Fig 1.
Iminosugars used in this study.
Iminosugars are sugar mimics with nitrogen substitution of the endocyclic oxygen. Sugars from which iminosugars are derived are presented to the left with d-glucose on top (white background) and d-galactose on the bottom (grey background). A lead clinical candidate for DENV antiviral activity, celgosivir, is a pro-drug of castanospermine, both of which possess d-glucose stereochemistry. A series of deoxynojirimycin (DNJ) derivatives with variable alkylation of the ring nitrogen was synthesized for comparison to equivalent galactose mimic deoxygalactonojirimycin (DGJ) derivatives.
Fig 2.
In vitro evaluation of iminosugar antiviral efficacy against DENV.
MDMΦs were infected with DENV (serotype 2, MOI = 1) and treated with a titration of iminosugars for 48 hours. Infectious virus titer was determined by plaque assay using LLC-MK2 (monkey kidney) cells. Counts were normalised to 100 percent for untreated samples. (a) DNJ-derived iminosugars (black) had antiviral efficacy with EC50s between 1.2 and 10.6 μM, whereas DGJ-derived iminosugars (red) were not effective up to the maximum non-toxic dose tested. (b) Parent compounds DNJ and castanospermine were compared to derivatives NN-DNJ and celgosivir, respectively. A four-parameter logistic nonlinear regression curve was determined to calculate EC50 values. Enhancement of antiviral activity by derivatization for NN-DNJ was 246 fold and for celgosivir 7 fold. Each donor was treated in triplicate, and plaque assays were conducted in triplicate on each sample obtained. A minimum of three donors were tested for each compound. Data are presented as mean ± SD.
Table 1.
Summary of in vitro antiviral properties of iminosugars.
Table 2.
In vitro enzyme inhibition of iminosugars.
Fig 3.
NB-DNJ reduces glycolipid production in a potent, dose-dependent manner in primary human MDMΦs.
Uninfected MDMΦs were treated with a titration of NB-DNJ for 48 hours, and glycolipids were isolated from whole cell lysates. Production of monosialodihexosylganglioside (GM3) was normalized to total protein content for each sample, and each treatment was normalized to untreated controls on a donor-specific basis. NB-DNJ inhibited production of GM3 in a dose-dependent manner that was approximately 10-fold more potent than the antiviral inhibition. Data are presented as mean ± SD from assay of three biological replicates assayed in technical singlicate.
Table 3.
GM3 inhibition in MDMΦ by treatment with MNTD iminosugar concentrations.
Fig 4.
Iminosugars possessing glucostereochemistry but not galactostereochemistry produce ER α-glucosidase associated FOS in MDMΦs.
MDMΦs were treated in technical duplicate for 48 hours with iminosugars. (a) Untreated cells do not produce detectable FOS representative of ER α-glucosidase inhibition. (b) Cells treated with 100 μM NB-DNJ iminosugar produce FOS representative of ER α-glucosidase I inhibition (Glc3Man5GlcNAc1) and ER α-glucosidase II inhibition (Glc1Man4GlcNAc1 and Glc1Man6GlcNAc1). (c-e) NB-DGJ (c), and NN-DGJ (d) all fail to produce detectable ER α-glucosidase associated FOS, but do produce FOS generated by inhibition of lysosomal β-N-acetylhexosaminidase. (e-f) MDMΦs were treated with a serial dilution of celgosivir (e) or NB-DNJ (f), and inhibition of α-glucosidase I was measured by accumulation of Glc3Man5GlcNAc1 (white circles) while inhibition of α-glucosidase II was measured by accumulation of Glc1Man4GlcNAc1 (black circles). For each donor, the maximal concentration of each oligosaccharide species reached was normalized to 100%. Assays were conducted in technical duplicate on a minimum of three donors, of which one representative replicate is presented (a-d) or of which the mean ± SD is presented (e-f).
Fig 5.
Glucostereochemistry iminosugars reduce secretion of DENV.
MDMΦs were treated in technical triplicate for 48 hours with a serial dilution of iminosugars. Treatment with NB-DNJ (a), and celgosivir (b) reduces infectious virus (solid lines) and total virus (dashed lines) secreted in a one-to-one ratio. The values for both RNA and infectious virus are means normalized to untreated samples within a donor. Each donor was treated in technical triplicate and qRT-PCR was conducted in technical duplicate, while LLC-MK2 plaque assays were conducted on nine technical replicates. A single representative donor is plotted for each drug as mean ± SD.
Fig 6.
Celgosivir reduces circulating virus in vivo immediately prior to normal time of death in untreated controls.
Iminosugar was given orally t.i.d. (celgosivir: 33.3 mg/kg/dose) in a lethal mouse model of antibody-enhanced DENV infection. Serum samples from terminal bleeds were collected from all mice 8 hours prior to normal time of death in infected, untreated controls. (a) Circulating viral load was significantly reduced by celgosivir treatment as assayed by qRT-PCR. Statistically significant differences (*, α<0.05) were assessed by non-parametric Mann-Whitney test with Bonferroni correction for multiple comparisons of RNA (see S1 Fig). Individual readings are plotted (triangles), with the median value for each treatment indicated by the horizontal line. (b) Infectious virus level in the same serum samples was assayed by BHK-21 plaque assay. Celgosivir reduced the average viral titer, but the differences were not statistically significant by ANOVA. Data are presented as mean ± SD. Three mice were tested for each compound, and both assays were conducted in technical duplicate.