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It has long been thought that iminosugar antiviral activity is a function of inhibition of endoplasmic reticulum-resident α-glucosidases, and on this basis, many iminosugars have been investigated as therapeutic agents for treatment of infection by a diverse spectrum of viruses, including dengue virus (DENV). However, iminosugars are glycomimetics possessing a nitrogen atom in place of the endocyclic oxygen atom, and the ubiquity of glycans in host metabolism suggests that multiple pathways can be targeted via iminosugar treatment. Successful treatment of patients with glycolipid processing defects using iminosugars highlights the clinical exploitation of iminosugar inhibition of enzymes other than ER α-glucosidases. Evidence correlating antiviral activity with successful inhibition of ER glucosidases together with the exclusion of alternative mechanisms of action of iminosugars in the context of DENV infection is limited. Celgosivir, a bicyclic iminosugar evaluated in phase Ib clinical trials as a therapeutic for the treatment of DENV infection, was confirmed to be antiviral in a lethal mouse model of antibody-enhanced DENV infection. In this study we provide the first evidence of the antiviral activity of celgosivir in primary human macrophages in vitro, in which it inhibits DENV secretion with an EC50 of 5 μM. We further demonstrate that monocyclic glucose-mimicking iminosugars inhibit isolated glycoprotein and glycolipid processing enzymes and that this inhibition also occurs in primary cells treated with these drugs. By comparison to bicyclic glucose-mimicking iminosugars which inhibit glycoprotein processing but do not inhibit glycolipid processing and galactose-mimicking iminosugars which do not inhibit glycoprotein processing but do inhibit glycolipid processing, we demonstrate that inhibition of endoplasmic reticulum-resident α-glucosidases, not glycolipid processing, is responsible for iminosugar antiviral activity against DENV. Our data suggest that inhibition of ER α-glucosidases prevents release of virus and is the primary antiviral mechanism of action of iminosugars against DENV.

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.

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.)

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.

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.

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 Glc1/3Man9GlcNAc2 yields Glc1/3-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] Three imino sugars (IHVR11029, 17028 and 19029), were identified through SAR study of 120 derivatives. [•] These three imino sugars demonstrated broad antiviral activities against HFVs in vitro, and in vivo. [•] All three compounds inhibited ER α-glucosidases in vitro and in vivo. [Copyright &y& Elsevier]