION: Vascular smooth muscle cells (VSMCs)-based foam cell formation is a crucial factor in the atherosclerosis process. We aimed to explore the mechanism of Golgi a-mannosidase II (GMII) effects on the VSMCs-based foam cell formation. MATERIAL AND METHODS: VSMCs were exposed to different concentrations of low-density lipoproteins (LDLs), lipopolysaccharide (LPS), and/or GMII inhibitor (swainsonine). The qRT-PCR and western blot were used for expression analysis. Oil Red O staining was used to verify changes of lipid droplets in VSMCs. The translocation of the SCAP from the endoplasmic reticulum (ER) to Golgi was detected by immunofluorescence (IF). RESULTS: LPS disrupted the LDLs-mediated regulation of LDL receptor (LDLr) and increased intracellular cholesterol ester, which was inversely inhibited by swainsonine. The activity of a-mannosidase II and GMII expression were decreased by LDLs but increased by the addition of LPS. Conversely, LPS-induced enhancement was reversed by swainsonine. Additionally, swainsonine reversed the LPS-induced increase of intracellular lipid droplets in the presence of LDLs. Expression analysis demonstrated that LDLr, SCAP, and SREBP2 were up-regulated by LPS, but reversed by swainsonine in LDLs-treated cells. IF staining revealed that swainsonine inhibited the translocation of SCAP to Golgi under inflammatory stress. CONCLUSIONS: Collectively, swainsonine restrained LDLr expression to suppress the formation of VSMCs-based foam cells by reducing SREBP2 and SCAP under inflammatory stress conditions, suggesting that GMII contributes to the formation of VSMCs-based foam cells under inflammatory stress.

Functionalized High Mannose-Specific Lectins for the Discovery of Type†I Mannosidase Inhibitors.

An engineered cyanovirin-N homologue that exhibits specificity for high mannose N-glycans has been constructed to aid type I α 1,2-mannosidase inhibitor discovery and development. Engineering the lectins C-terminus permitted facile functionalization with fluorophores via a sortase and click strategy. The resulting lectin constructs exhibit specificity for cells presenting high mannose N-glycans. Importantly, these lectin constructs can also be applied to specifically assess changes in cell surface glycosylation induced by type I mannosidase inhibitors. Testing the utility of these lectin constructs led to the discovery of type I mannosidase inhibitors with nanomolar potency. Cumulatively, these findings reveal the specificity and utility of the functionalized cyanovirin-N homologue constructs, and highlight their potential in analytical contexts that require high mannose-specific lectins.

A class of low-cost alternatives to kifunensine for increasing high mannose N-linked glycosylation for monoclonal antibody production in Chinese hamster ovary cells.

N-linked glycosylation of therapeutic monoclonal antibodies is an important product quality attribute for drug safety and efficacy. An increase in the percent of high mannose N-linked glycosylation may be required for drug efficacy or to match the glycosylation profile of the innovator drug during the development of a biosimilar. In this study, the addition of several chemical additives to a cell culture process resulted in high mannose N-glycans on monoclonal antibodies produced by Chinese hamster ovary (CHO) cells without impacting cell culture performance. The additives, which include known mannosidase inhibitors (kifunensine and deoxymannojirimycin) as well as novel inhibitors (tris, bis-tris, and 1-amino-1-methyl-1,3-propanediol), contain one similar molecular structure: 2-amino-1,3-propanediol, commonly referred to as serinol. The shared chemical structure provides insight into the binding and inhibition of mannosidase in CHO cells. One of the novel inhibitors, tris, is safer compared to kifunensine, 35x as cost-effective, and stable at room temperature. In addition, tris and bis-tris provide multiple low-cost alternatives to kifunensine for manipulating glycosylation in monoclonal antibody production in a cell culture process with minimal impact to productivity or cell health.

Manno-epi-cyclophellitols Enable Activity-Based Protein Profiling of Human α-Mannosidases and Discovery of New Golgi Mannosidase II Inhibitors.

Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitol aziridine derivatives enabled activity-based protein profiling of α-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.

Synthesis and mannosidase inhibitory profile of a small library of aminocyclitols from shikimic acid-derived scaffolds.

A short synthetic route to a small library of aminocyclitols 14·HCl-19·HCl has been elaborated from the common shikimic acid-derived scaffolds 20 and 21. The developed strategy features three oxidative processes ‒ ozonolysis, dihydroxylation and epoxidation ‒ as the key transformations. The stereochemistry of the newly created stereocentres was confirmed either via crystallographic analysis or by means of NOESY experiments conducted on advanced intermediates. Glycosidase inhibition study revealed no glucosidase inhibition and only weak inhibitory activity against recombinant Drosophila melanogaster Golgi mannosidase (GMIIb).

A novel Golgi mannosidase inhibitor: Molecular design, synthesis, enzyme inhibition, and inhibition of spheroid formation.

Effective chemotherapy for solid cancers is challenging due to a limitation in permeation that prevents anticancer drugs from reaching the center of the tumor, therefore unable to limit cancer cell growth. To circumvent this issue, we planned to apply the drugs directly at the center by first collapsing the outer structure. For this, we focused on cell-cell communication (CCC) between N-glycans and proteins at the tumor cell surface. Mature N-glycans establish CCC; however, CCC is hindered when numerous immature N-glycans are present at the cell surface. Inhibition of Golgi mannosidases (GMs) results in the transport of immature N-glycans to the cell surface. This can be employed to disrupt CCC. Here, we describe the molecular design and synthesis of an improved GM inhibitor with a non-sugar mimic scaffold that was screened from a compound library. The synthesized compounds were tested for enzyme inhibition ability and inhibition of spheroid formation using cell-based methods. Most of the compounds designed and synthesized exhibited GM inhibition at the cellular level. Of those, AR524 had higher inhibitory activity than a known GM inhibitor, kifunensine. Moreover, AR524 inhibited spheroid formation of human malignant cells at low concentration (10 µM), based on the disruption of CCC by GM inhibition.

In Vivo Glycan Engineering via the Mannosidase I Inhibitor (Kifunensine) Improves Efficacy of Rituximab Manufactured in Nicotiana benthamiana Plants.

N-glycosylation has been shown to affect the pharmacokinetic properties of several classes of biologics, including monoclonal antibodies, blood factors, and lysosomal enzymes. In the last two decades, N-glycan engineering has been employed to achieve a N-glycosylation profile that is either more consistent or aligned with a specific improved activity (i.e., effector function or serum half-life). In particular, attention has focused on engineering processes in vivo or in vitro to alter the structure of the N-glycosylation of the Fc region of anti-cancer monoclonal antibodies in order to increase antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we applied the mannosidase I inhibitor kifunensine to the Nicotiana benthamiana transient expression platform to produce an afucosylated anti-CD20 antibody (rituximab). We determined the optimal concentration of kifunensine used in the infiltration solution, 0.375 µM, which was sufficient to produce exclusively oligomannose glycoforms, at a concentration 14 times lower than previously published levels. The resulting afucosylated rituximab revealed a 14-fold increase in ADCC activity targeting the lymphoma cell line Wil2-S when compared with rituximab produced in the absence of kifunensine. When applied to the cost-effective and scalable N. benthamiana transient expression platform, the use of kifunensine allows simple in-process glycan engineering without the need for transgenic hosts.

N-glycan Remodeling Using Mannosidase Inhibitors to Increase High-mannose Glycans on Acid α-Glucosidase in Transgenic Rice Cell Cultures.

Glycoengineering of plant expression systems is a prerequisite for the production of biopharmaceuticals that are compatible with animal-derived glycoproteins. Large amounts of high-mannose glycans such as Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 (Man7/8/9), which can be favorably modified by chemical conjugation of mannose-6-phosphate, are desirable for lysosomal enzyme targeting. This study proposed a rice cell-based glycoengineering strategy using two different mannosidase inhibitors, kifunensine (KIF) and swainsonine (SWA), to increase Man7/8/9 glycoforms of recombinant human acid α-glucosidase (rhGAA), which is a therapeutic enzyme for Pompe disease. Response surface methodology was used to investigate the effects of the mannosidase inhibitors and to evaluate the synergistic effect of glycoengineering on rhGAA. Both inhibitors suppressed formation of plant-specific complex and paucimannose type N-glycans. SWA increased hybrid type glycans while KIF significantly increased Man7/8/9. Interestingly, the combination of KIF and SWA more effectively enhanced synthesis of Man7/8/9, especially Man9, than KIF alone. These changes show that SWA in combination with KIF more efficiently inhibited ER α-mannosidase II, resulting in a synergistic effect on synthesis of Man7/8/9. In conclusion, combined KIF and SWA treatment in rice cell culture media can be an effective method for the production of rhGAA displaying dominantly Man7/8/9 glycoforms without genetic manipulation of glycosylation.

N-Benzyl Substitution of Polyhydroxypyrrolidines: The Way to Selective Inhibitors of Golgi α-Mannosidase†II.

Inhibition of the biosynthesis of complex N-glycans in the Golgi apparatus influences progress of tumor growth and metastasis. Golgi α-mannosidase II (GMII) has become a therapeutic target for drugs with anticancer activities. One critical task for successful application of GMII drugs in medical treatments is to decrease their unwanted co-inhibition of lysosomal α-mannosidase (LMan), a weakness of all known potent GMII inhibitors. A series of novel N-substituted polyhydroxypyrrolidines was synthesized and tested with modeled GH38 α-mannosidases from Drosophila melanogaster (GMIIb and LManII). The most potent structures inhibited GMIIb (Ki =50-76 µm, as determined by enzyme assays) with a significant selectivity index of IC50 (LManII)/IC50 (GMIIb) >100. These compounds also showed inhibitory activities in in vitro assays with cancer cell lines (leukemia, IC50 =92-200 µm) and low cytotoxic activities in normal fibroblast cell lines (IC50 >200 µm). In addition, they did not show any significant inhibitory activity toward GH47 Aspergillus saitoiα1,2-mannosidase. An appropriate stereo configuration of hydroxymethyl and benzyl functional groups on the pyrrolidine ring of the inhibitor may lead to an inhibitor with the required selectivity for the active site of a target α-mannosidase.

An atypical interaction explains the high-affinity of a non-hydrolyzable S-linked 1,6-α-mannanase inhibitor.

The non-hydrolyzable S-linked azasugars, 1,6-α-mannosylthio- and 1,6-α-mannobiosylthioisofagomine, were synthesized and shown to bind with high affinity to a family 76 endo-1,6-α-mannanase from Bacillus circulans. X-ray crystallography showed an atypical interaction of the isofagomine nitrogen with the catalytic acid/base. Molecular dynamics simulations reveal that the atypical binding results from sulfur perturbing the most stable form away from the nucleophile interaction preferred for the O-linked congener.

Enterocyte glycosylation is responsive to changes in extracellular conditions: implications for membrane functions.

Epithelial cells in the lining of the intestines play critical roles in maintaining homeostasis while challenged by dynamic and sudden changes in luminal contents. Given the high density of glycosylation that encompasses their extracellular surface, environmental changes may lead to extensive reorganization of membrane-associated glycans. However, neither the molecular details nor the consequences of conditional glycan changes are well understood. Here we assessed the sensitivity of Caco-2 and HT-29 membrane N-glycosylation to variations in (i) dietary elements, (ii) microbial fermentation products and (iii) cell culture parameters relevant to intestinal epithelial cell growth and survival. Based on global LC-MS glycomic and statistical analyses, the resulting glycan expression changes were systematic, dependent upon the conditions of each controlled environment. Exposure to short chain fatty acids produced significant increases in fucosylation while further acidification promoted hypersialylation. Notably, among all conditions, increases of high mannose type glycans were identified as a major response when extracellular fructose, galactose and glutamine were independently elevated. To examine the functional consequences of this discrete shift in the displayed glycome, we applied a chemical inhibitor of the glycan processing mannosidase, globally intensifying high mannose expression. The data reveal that upregulation of high mannose glycosylation has detrimental effects on basic intestinal epithelium functions by altering permeability, host-microbe associations and membrane protein activities.

Selective Manipulation of Discrete Mannosidase Activities in the Endoplasmic Reticulum by Using Reciprocally Selective Inhibitors.

Within the endoplasmic reticulum, immature glycoproteins are sorted into secretion and degradation pathways through the sequential trimming of mannose residues from Man9 GlcNAc2 to Man5 GlcNAc2 by the combined actions of assorted α-1,2-mannosidases. It has been speculated that specific glycoforms encode signals for secretion and degradation. However, it is unclear whether the specific signal glycoforms are produced by random mannosidase action or are produced regioselectively in a sequenced manner by specific α-1,2-mannosidases. Here, we report the identification of a set of selective mannosidase inhibitors and development of conditions for their use that enable production of distinct pools of Man8 GlcNAc2 isomers from a structurally defined synthetic Man9 GlcNAc2 substrate in an endoplasmic reticulum fraction. Glycan processing analysis with these inhibitors provides the first biochemical evidence for selective production of the signal glycoforms contributing to traffic control in glycoprotein quality control.

Conformational Behaviour of Azasugars Based on Mannuronic Acid.

A set of mannuronic-acid-based iminosugars, consisting of the C-5-carboxylic acid, methyl ester and amide analogues of 1deoxymannorjirimicin (DMJ), was synthesised and their pH-dependent conformational behaviour was studied. Under acidic conditions the methyl ester and the carboxylic acid adopted an "inverted" 1 C4 chair conformation as opposed to the "normal" 4 C1 chair at basic pH. This conformational change is explained in terms of the stereoelectronic effects of the ring substituents and it parallels the behaviour of the mannuronic acid ester oxocarbenium ion. Because of this solution-phase behaviour, the mannuronic acid ester azasugar was examined as an inhibitor for a Caulobacter GH47 mannosidase that hydrolyses its substrates by way of a reaction itinerary that proceeds through a 3 H4 transition state. No binding was observed for the mannuronic acid ester azasugar, but sub-atomic resolution data were obtained for the DMJ⋅CkGH47 complex, showing two conformations-3 S1 and 1 C4 -for the DMJ inhibitor.

The Endoplasmic Reticulum Alpha-Glycosidases as Potential Targets for Virus Control.

Protein glycosylation is a widely distributed posttranslational modification, though not exclusive to eukaryotic cells. The addition of glycans to proteins plays crucial roles in protein folding and secretion, cell-cell interaction, functional specificity and structural properties of both secreted and membrane-bound proteins. In this review, we emphasize the N-linked glycosylation pathway found in eukaryotic cells, the contribution of processing α-glycosidases, and the use of such enzymes as potential drug targets to control some medically relevant viral infections. Thus far, some inhibitors of the endoplasmic reticulum α -glucosidases such as castanospermine, 1-deoxyjirimycin and derivative molecules have been shown to control viral particles in both in vitro and in vivo models. Nonetheless, the mechanism used for these molecules to inhibit specific viral groups, without affecting the host cells, remains unknown. Furthermore, certain α-mannosidase inhibitors have proven to be helpful in cancer therapy, either improving the sensitivity to chemotherapeutic drugs or reducing metastasis of the tumor. Undeniably promising, the use of α-glycosidase inhibitors rises as an alternative to control both viral infections and cancer. Despite the significant progress in the field, it remains to be demonstrated whether those inhibitors are good candidates to control other pathogens and if so, a careful treatment of the data must be done before extrapolating their use to other systems.

Contribution of Shape and Charge to the Inhibition of a Family GH99 endo-α-1,2-Mannanase.

Inhibitor design incorporating features of the reaction coordinate and transition-state structure has emerged as a powerful approach for the development of enzyme inhibitors. Such inhibitors find use as mechanistic probes, chemical biology tools, and therapeutics. Endo-α-1,2-mannosidases and endo-α-1,2-mannanases, members of glycoside hydrolase family 99 (GH99), are interesting targets for inhibitor development as they play key roles in N-glycan maturation and microbiotal yeast mannan degradation, respectively. These enzymes are proposed to act via a 1,2-anhydrosugar "epoxide" mechanism that proceeds through an unusual conformational itinerary. Here, we explore how shape and charge contribute to binding of diverse inhibitors of these enzymes. We report the synthesis of neutral dideoxy, glucal and cyclohexenyl disaccharide inhibitors, their binding to GH99 endo-α-1,2-mannanases, and their structural analysis by X-ray crystallography. Quantum mechanical calculations of the free energy landscapes reveal how the neutral inhibitors provide shape but not charge mimicry of the proposed intermediate and transition state structures. Building upon the knowledge of shape and charge contributions to inhibition of family GH99 enzymes, we design and synthesize α-Man-1,3-noeuromycin, which is revealed to be the most potent inhibitor (KD 13 nM for Bacteroides xylanisolvens GH99 enzyme) of these enzymes yet reported. This work reveals how shape and charge mimicry of transition state features can enable the rational design of potent inhibitors.

The toxicology mechanism of endophytic fungus and swainsonine in locoweed.

Locoweed is a perennial herbaceous plant included in Astragalus spp. and Oxytropis spp. that contains the toxic indolizidine alkaloid swainsonine. The livestock that consume locoweed can suffer from a type of toxicity called locoism. There are aliphaticnitro compounds, selenium, selenium compounds, and alkaloids in locoweed. The toxic component in locoweed has been identified as swainsonine, an indolizidine alkaloid. Swainsonine inhibits lysosomal a-mannosidase and mannosidase II, resulting in altered oligosaccharide degradation and incomplete glycoprotein processing. Corresponding studies on endophytic fungi producing swainsonine have been isolated from a variety of locoweed, and these endophytic fungi and locoweed have a close relationship. Endophytic fungi can promote the growth of locoweed and increase swainsonine production. As a result, livestock that consume locoweed exhibit several symptoms, including dispirited behavior, staggering gait, chromatopsia, trembling, ataxia, and cellular vacuolar degeneration of most tissues by pathological observation. Locoism results in significant annual economic losses. Therefore, in this paper, we review the current research on locoweed, including that on locoweed species distribution in China, endophyte fungus in locoweed, the toxicology mechanism of locoweed, and the swainsonine effect on reproduction.

Synthesis of modified D-mannose core derivatives and their impact on GH38 α-mannosidases.

Nine new compounds having five- and modified six-member carbohydrate core derived from D-lyxose or D-mannose, and non-hydrolysable aglycones (benzylsulfonyl or aryl(alkyl)triazolyl) were synthesised to investigate their ability to inhibit the recombinant Drosophila melanogaster homologs of two human GH38 family enzymes: Golgi mannosidase II (dGMIIb) and lysosomal mannosidase (dLMII). Two compounds were weak selective dGMIIb inhibitors showing IC50 at mM level. Moreover, it was found that another GH38 enzyme, commercial jack bean α-mannosidase, was inhibited by triazole conjugates regardless of the carbohydrate core while the corresponding sulfones were inactive.

A swainsonine survey of North American Astragalus and Oxytropis taxa implicated as locoweeds.

Swainsonine, an indolizidine alkaloid with significant physiological activity, is an α-mannosidase and mannosidase II inhibitor that causes lysosomal storage disease and alters glycoprotein processing. Swainsonine is found in a number of plant species worldwide, and causes severe toxicosis in livestock grazing these plants, leading to a chronic wasting disease characterized by weight loss, depression, altered behavior, decreased libido, infertility, and death. Swainsonine has been detected in 19 Astragalus and 2 Oxytropis species in North America by thin layer chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry and a jack bean α-mannosidase inhibition assay. In addition, 5 species in North America are presumed to contain swainsonine based upon reports from field cases. Many of these plant species have not been analyzed for swainsonine using modern instrumentation such as gas or liquid chromatography coupled with mass spectrometry. To provide clarification, 22 Astragalus species representing 93 taxa and 4 Oxytropis species representing 18 taxa were screened for swainsonine using both liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Swainsonine was detected in 48 Astragalus taxa representing 13 species and 5 Oxytropis taxa representing 4 species. Forty of the fifty-three swainsonine-positive taxa had not been determined to contain swainsonine previously using liquid or gas chromatography coupled with mass spectrometry. The list of swainsonine-containing taxa reported here will serve as a reference for risk assessment and diagnostic purposes.