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

Abstract

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.

Structures of mammalian ER α-glucosidase II capture the binding modes of broad-spectrum iminosugar antivirals

“…At higher substrate concentrations (2-10 mM), the best diglucoside substrate, the α(1,3)-linked nigerose, shows kinetics indicative of substrate inhibition (41), compatible with two overlapping substrate-binding sites of different affinity (45,46). Our crystal structures reveal a high degree of structural similarity between the Mmα-GluII catalytic pocket −1 subsite and the ones of intestinal α-glucosidases, and rationalize the observed reactivity against maltose (45)(46)(47) and the cross-reactivity of inhibitors within this family of enzymes (48). Most residues lining the Mmα-GluII α-subunit −1 and +1 subsites are identical to the ones in the human intestinal MGAM and SI enzymes ( Fig.…”

“…4D and SI Appendix, Fig. S8D), which has a longer alkyl chain, and is the most potent of these DNJ derivatives [MON-DNJ has an IC 50 against isolated Mmα-GluII of about 1.8 ± 0.3 μM (51) vs. 5.2 ± 1.0 μM for NB-DNJ and 11.4 ± 4.3 μM for DNJ (48)]. MON-DNJ displaces the whole loop, α 523-528, which changes conformation and whose terminal hairpin, α 525-527, is disordered in the crystal after MON-DNJ soaking.…”

Minimal In Vivo Efficacy of Iminosugars in a Lethal Ebola Virus Guinea Pig Model

“…Both drugs were able to inhibit ER α-glucosidase II in vivo , although only N B-DNJ inhibited α-glucosidase I. This effect is probably due to the 15x higher dose of N B-DNJ that was administered, and given that M O N-DNJ is only 3–4 fold more potent than N B-DNJ against α-glucosidase I and α-glucosidase II in vitro ([36, 47, 49]), this may explain why N B-DNJ showed some effect yet M O N-DNJ did not. This also could imply that inhibition of ER α-glucosidase I is needed to achieve an antiviral effect against EBOV.…”

Iminosugars: Promising therapeutics for influenza infection

“…Modifications, such as alkyl chain addition, affect biological properties such as uptake by cells and organelles thereby impacting antiviral efficacy and cytotoxicity (Norton et al., 2007; Sayce et al., 2016). Structural mimicry of terminal sugar moieties in natural substrates underlies iminosugar biological activity.…”