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.

长期以来，学界普遍认为亚氨基糖（iminosugar）的抗病毒活性源于对内质网（endoplasmic reticulum, ER）驻留α-葡萄糖苷酶的抑制作用。基于这一理论基础，诸多亚氨基糖被开发为广谱病毒感染治疗药物，其中涵盖登革病毒（DENV）。然而，亚氨基糖属于糖模拟物，其结构以氮原子取代了环内氧原子；而聚糖在宿主代谢中广泛分布，这提示亚氨基糖治疗可靶向多条通路。此前已有研究通过亚氨基糖成功治疗糖脂加工缺陷患者，这凸显了亚氨基糖可靶向内质网α-葡萄糖苷酶以外的酶类开展临床转化。目前，关于抗病毒活性与内质网葡萄糖苷酶抑制作用相关的证据，以及登革病毒感染背景下排除亚氨基糖其他作用机制的相关证据均较为匮乏。塞格司韦（Celgosivir）作为一种双环亚氨基糖，曾作为登革病毒感染治疗药物进入Ib期临床试验，其在抗体增强型登革病毒感染致死性小鼠模型中被证实具有抗病毒活性。本研究首次证实了塞格司韦在体外原代人巨噬细胞中的抗病毒活性：该药物以5 μM的半最大效应浓度（EC50）抑制登革病毒的分泌。我们进一步证明，单环葡萄糖模拟型亚氨基糖可抑制纯化的糖蛋白与糖脂加工酶，且该抑制效应在经此类药物处理的原代细胞中同样存在。通过对比两类亚氨基糖的作用特征：一类是可抑制糖蛋白加工但不影响糖脂加工的双环葡萄糖模拟型亚氨基糖，另一类是不抑制糖蛋白加工但可阻断糖脂加工的半乳糖模拟型亚氨基糖，我们证实，亚氨基糖抗登革病毒活性的核心机制是抑制内质网驻留α-葡萄糖苷酶，而非糖脂加工通路。本研究数据表明，抑制内质网α-葡萄糖苷酶可阻断病毒释放，这正是亚氨基糖抗登革病毒的主要抗病毒作用机制。