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The COVID-19 pandemic highlights the need for novel antiviral drug discovery approaches that could dramatically shorten timelines from compound discovery to clinical development. At the beginning of the pandemic, repurposing approaches were at the forefront of early research efforts to screen for antiviral activity against SARS-CoV-2 in over 2500 compounds. Here, we report cellular screening results of 100 FDA-approved and experimental compounds against SARS-CoV-2 in the human Calu-3 cell line. We observed 13 compounds showing antiviral activity against SARS-CoV-2, including seven FDA-approved compounds (remdesivir, boceprevir, amiloride, nafamostat, cisplatin, silmitasertib, and miglustat), and six compounds in pre-clinical and clinical development (tarloxotinib, lucerastat (NB-DGJ), MON-DNJ, NAP-DNJ, NN-DGJ and NN-DNJ). Further, we observed that our screening hits include several host-targeting antivirals, namely iminosugars, that are largely non-toxic and offer a large therapeutic window. The most-developed iminosugar MON-DNJ (UV-4B), which has been evaluated in a Phase 1 clinical trial, shows antiviral activity against SARS-CoV-2 wild type as well as alpha, beta, gamma, delta, and Omicron variants. Its activity also extended to another betacoronavirus HCoV OC43, but not alphacoronavirus HCoV 229E. Our cellular screening results add to the body of knowledge on antivirals against coronaviruses and confirm the antiviral efficacy of iminosugars in cellular assays using the human lung-cell line Calu-3., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.

Mucopolysaccharidosis type IIIB is a devastating neurological disease caused by a lack of the lysosomal enzyme, α-N-acetylglucosaminidase (NAGLU), leading to a toxic accumulation of heparan sulfate. Herein we explored a pharmacological chaperone approach to enhance the residual activity of NAGLU in patient fibroblasts. Capitalizing on the three-dimensional structures of two modest homoiminosugar-based NAGLU inhibitors in complex with bacterial homolog of NAGLU, CpGH89, we have synthesized a library of 17 iminosugar C-glycosides mimicking N-acetyl-D-glucosamine and bearing various pseudo-anomeric substituents of both α- and β-configuration. Elaboration of the aglycon moiety results in low micromolar selective inhibitors of human recombinant NAGLU, but surprisingly it is the non-functionalized and wrongly configured β-homoiminosugar that was proved to act as the most promising pharmacological chaperone, promoting a 2.4 fold activity enhancement of mutant NAGLU at its optimal concentration., (© 2021 Wiley-VCH GmbH.)

Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of March 29, 2021, has claimed 2 776 175 lives worldwide. Vaccine development efforts focus on the viral trimeric spike glycoprotein as the main target of the humoral immune response. Viral spikes carry glycans that facilitate immune evasion by shielding specific protein epitopes from antibody neutralization, and antigen efficacy is influenced by spike glycoprotein production in vivo. Therefore, immunogen integrity is important for glycoprotein-based vaccine candidates. Here, we show how site-specific glycosylation differs between virus-derived spikes, wild-type, non-stabilized spikes expressed from a plasmid with a CMV promoter and tPA signal sequence, and commonly used recombinant, engineered spike glycoproteins. Furthermore, we show that their distinctive cellular secretion pathways result in different protein glycosylation and secretion patterns, including shedding of spike monomeric subunits for the non-stabilized wild-type spike tested, which may have implications for the resulting immune response and vaccine design., Competing Interests: The authors declare the following competing financial interest(s): W.B.S. is a shareholder and consultant to Refeyn Ltd. All other authors declare no conflict of interest., (© 2021 The Authors. Published by American Chemical Society.)

UDP-glucose:glycoprotein glucosyltransferase (UGGT) flags misfolded glycoproteins for ER retention. We report crystal structures of full-length Chaetomium thermophilum UGGT (CtUGGT), two CtUGGT double-cysteine mutants, and its TRXL2 domain truncation (CtUGGT-ΔTRXL2). CtUGGT molecular dynamics (MD) simulations capture extended conformations and reveal clamping, bending, and twisting inter-domain movements. We name "Parodi limit" the maximum distance on the same glycoprotein between a site of misfolding and an N-linked glycan that can be reglucosylated by monomeric UGGT in vitro, in response to recognition of misfold at that site. Based on the MD simulations, we estimate the Parodi limit as around 70-80 Å. Frequency distributions of distances between glycoprotein residues and their closest N-linked glycosylation sites in glycoprotein crystal structures suggests relevance of the Parodi limit to UGGT activity in vivo. Our data support a "one-size-fits-all adjustable spanner" UGGT substrate recognition model, with an essential role for the UGGT TRXL2 domain., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc 3 Man 9 GlcNAc 2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo -acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Iminosugars have therapeutic potential against a range of diseases, due to their efficacy as glycosidase inhibitors. A major challenge in the development of iminosugar drugs lies in making a compound that is selective for the glycosidase associated with a given disease. We report the synthesis of ToP-DNJ, an antiviral iminosugar-tocopherol conjugate. Tocopherol was incorporated into the design of the iminosugar in order to direct the drug to the liver and immune cells, specific tissues of interest for antiviral therapy. ToP-DNJ inhibits ER α-glucosidase II at low micromolar concentrations and selectively accumulates in the liver in vivo. In cellular assays, the drug showed efficacy exclusively in immune cells of the myeloid lineage. Taken together, these data demonstrate that inclusion of a native metabolite into an iminosugar provides selectivity with respect to target enzyme, target cell, and target tissue.

Targeting the host-cell endoplasmic reticulum quality control (ERQC) pathway is an effective broad-spectrum antiviral strategy. The two ER resident α-glucosidases whose sequential action permits entry in this pathway are the targets of glucomimetic inhibitors. Knowledge of the molecular details of the ER α-glucosidase II (α-Glu II) structure was limited. We determined crystal structures of a trypsinolytic fragment of murine α-Glu II, alone and in complex with key catalytic cycle ligands, and four different broad-spectrum antiviral iminosugar inhibitors, two of which are currently in clinical trials against dengue fever. The structures highlight novel portions of the enzyme outside its catalytic pocket which contribute to its activity and substrate specificity. These crystal structures and hydrogen-deuterium exchange mass spectrometry of the murine ER alpha glucosidase II heterodimer uncover the quaternary arrangement of the enzyme's α- and β-subunits, and suggest a conformational rearrangement of ER α-Glu II upon association of the enzyme with client glycoproteins.

Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc that terminates in two β-sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first β-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold., Competing Interests: The authors declare no conflict of interest.

Many viruses require the host endoplasmic reticulum protein-folding machinery in order to correctly fold one or more of their glycoproteins. Iminosugars with glucose stereochemistry target the glucosidases which are key for entry into the glycoprotein folding cycle. Viral glycoproteins are thus prevented from interacting with the protein-folding machinery leading to misfolding and an antiviral effect against a wide range of different viral families. As iminosugars target host enzymes, they should be refractory to mutations in the virus. Iminosugars therefore have great potential for development as broad-spectrum antiviral therapeutics. We outline the mechanism giving rise to the antiviral activity of iminosugars, the current progress in the development of iminosugar antivirals and future prospects for this field., (© 2017 The Author(s).)

The antiviral properties of iminosugars have been reported previously in vitro and in small animal models against Ebola virus (EBOV); however, their effects have not been tested in larger animal models such as guinea pigs. We tested the iminosugars N-butyl-deoxynojirimycin (NB-DNJ) and N-(9-methoxynonyl)-1deoxynojirimycin (MON-DNJ) for safety in uninfected animals, and for antiviral efficacy in animals infected with a lethal dose of guinea pig adapted EBOV. 1850 mg/kg/day NB-DNJ and 120 mg/kg/day MON-DNJ administered intravenously, three times daily, caused no adverse effects and were well tolerated. A pilot study treating infected animals three times within an 8 hour period was promising with 1 of 4 infected NB-DNJ treated animals surviving and the remaining three showing improved clinical signs. MON-DNJ showed no protective effects when EBOV-infected guinea pigs were treated. On histopathological examination, animals treated with NB-DNJ had reduced lesion severity in liver and spleen. However, a second study, in which NB-DNJ was administered at equally-spaced 8 hour intervals, could not confirm drug-associated benefits. Neither was any antiviral effect of iminosugars detected in an EBOV glycoprotein pseudotyped virus assay. Overall, this study provides evidence that NB-DNJ and MON-DNJ do not protect guinea pigs from a lethal EBOV-infection at the dose levels and regimens tested. However, the one surviving animal and signs of improvements in three animals of the NB-DNJ treated cohort could indicate that NB-DNJ at these levels may have a marginal beneficial effect. Future work could be focused on the development of more potent iminosugars., Competing Interests: This work has been supported by Oxford Glycobiology Institute Endowment and a research grant from Emergent BioSolutions, Inc. (formerly Unither Virology LLC). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The biosynthesis of enveloped viruses depends heavily on the host cell endoplasmic reticulum (ER) glycoprotein quality control (QC) machinery. This dependency exceeds the dependency of host glycoproteins, offering a window for the targeting of ERQC for the development of broad-spectrum antivirals. We determined small-angle X-ray scattering (SAXS) and crystal structures of the main ERQC enzyme, ER α-glucosidase II (α-GluII; from mouse), alone and in complex with key ligands of its catalytic cycle and antiviral iminosugars, including two that are in clinical trials for the treatment of dengue fever. The SAXS data capture the enzyme's quaternary structure and suggest a conformational rearrangement is needed for the simultaneous binding of a monoglucosylated glycan to both subunits. The X-ray structures with key catalytic cycle intermediates highlight that an insertion between the +1 and +2 subsites contributes to the enzyme's activity and substrate specificity, and reveal that the presence of d-mannose at the +1 subsite renders the acid catalyst less efficient during the cleavage of the monoglucosylated substrate. The complexes with iminosugar antivirals suggest that inhibitors targeting a conserved ring of aromatic residues between the α-GluII +1 and +2 subsites would have increased potency and selectivity, thus providing a template for further rational drug design.

The antiviral activity of UV-4 was previously demonstrated against dengue virus serotype 2 (DENV2) in multiple mouse models. Herein, step-wise minimal effective dose and therapeutic window of efficacy studies of UV-4B (UV-4 hydrochloride salt) were conducted in an antibody-dependent enhancement (ADE) mouse model of severe DENV2 infection in AG129 mice lacking types I and II interferon receptors. Significant survival benefit was demonstrated with 10-20 mg/kg of UV-4B administered thrice daily (TID) for seven days with initiation of treatment up to 48 h after infection. UV-4B also reduced infectious virus production in in vitro antiviral activity assays against all four DENV serotypes, including clinical isolates. A set of purified enzyme, in vitro, and in vivo studies demonstrated that inhibition of endoplasmic reticulum (ER) α-glucosidases and not the glycosphingolipid pathway appears to be responsible for the antiviral activity of UV-4B against DENV. Along with a comprehensive safety package, these and previously published data provided support for an Investigational New Drug (IND) filing and Phases 1 and 2 clinical trials for UV-4B with an indication of acute dengue disease., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Glycosphingolipids (GSLs) are essential constituents of cell membranes and lipid rafts and can modulate signal transduction events. The contribution of GSLs in osteoclast (OC) activation and osteolytic bone diseases in malignancies such as the plasma cell dyscrasia multiple myeloma (MM) is not known. Here, we tested the hypothesis that pathological activation of OCs in MM requires de novo GSL synthesis and is further enhanced by myeloma cell-derived GSLs. Glucosylceramide synthase (GCS) inhibitors, including the clinically approved agent N-butyl-deoxynojirimycin (NB-DNJ), prevented OC development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of transcriptional activator NFATc1. GM3 was the prevailing GSL produced by patient-derived myeloma cells and MM cell lines, and exogenous addition of GM3 synergistically enhanced the ability of the pro-osteoclastogenic factors RANKL and insulin-like growth factor 1 (IGF-1) to induce osteoclastogenesis in precursors. In WT mice, administration of GM3 increased OC numbers and activity, an effect that was reversed by treatment with NB-DNJ. In a murine MM model, treatment with NB-DNJ markedly improved osteolytic bone disease symptoms. Together, these data demonstrate that both tumor-derived and de novo synthesized GSLs influence osteoclastogenesis and suggest that NB-DNJ may reduce pathological OC activation and bone destruction associated with MM.

Iminosugars are capable of targeting the life cycles of multiple viruses by blocking host endoplasmic reticulum α-glucosidase enzymes that are required for competent replication of a variety of enveloped, glycosylated viruses. Iminosugars as a class are approved for use in humans with diseases such as diabetes and Gaucher's disease, providing evidence for safety of this class of compounds. The in vitro antiviral activity of iminosugars has been described in several publications with a subset of these demonstrating in vivo activity against flaviviruses, herpesviruses, retroviruses and filoviruses. Although there is compelling non-clinical in vivo evidence of antiviral efficacy, the efficacy of iminosugars as antivirals has yet to be demonstrated in humans. In the current study, we report a novel iminosugar, UV-12, which has efficacy against dengue and influenza in mouse models. UV-12 exhibits drug-like properties including oral bioavailability and good safety profile in mice and guinea pigs. UV-12 is an example of an iminosugar with activity against multiple virus families that should be investigated in further safety and efficacy studies and demonstrates potential value of this drug class as antiviral therapeutics.

The glycosidase inhibitory properties of synthetic C-alkyl and N-alkyl six-membered iminosugars have been extensively studied leading to therapeutic candidates. The related seven-membered iminocyclitols have been less examined despite the report of promising structures. Using an in house ring enlargement/C-alkylation as well as cross-metathesis methodologies as the key steps, we have undertaken the synthesis and biological evaluation of a library of fourteen 2C- and eight N-alkyl tetrahydroxylated azepanes starting from an easily available glucopyranose-derived azidolactol. Four, six, nine and twelve carbon atom alkyl chains have been introduced. The study of two distinct D-gluco and L-ido stereochemistries for the tetrol pattern as well as R and S configurations for the C-2 carbon bearing the C-alkyl chain is reported. We observed that C-alkylation of the L-ido tetrahydroxylated azepane converts it from an α-L-fucosidase to a β-glucosidase and β-galactosidase inhibitor while N-alkylation of the D-gluco iminosugar significantly improves its inhibition profile leading to potent β-glucosidase, β-galactosidase, α-L-rhamnosidase and β-glucuronidase inhibitors whatever the stereochemistry of the alkyl chain. Interestingly, the N-alkyl chain length usually parallels the azepane inhibitor potency as exemplified by the identification of a potent glucocerebrosidase inhibitor (Ki 1 μM) bearing a twelve carbon atom chain. Additionally, several C-alkyl azepanes demonstrated promising F508del-CFTR correction unlike the parent tetrahydroxyazepanes. None of the C-alkyl and N-alkyl azepanes did inhibit ER α-glucosidases I or II.

Background: UDP-GlcNAc 2-epimerase/ManNAc 6-kinase (GNE) is a bifunctional enzyme responsible for the first committed steps in the synthesis of sialic acid, a common terminal monosaccharide in both protein and lipid glycosylation. GNE mutations are responsible for a rare autosomal recessive neuromuscular disorder, GNE myopathy (also called hereditary inclusion body myopathy). The connection between the impairment of sialic acid synthesis and muscle pathology in GNE myopathy remains poorly understood., Methods: Glycosphingolipid (GSL) analysis was performed by HPLC in multiple models of GNE myopathy, including patients' fibroblasts and plasma, control fibroblasts with inhibited GNE epimerase activity through a novel imino sugar, and tissues of Gne(M712T/M712T) knock-in mice., Results: Not only neutral GSLs, but also sialylated GSLs, were significantly increased compared to controls in all tested models of GNE myopathy. Treatment of GNE myopathy fibroblasts with N-acetylmannosamine (ManNAc), a sialic acid precursor downstream of GNE epimerase activity, ameliorated the increased total GSL concentrations., Conclusion: GNE myopathy models have increased total GSL concentrations. ManNAc supplementation results in decrease of GSL levels, linking abnormal increase of total GSLs in GNE myopathy to defects in the sialic acid biosynthetic pathway. These data advocate for further exploring GSL concentrations as an informative biomarker, not only for GNE myopathy, but also for other disorders of sialic acid metabolism.

Deoxynojirimycin (DNJ) based imino sugars display antiviral activity in the tissue culture surrogate model of Hepatitis C (HCV), bovine viral diarrhoea virus (BVDV), mediated by inhibition of ER α-glucosidases. Here, the antiviral activities of neoglycoconjugates derived from deoxynojirimycin, and a novel compound derived from deoxygalactonojirimycin, by click chemistry with functionalised adamantanes are presented. Their antiviral potency, in terms of both viral infectivity and virion secretion, with respect to their effect on α-glucosidase inhibition, are reported. The distinct correlation between the ability of long alkyl chain derivatives to inhibit ER α-glucosidases and their anti-viral effect is demonstrated. Increasing alkyl linker length between DNJ and triazole groups increases α-glucosidase inhibition and reduces the production of viral progeny RNA and the maturation of the envelope polypeptide. Disruption to viral glycoprotein processing, with increased glucosylation on BVDV E2 species, is representative of α-glucosidase inhibition, whilst derivatives with longer alkyl linkers also show a further decrease in infectivity of secreted virions, an effect proposed to be distinct from α-glucosidase inhibition., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The Ho crossed aldol condensation provides access to a series of carbon branched iminosugars as exemplified by the synthesis of enantiomeric pairs of isoDMDP, isoDGDP, and isoDAB, allowing comparison of their biological activities with three linear isomeric natural products DMDP, DGDP, and DAB and their enantiomers. L-IsoDMDP [(2S,3S,4R)-2,4-bis(hydroxymethyl)pyrrolidine-3,4-diol], prepared in 11 steps in an overall yield of 45% from d-lyxonolactone, is a potent specific competitive inhibitor of gut disaccharidases [K(i) 0.081 μM for rat intestinal maltase] and is more effective in the suppression of hyperglycaemia in a maltose loading test than miglitol, a drug presently used in the treatment of late onset diabetes. The partial rescue of the defective F508del-CFTR function in CF-KM4 cells by L-isoDMDP is compared with miglustat and isoLAB in an approach to the treatment of cystic fibrosis.

Endoplasmic reticulum-associated degradation (ERAD) is a key cellular process whereby misfolded proteins are removed from the endoplasmic reticulum (ER) for subsequent degradation by the ubiquitin/proteasome system. In the present work, analysis of the released, free oligosaccharides (FOS) derived from all glycoproteins undergoing ERAD, has allowed a global estimation of the mechanisms of this pathway rather than following model proteins through degradative routes. Examining the FOS produced in endomannosidase-compromised cells following α-glucosidase inhibition has revealed a mechanism for clearing Golgi-retrieved glycoproteins that have failed to enter the ER quality control cycle. The Glc3Man7GlcNAc2 FOS species has been shown to be produced in the ER lumen by a mechanism involving a peptide: N-glycanase-like activity, and its production was sensitive to disruption of Golgi-ER trafficking. The detection of this oligosaccharide was unaffected by the overexpression of EDEM1 or cytosolic mannosidase, both of which increased the production of previously characterised cytosolically localised FOS. The lumenal FOS identified are therefore distinct in their production and regulation compared to FOS produced by the conventional route of misfolded glycoproteins directly removed from the ER. The production of such lumenal FOS is indicative of a novel degradative route for cellular glycoproteins that may exist under certain conditions.

Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is mediated in part through the activatory Fc receptor, FcγRIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein-carbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for FcγRIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 Å, which revealed localized destabilization of this glycan-protein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on FcγRIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to FcγRIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man5GlcNAc2) glycoform, the individual effect of each mutation on FcγRIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIB). These data indicate that destabilization of the glycan-protein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for FcγR binding. Engineering of the protein-carbohydrate interface thus provides an independent parameter in the engineering of Fc effector functions and a route to the synthesis of new classes of Fc domain with novel combinations of affinities for activatory and inhibitory Fc receptors.

Host cellular endoplasmic reticulum α-glucosidases I and II are essential for the maturation of viral glycosylated envelope proteins that use the calnexin mediated folding pathway. Inhibition of these glycan processing enzymes leads to the misfolding and degradation of these viral glycoproteins and subsequent reduction in virion secretion. We previously reported that, CM-10-18, an imino sugar α-glucosidase inhibitor, efficiently protected the lethality of dengue virus infection of mice. In the current study, through an extensive structure-activity relationship study, we have identified three CM-10-18 derivatives that demonstrated superior in vitro antiviral activity against representative viruses from four viral families causing hemorrhagic fever. Moreover, the three novel imino sugars significantly reduced the mortality of two of the most pathogenic hemorrhagic fever viruses, Marburg virus and Ebola virus, in mice. Our study thus proves the concept that imino sugars are promising drug candidates for the management of viral hemorrhagic fever caused by variety of viruses., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The aim of the present study was to evaluate the ability of the iminosugar drug UV-4 to provide in vivo protection from lethal dengue virus (DENV) challenge. This study utilized a well-described model of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS)-like lethal disease in AG129 mice lacking the type I and II interferon receptors. Herein, we present UV-4 as a potent iminosugar for controlling DENV infection and disease in this mouse model. Specifically, administration of UV-4 reduced mortality, as well as viremia and viral RNA in key tissues, and cytokine storm. In addition, UV-4 treatment can be delayed, and it does not alter the anti-DENV antibody response. These results have set the foundation for development of UV-4 as a DENV-specific antiviral in phase I human clinical trials., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Removal of α-glucose residues from nascent glycoproteins in the early secretory pathway is a requirement for further N-glycan maturation. Although deglucosylation is a stepwise process mediated by endoplasmic reticulum-associated glucosidases I and II for most glycoproteins, Golgi endo-α-mannosidase provides a backup mechanism for glycoprotein deglucosylation. Although conserved in mammals, in certain cell lines, endomannosidase activity in vitro appears to differ from its activity in cells following glucosidase inhibition. Here, we show that in bovine cells this is explained by restricted substrate specificity allowing processing of Glc(1)Man(7)GlcNAc(1/2) and Glc(1)Man(5)GlcNAc(1/2) but not fully glucosylated glycans that build up when glucosidases are inhibited. Our data further demonstrate that such specificity is determined genetically rather than post-translationally. We also demonstrate that the bovine endomannosidase is transcriptionally upregulated by comparison with glucosidase II in Madin-Darby bovine kidney cells and speculate that this is to compensate for the reduced catalytic activity as measured in the recombinant form of the enzyme.

Hydrophilic interaction liquid chromatography (HILIC) of fluorescently labelled oligosaccharides is used in many laboratories to analyse complex oligosaccharide mixtures. Separations are routinely performed using a TSK gel-Amide 80 HPLC column, and retention times of different oligosaccharide species are converted to glucose unit (GU) values that are determined with reference to an external standard. However, if retention times were to be compared with an internal standard, consistent and more accurate GU values would be obtained. We present a method to perform internal standard-calibrated HILIC of fluorescently labelled oligosaccharides. The method relies on co-injection of 4-aminobenzoic acid ethyl ester (4-ABEE)-labelled internal standard and detection by UV absorption, with 2-AA (2-aminobenzoic acid)-labelled oligosaccharides. 4-ABEE is a UV chromophore and a fluorophore, but there is no overlap of the fluorescent spectrum of 4-ABEE with the commonly used fluorescent reagents. The dual nature of 4-ABEE allows for accurate calculation of the delay between UV and fluorescent signals when determining the GU values of individual oligosaccharides. The GU values obtained are inherently more accurate as slight differences in gradients that can influence retention are negated by use of an internal standard. Therefore, this paper provides the first method for determination of HPLC-derived GU values of fluorescently labelled oligosaccharides using an internal calibrant., (Copyright © 2012 Elsevier B.V. All rights reserved.)

N-linked glycans play key roles in protein folding, stability, and function. Biosynthetic modification of N-linked glycans, within the endoplasmic reticulum, features sequential trimming and readornment steps. One unusual enzyme, endo-α-mannosidase, cleaves mannoside linkages internally within an N-linked glycan chain, short circuiting the classical N-glycan biosynthetic pathway. Here, using two bacterial orthologs, we present the first structural and mechanistic dissection of endo-α-mannosidase. Structures solved at resolutions 1.7-2.1 Å reveal a (β/α)(8) barrel fold in which the catalytic center is present in a long substrate-binding groove, consistent with cleavage within the N-glycan chain. Enzymatic cleavage of authentic Glc(1/3)Man(9)GlcNAc(2) yields Glc(1/3)-Man. Using the bespoke substrate α-Glc-1,3-α-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-α-mannosidase inhibitor α-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, α-Glc-1,3-isofagomine, and with the reducing-end product α-1,2-mannobiose structurally define the -2 to +2 subsites of the enzyme. These structural and mechanistic data provide a foundation upon which to develop new enzyme inhibitors targeting the hijacking of N-glycan synthesis in viral disease and cancer.

Noeuromycin is a highly potent albeit unstable glycosidase inhibitor due to its hemiaminal function. While stable D-gluco-like analogs have been reported, no data are available for D-manno-like structures. A series of tri- and tetrahydroxylated seven-membered iminosugars displaying either a D-manno-or a L-gulo-like configuration, were synthesized from methyl α-D-mannopyranoside using a reductive amination-mediated ring expansion as the key step. Screening towards a range of commercial glycosidases demonstrated their potency as competitive glycosidase inhibitors while cellular assay showed selective albeit weak glycoprotein processing mannosidase inactivation., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Valienol-derived allylic C-1 bromides have been used as carbaglycosyl donors for α-xylo configured valienamine pseudodisaccharide synthesis. We synthesised valienamine analogues of the Glc(α1→3)Glc and Glc(α1→3)Man disaccharides representing the linkages cleaved by α-Glucosidase II in N-glycan biosynthesis. These (N1→3)-linked pseudodisaccharides were found to have some α-Glucosidase II inhibitory activity, while two other (N1→6)-linked valienamine pseudodisaccharides failed to inhibit the enzyme., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

During quality control in the ER (endoplasmic reticulum), nascent glycoproteins are deglucosylated by ER glucosidases I and II. In the post-ER compartments, glycoprotein endo-α-mannosidase provides an alternative route for deglucosylation. Previous evidence suggests that endomannosidase non-selectively deglucosylates glycoproteins that escape quality control in the ER, facilitating secretion of aberrantly folded as well as normal glycoproteins. In the present study, we employed FOS (free oligosaccharides) released from degrading glycoproteins as biomarkers of ERAD (ER-associated degradation), allowing us to gain a global rather than single protein-centred view of ERAD. Glucosidase inhibition was used to discriminate between glucosidase- and endomannosidase-mediated ERAD pathways. Endomannosidase expression was manipulated in CHO (Chinese-hamster ovary)-K1 cells, naturally lacking a functional version of the enzyme, and HEK (human embryonic kidney)-293T cells. Endomannosidase was shown to decrease the levels of total FOS, suggesting decreased rates of ERAD. However, following pharmacological inhibition of ER glucosidases I and II, endomannosidase expression resulted in a partial switch between glucosylated FOS, released from ER-confined glycoproteins, to deglucosylated FOS, released from endomannosidase-processed glycoproteins transported from the Golgi/ERGIC (ER/Golgi intermediate compartment) to the ER. Using this approach, we have identified a previously unknown pathway of glycoprotein flow, undetectable by the commonly employed methods, in which secretory cargo is targeted back to the ER after being processed by endomannosidase., (© The Authors Journal compilation © 2011 Biochemical Society)

A series of neoglycoconjugates derived from deoxynojirimycin has been prepared by click connection with functionalised adamantanes. They have been assayed as glycosidase inhibitors, as inhibitors of the glycoenzymes relevant to the treatment of Gaucher disease, as well as correctors of the defective ion-transport protein involved in cystic fibrosis. We have demonstrated that it is possible to selectively either strongly inhibit ER-α-glucosidases and ceramide glucosyltransferase or restore the activity of CFTR in CF-KM4 cells by varying the length of the alkyl chain linking DNJ and adamantane.

The syntheses of 4-C-Me-DAB [1,4-dideoxy-1,4-imino-4-C-methyl-d-arabinitol] from l-erythronolactone and of 4-C-Me-LAB [from d-erythronolactone] require only a single acetonide protecting group. The effect of pH on the NMR spectra of 4-C-Me-DAB [pK(a) of the salt around 8.4] is discussed and illustrates the need for care in analysis of both coupling constants and chemical shift. 4-C-Me-DAB (for rat intestinal sucrase K(i) 0.89 μM, IC(50) 0.41 μM) is a competitive - whereas 4-C-Me-LAB (for rat intestinal sucrase K(i) 0.95 μM, IC(50) 0.66 μM) is a non-competitive - specific and potent α-glucosidase inhibitor. A rationale for the α-glucosidase inhibition by DAB, LAB, 4-C-Me-DAB, 4-C-Me-LAB, and isoDAB - but not isoLAB - is provided. Both are inhibitors of endoplasmic reticulum (ER) resident α-glucosidase I and II.

Small molecule inhibitors of glycoconjugate metabolism are being exploited for therapeutic benefit in a number of human disorders. As examples of this class of compound, imino sugars, as monosaccharide mimics, have a number of advantages for compound design and synthesis to define biological activity. As polyhydroxylated molecules, each chiral centre offers manipulation to generate isomers with restricted or enhanced mimicry, and the endocyclic nitrogen atom is readily modified to gain selectivity, increase potency or improve pharmacodynamics. This review focuses on the discovery of imino sugars that have considerable potential for treating a diverse range of diseases, from lysosomal storage disorders diabetes and cystic fibrosis to viral pathogenesis, and addresses the mechanism of action that is dictated by structural modification.

Diglycose derivatives, consisting of two monosaccharides linked at non-anomeric positions by a bridging nitrogen atom, have been synthesised. Conversion of one of the precursor monosaccharide coupling components into an unsaturated derivative enhances its electrophilicity at the allylic position, facilitating coupling reactions. Mitsunobu coupling between nosylamides and 2,3-unsaturated-4-alcohols gave the 4-amino-pseudodisaccharides with inversion of configuration as single regio- and diastereoisomers. A palladium-catalysed coupling between an amine and a 2,3-unsaturated 4-trichloroacetimidate gave a 2-amino-pseudodisaccharide as the major product, along with other minor products. Derivatisation of the C=C double bond in pseudodisaccharides allowed the formation of Man(N4-6)Glc and Man(N4-6)Man diglycosides. The amine-linked diglycosides were found to show weak glycosidase inhibitory activity.

Robust assays for the isolation and characterization of urinary FOS (free oligosaccharides) have been developed to screen patients for altered protein and/or lipid glycosylation. A FOS analysis can therefore identify potential biomarkers for hepatocellular carcinoma, since variations in glycosylation as a result of tumorigenecity should be detectable in the FOS of patients. HCC (hepatocellular carcinoma) accounts for 80-90% of all liver cancers. It occurs more often in men than women and occurs mostly in people 50-60 years old. The disease is more common in parts of Africa and Asia than in North or South America and Europe. Using a combination of solid-phase extraction techniques and affinity chromatography, followed by separation of urinary FOS by NP (normal phase)-HPLC and HIAX (hydrophilic interaction and anion-exchange)-HPLC, more than 200 different species have been identified in patient samples. The high incidence of small sialylated oligosaccharides in HCC patients suggests that pro-inflammatory markers may be detected as early indicators of disease progression. In addition, the methods developed here to isolate and analyse excreted glycoprotein- and glycosphingolipid-bound oligosaccharides have been used to characterize changes in metabolic processes that underlie a number of human genetic disorders. The ability to predict disease status in microlitre amounts of readily available non-invasive urine samples indicates that rapid methods for screening can be developed.

Multiple isoforms of mammalian alpha-mannosidases are active in the pathways of N-linked glycoprotein synthesis and catabolism. They differ in specificity, function and location within the cell and can be selectively inhibited by imino sugar monosaccharide mimics. Previously, a series of structurally related novel 7-membered iminocyclitols were synthesised and found to be inhibitors of alpha-mannosidase using in vitro assays. The present study aimed to delineate alpha-mannosidases hydrolytic pathways in azepane inhibitor treated cells by the analysis of free oligosaccharides (FOS) as markers of endoplasmic reticulum (ER), Golgi, lysosomal and cytosolic alpha-mannosidase activities. Two compounds were identified as potent and selective cytosolic alpha-mannosidase inhibitors. Two related compounds were shown to be potent inhibitors of lysosomal alpha-mannosidase with different potencies towards alpha1,6 mannosidase. The specificities of these novel 7-membered imino sugars are related to differences in their structure and D: -mannose-like stereochemistry. Specific ER-mannosidase inhibition by kifunensine also reveals significant non-proteasomal degradation following FOS analysis and appears to be cell line dependent. The availability of more selective inhibitors allows the pathways of N-linked oligosaccharide metabolism to be dissected.

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.

Analogues of the alpha-Glcp-(1-->3)-alpha-Glcp and alpha-Glcp-(1-->3)-alpha-Manp disaccharides (representing the two alpha-gluco linkages cleaved by alpha-Glucosidase II in N-glycan biosynthesis) in which the non-reducing-end sugar is replaced by a carbasugar and the inter-glycosidic oxygen by a sulfur were synthesised. The key coupling step was an S(N)2 displacement of an equatorial triflate at C-1 of the carbasugar by C-3 gluco or manno thiolates with inversion of configuration to give thioether pseudodisaccharides with axial substitution at C-1 of the carbasugar. The deprotected pseudodisaccharides failed to inhibit the action of alpha-Glucosidase II as measured both by an in vitro assay and by free oligosaccharide (FOS) analysis from cell studies.

The inhibition of ER (endoplasmic reticulum) alpha-glucosidases I and II by imino sugars, including NB-DNJ (N-butyl-deoxynojirimycin), causes the retention of glucose residues on N-linked oligosaccharides. Therefore, normal glycoprotein trafficking and processing through the glycosylation pathway is abrogated and glycoproteins are directed to undergo ERAD (ER-associated degradation), a consequence of which is the production of cytosolic FOS (free oligosaccharides). Following treatment with NB-DNJ, FOS were extracted from cells, murine tissues and human plasma and urine. Improved protocols for analysis were developed using ion-exchange chromatography followed by fluorescent labelling with 2-AA (2-aminobenzoic acid) and purification by lectin-affinity chromatography. Separation of 2-AA-labelled FOS by HPLC provided a rapid and sensitive method that enabled the detection of all FOS species resulting from the degradation of glycoproteins exported from the ER. The generation of oligosaccharides derived from glucosylated protein degradation was rapid, reversible, and time- and inhibitor concentration-dependent in cultured cells and in vivo. Long-term inhibition in cultured cells and in vivo indicated a slow rate of clearance of glucosylated FOS. In mouse and human urine, glucosylated FOS were detected as a result of transrenal excretion and provide unique and quantifiable biomarkers of ER-glucosidase inhibition.