Pathogen-induced inflammation is attenuated by the iminosugar MON-DNJ via modulation of the unfolded protein response.

Sepsis is a life-threatening condition involving a dysregulated immune response to infectious agents that cause injury to host tissues and organs. Current treatments are limited to early administration of antibiotics and supportive care. While appealing, the strategy of targeted inhibition of individual molecules in the inflammatory cascade has not proved beneficial. Non-targeted, systemic immunosuppression with steroids has shown limited efficacy and raises concern for secondary infection. Iminosugars are a class of small molecule glycomimetics with distinct inhibition profiles for glycan processing enzymes based on stereochemistry. Inhibition of host endoplasmic reticulum resident glycoprotein processing enzymes has demonstrated efficacy as a broad-spectrum antiviral strategy, but limited consideration has been given to the effects on host glycoprotein production and consequent disruption of signalling cascades. This work demonstrates that iminosugars inhibit dengue virus, bacterial lipopolysaccharide and fungal antigen-stimulated cytokine responses in human macrophages. In spite of decreased inflammatory mediator production, viral replication is suppressed in the presence of iminosugar. Transcriptome analysis reveals the key interaction of pathogen-induced endoplasmic reticulum stress, the resulting unfolded protein response and inflammation. Our work shows that iminosugars modulate these interactions. Based on these findings, we propose a new therapeutic role for iminosugars as treatment for sepsis-related inflammatory disorders associated with excess cytokine secretion.

Mechanisms of Antiviral Activity of Iminosugars Against Dengue Virus.

The antiviral mechanism of action of iminosugars against many enveloped viruses, including dengue virus (DENV), HIV, influenza and hepatitis C virus, is believed to be mediated by inducing misfolding of viral N-linked glycoproteins through inhibition of host endoplasmic reticulum-resident α-glucosidase enzymes. This leads to reduced secretion and/or infectivity of virions and hence lower viral titres, both in vitro and in vivo. Free oligosaccharide analysis from iminosugar-treated cells shows that antiviral activity correlates with production of mono- and tri-glucosylated sugars, indicative of inhibition of ER α-glucosidases. We demonstrate that glucose-mimicking iminosugars inhibit isolated glycoprotein and glycolipid processing enzymes and that this inhibition also occurs in primary cells treated with these drugs. Galactose-mimicking iminosugars that have been tested do not inhibit glycoprotein processing but do inhibit glycolipid processing, and are not antiviral against DENV. By comparison, the antiviral activity of glucose-mimetic iminosugars that inhibit endoplasmic reticulum-resident α-glucosidases, but not glycolipid processing, demonstrates that inhibition of α-glucosidases is responsible for iminosugar antiviral activity against DENV. This monograph will review the investigations of many researchers into the mechanisms of action of iminosugars and the contribution of our current understanding of these mechanisms for optimising clinical delivery of iminosugars. The effects of iminosugars on enzymes other than glucosidases, the induction of ER stress and viral receptors will be also put into context. Data suggest that inhibition of α-glucosidases results in inhibited release of virus and is the primary antiviral mechanism of action of iminosugars against DENV.

Exploring the Potential of Iminosugars as Antivirals for Crimean-Congo Haemorrhagic Fever Virus, Using the Surrogate Hazara Virus: Liquid-Chromatography-Based Mapping of Viral N-Glycosylation and In Vitro Antiviral Assays.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a pathogen of increasing public health concern, being a widely distributed arbovirus and the causative agent of the potentially fatal Crimean-Congo haemorrhagic fever. Hazara virus (HAZV) is a genetically and serologically related virus that has been proposed as a surrogate for antiviral and vaccine testing for CCHFV. Glycosylation analysis of HAZV has been limited; first, we confirmed for the first time the occupation of two N-glycosylation sites in the HAZV glycoprotein. Despite this, there was no apparent antiviral efficacy of a panel of iminosugars against HAZV, as determined by quantification of the total secretion and infectious virus titres produced following infection of SW13 and Vero cells. This lack of efficacy was not due to an inability of deoxynojirimycin (DNJ)-derivative iminosugars to access and inhibit endoplasmic reticulum α-glucosidases, as demonstrated by free oligosaccharide analysis in uninfected and infected SW13 and uninfected Vero cells. Even so, iminosugars may yet have potential as antivirals for CCHFV since the positions and importance of N-linked glycans may differ between the viruses, a hypothesis requiring further evaluation.

Antiviral effects of deoxynojirimycin (DNJ)-based iminosugars in dengue virus-infected primary dendritic cells.

Dendritic cells (DCs) are important targets for dengue virus (DENV) infection and play a significant role in the early immune response. Antiviral effects of iminosugars against DENV in primary cells have been demonstrated previously in monocyte-derived macrophages (MDMΦs). Given the important role played by DCs in innate immune defense against DENV, the antiviral effects of three deoxynojirimycin (DNJ) derivatives (NN-DNJ, EOO-DNJ and 2THO-DNJ) and a deoxygalactonojirimycin (DGJ) negative control were evaluated in DENV-infected primary human monocyte-derived immature DCs (imDCs). DNJ- but not DGJ-derivatives elicited antiviral activity in DENV-infected imDCs, similar to that observed in MDMΦs. The DNJ-derivatives inhibited DENV secretion in a dose-dependent manner. Endoplasmic reticulum (ER) α-glucosidase I inhibition by DNJ-derived iminosugars, at concentrations of 3.16 µM, correlated with a reduction in the specific infectivity of virions that were still secreted, as well as a reduction in DENV-induced tumour necrosis factor alpha secretion. This suggests iminosugar-mediated ER α-glucosidase I inhibition may give rise to further benefits during DENV infection, beyond the reduction in viral secretion associated with ER α-glucosidase II inhibition.

Iminosugars Inhibit Dengue Virus Production via Inhibition of ER Alpha-Glucosidases--Not Glycolipid Processing Enzymes.

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.

Inhibition of endoplasmic reticulum glucosidases is required for in vitro and in vivo dengue antiviral activity by the iminosugar UV-4.

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.