ToP-DNJ, a Selective Inhibitor of Endoplasmic Reticulum α-Glucosidase II Exhibiting Antiflaviviral Activity.

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.

Synthesis and characterization of novel, conjugated, fluorescent DNJ derivatives for α-glucosidase recognition.

A series of five new fluorescent deoxynojirimycin (DNJ) conjugates were synthesized and evaluated for their inhibitory effect (IC50) on several α- and ß-glucosidases. Three of the conjugates showed enhanced activity. The two synthetic conjugates, DNJ-CF31 and DNJ-Me 2, exhibited improved α-glucosidase inhibitory effects compared to DNJ and miglitol. Interestingly, conjugates 1 and 2 showed strong inhibition of almond-derived ß-glucosidase, in contrast to the inhibition tendencies of other inhibitors. Conjugate 5 strongly inhibited rat intestinal maltase, even at 0.10µM. A docking study indicated that all five conjugates bind to the active site of α-glucosidase (PDB: 3L4V, derived from Homo sapiens). The DNJ portion of the conjugate fits into the cavity of the enzyme, and the fluorescent part locates randomly on the outside surface. Thus, it is likely that these conjugates can specifically recognize intestinal cells, specifically the α-glucosidase on cell membranes.

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.

Synthesis and biological evaluation of N-(2-fluorophenyl)-2ß-deoxyfuconojirimycin acetamide as a potent inhibitor for α-l-fucosidases.

In this study we revealed that the addition of an N-phenylacetamide substituent to the C-1 position of 1-deoxyfuconojirimycin (DFJ) can lead to highly potent inhibitors of α-l-fucosidases. A structure-activity relationship study showed that a fluoro group on the phenyl ring greatly increased its potency and selectivity. In contrast the addition of two or three fluoro groups decreased their inhibition potency. Consequently, N-(2-fluorophenyl)-2ß-DFJ acetamide (18j) was found to display very potent and selective inhibition of bovine kidney, rat epididymis, and human lysosome α-l-fucosidases, with IC50 value of 0.012, 0.044, and 0.0079µM respectively. It is noteworthy that our designed N-phenyl-2ß-DFJ acetamide derivative exhibited about 18-fold stronger effects on human lysosomal α-l-fucosidase than original DFJ and it occupied the active-site of this enzyme. We therefore expect that this compound may find applications in new therapeutic trials against genetic deficiency disorders.

NHC-mediated cross-coupling of sugar-derived cyclic nitrones with enals: general and efficient synthesis of polyhydroxylated pyrrolizidines and indolizidines.

A general and efficient method for the synthesis of polyhydroxylated pyrrolizidines and indolizidines has been developed based on the NHC-catalyzed cross-coupling of sugar-derived cyclic nitrones with enals, which afforded the key intermediates, γ-hydroxyl amino esters, in good to excellent yields. Thus, a variety of polyhydroxylated pyrrolizidines and indolizidines have been synthesized and assayed against various glycosidases, which showed that aryl or alkyl substituents at C-7 of pyrrolizidines or at C-8 of indolizidines reduced the potency of the glycosidase inhibition of these bicyclic iminosugars.