Polymeric Prodrugs Targeting Polyamine Metabolism Inhibit Zika Virus Replication

Abstract

The Zika virus (ZIKV) is primarily transmitted via an infected mosquito bite, during sexual intercourse, or in utero mother to child transmission. When a fetus is infected, both neurological malformations and deficits in brain development are frequently manifested. As such, there is a need for vaccines or drugs that may be used to cure ZIKV infections. Metabolic pathways play a crucial role in cell differentiation and development. More importantly, polyamines play a key role in replication and translation of several RNA viruses, including ZIKV, Dengue virus, and Chikungunya virus. Here, we present polyamine analogues (BENSpm and PG11047) and their corresponding polymer prodrug derivatives for inhibiting ZIKV infection by intersecting with polyamine catabolism pathways. We tested the compounds against ZIKV African (MR766) and Asian (PRVABC59) strains in human kidney epithelial (Vero) and glioblastoma derived (SNB-19) cell lines. Our results demonstrate potent inhibition of ZIKV viral replication in both cell lines tested. This antiviral effect was mediated by the upregulation of two polyamine catabolic enzymes, spermine oxidase, and spermidine (SMOX)/spermine N1-acetyltransferase (SAT1) as apparent reduction of the ZIKV infection following heterologous expression of SMOX and SAT1. On the basis of these observations, we infer potential use of these polyamine analogues to treat ZIKV infections.

Keywords: SAT1; SMOX; Zika virus; bisethylnorspermine; polyamine metabolism; polyamines; prodrugs; virus replication.

Figure 1.

Chemical structures of polyamine analogs and their polymeric prodrugs. BENSpm, PG11047, DSS-BEN, and DSS-PG. Chemical structure of a first-generation polyamine analogue, BENSpm (A), a second-generation polyamine analogue PG-11047 (B), polyamine drugs DSS-BEN (C), and DSS-PG (D) derived from parental drugs, respectively.

Figure 2.

Cytotoxicity of the tested compounds in noninfected Vero cells and SNB-19 cells. In vitro cytotoxicity assay of polyamine drugs (BENSpm, PG11047, DSS-BEN, and DSS-PG) of different concentrations after incubation for 72 h was performed in Vero cell line (A) and in SNB19 cell line (B) by the CellTiter-Blue assay. The CC50 value was calculated using GraphPad Prism version 7.0 software. The results are shown as the mean percentage relative cell viability with three independent experiments in triplicate.

Figure 3.

Evaluation of anti-ZIKV activity of polyamine drugs on viral RNA loads in Vero cells. The Vero cells were treated with (A) BENSpm, (B) PG11047, (C) DSS-BEN, and (D) DSS-PG at indicated concentrations. After a 16 h treatment, cells were infected with ZIKV at 0.1 MOI (PRVABC59 or MR766) for 24–48 h. Viral RNA levels were measured in cell culture supernatant from Vero cells. Control means untreated but ZIKV infected. One-way ANOVA was used, and *** indicates p ≤ 0.001. Error bars represent the standard error of the mean.

Figure 4.

Effect of polyamine drug treatment on ZIKV foci formation in Vero cells. Vero cells were treated with serial diluted (5-fold) polyamine drug concentrations of (A and C) BENSpm and PG11047 and (B and D) DSS-BEN and DSS-PG for 16 h and infected with an Asian strain, PRVABC59 (A and B), and an African strain, MR766 (C and D), at 0.1 MOI. After 24 h, the anti-flavivirus group antigen antibody (clone D1–4G2–4-15; mouse) was used to visualize infected cells (foci) in polymeric prodrugs treated and also in untreated but infected controls. The IC₅₀ value was calculated using GraphPad Prism version 7.0 software. Error bars represent the standard error of the mean.

Figure 5.

Treatment effect of polyamine drugs on ZIKV foci formation in SNB-19 cells. SNB19 cells were treated with serially diluted (5-fold) polyamine drug concentrations of (A and C) BENSpm and PG11047 and (B and D) DSS-BEN and DSS-PG for 16 h and infected with an Asian strain, PRVABC59 (A and B), and an African strain, MR766 (C and D), at 0.2 MOI. After 24 h, the anti-flavivirus group antigen antibody (clone D1–4G2–4-15; mouse) was used to visualize infected cells (foci) in polymeric prodrug compounds treated and also in untreated but infected controls. The IC₅₀ value was calculated using GraphPad Prism version 7.0 software. Error bars represent the standard error of the mean.

Figure 6.

Treatment effect of polyamine drug timing on ZIKV replication in both cell lines. Cells were treated with BENSpm (13.2 μg/mL), PG11047 (13.2 μg/mL), DSS-BEN (30 μg/mL), and DSS-PG (30 μg/mL) at specified times (A). At 16 and 4 h before infection (–16 h, –4 h), during virus adsorption (0 h), and after infection (+4 h), polyamine prodrugs were added to Vero cells (B and C) or SNB-19 cells (D and E). Cells were grown in a 24-well plate and infected with ZIKV (MR766 (B and D) or PRVABC59 (C and E) at a MOI of 0.1 and 0.2, respectively. Titers were determined at 24 h p.i.; the representative results are presented. The statistical analysis was performed using GraphPad Prism Software Version 7, and two-way ANOVA was used to calculate the statistical significance (*p < 0.05, **p < 0.01, and ***p < 0.001).

Figure 7.

Mechanisms of antiviral activity of polyamine drugs. Cells were treated with BENSpm, PG11047, DSS-BEN, or DSS-PG (BENSpm and PG11047 at 13.2 μg/mL, DSS-BEN and DSS-PG at 30 μg/mL) for 24 h. Intracellular concentration of BENSpm or PG11047 was determined by HPLC (n = 3) in Vero cells (A) and SNB-19 cells (D). Relative changes in the expression of SMOX and SAT1 mRNA in Vero cells (B) and SNB-19 cells (E). mRNA levels were measured by qRT-PCR. Results are expressed as the fold induction of specific mRNA in treated cells relative to the PBS-treated group (n = 3). Intracellular polyamine concentration determined by HPLC (n = 3) in Vero cells (C) and SNB-19 cells (F). Putrescine upon BENSpm and PG11047 treatment was not detectable. ***p < 0.001 and **p < 0.01, versus PBS.

Figure 8.

Effects of heterologous expression of SAT1 and SMOX proteins on ZIKV replication. (A) Vero cells were mock-transfected or transfected with either FLAG-tagged SAT1 or HA-tagged SMOX expressing constructs and were infected with either MR766 or PRVABC59 at a MOI of 1. At 24 hpi, cells were processed for the immunofluorescence assay to visualize heterologous expression of proteins (green), virus infection (red), and cell nuclei (blue). Representative images are presented. (B) Stained cells were analyzed and quantified for the percent infected and cotransfected infected cells under each condition. Error bars indicate standard deviations calculated from 4 replicate wells in a 96-well plate. ***p ≤ 0.001 using a two-way ANOVA.