Chloroquine and Sulfadoxine Derivatives Inhibit ZIKV Replication in Cervical Cells

Abstract

Despite the severe morbidity caused by Zika fever, its specific treatment is still a challenge for public health. Several research groups have investigated the drug repurposing of chloroquine. However, the highly toxic side effect induced by chloroquine paves the way for the improvement of this drug for use in Zika fever clinics. Our aim is to evaluate the anti-Zika virus (ZIKV) effect of hybrid compounds derived from chloroquine and sulfadoxine antimalarial drugs. The antiviral activity of hybrid compounds (C-Sd1 to C-Sd7) was assessed in an in-vitro model of human cervical and Vero cell lines infected with a Brazilian (BR) ZIKV strain. First, we evaluated the cytotoxic effect on cultures treated with up to 200 µM of C-Sds and observed CC₅₀ values that ranged from 112.0 ± 1.8 to >200 µM in cervical cells and 43.2 ± 0.4 to 143.0 ± 1.3 µM in Vero cells. Then, the cultures were ZIKV-infected and treated with up to 25 µM of C-Sds for 48 h. The treatment of cervical cells with C-Sds at 12 µM induced a reduction of 79.8% ± 4.2% to 90.7% ± 1.5% of ZIKV-envelope glycoprotein expression in infected cells as compared to 36.8% ± 2.9% of infection in vehicle control. The viral load was also investigated and revealed a reduction of 2- to 3-logs of ZIKV genome copies/mL in culture supernatants compared to 6.7 ± 0.7 × 10⁸ copies/mL in vehicle control. The dose-response curve by plaque-forming reduction (PFR) in cervical cells revealed a potent dose-dependent activity of C-Sds in inhibiting ZIKV replication, with PFR above 50% and 90% at 6 and 12 µM, respectively, while 25 µM inhibited 100% of viral progeny. The treatment of Vero cells at 12 µM led to 100% PFR, confirming the C-Sds activity in another cell type. Regarding effective concentration in cervical cells, the EC₅₀ values ranged from 3.2 ± 0.1 to 5.0 ± 0.2 µM, and the EC₉₀ values ranged from 7.2 ± 0.1 to 11.6 ± 0.1 µM, with selectivity index above 40 for most C-Sds, showing a good therapeutic window. Here, our aim is to investigate the anti-ZIKV activity of new hybrid compounds that show highly potent efficacy as inhibitors of ZIKV in-vitro infection. However, further studies will be needed to investigate whether these new chemical structures can lead to the improvement of chloroquine antiviral activity.

Keywords: Zika virus; antiviral effect; chloroquine; human cervical cells; hybrid compounds; sulfadoxine.

Figure 1

Kinetics of cellular viability of C-Sds hybrids and mefloquine were evaluated by PrestoBlue assay. The cervical and Vero cell lines were treated with C-Sds and mefloquine at different concentrations for 48 h. The graphs represent the mean ± standard deviation of the percentage of viable (A) cervical cells and (B) Vero cells. (C) The table shows the values of CC₅₀/48 h of C-Sds and mefloquine. The data are representative of 3–5 experiments run in duplicate.

Figure 2

Kinetics of the Brazilian ZIKV strain (ZIKV-BR) infection in human cervical cell line. The cells were mock-infected or ZIKV-infected at MOI 1 and 10 for 24, 48, and 72 h. (A) ZIKV–envelope glycoprotein expression (green) was detected by immunofluorescence and images were capture in differential interference contrast microscopy. (B) The graph represents the mean ± standard deviation of the percentage of infected cells during the kinetics of infection. The supernatants of cell cultures were collected to evaluate (C) viral load by RT-qPCR and (D) plaque-forming by plaque assay. The data are representative of 3–5 experiments run in duplicate. Bars, 50 µm.

Figure 3

Evaluation of the C-Sds antiviral effect by immunofluorescence and RT-qPCR. The cervical cells were mock-infected or ZIKV-infected at MOI 10 and treated with 12 µM of C-Sds or mefloquine for 48 h. The images are shown in DIC (differential interference contrast) microscopy; (A) fluorescent intracellular staining shows the ZIKV–envelope glycoprotein (green). The graphs represent the mean ± standard deviation of (B) the percentage of infected cells and (C) copies of the ZIKV genome/mL. The data are representative of 3–5 experiments run in duplicate. The statistical significance was determined by one-way ANOVA, followed by Dunnett’s multiple-comparisons test. **** p < 0.0001 for all C-Sds compared to control. Bars, 50 µm.

Figure 4

Evaluation of anti-ZIKV effect in cervical and Vero cell lines by plaque assay. The cervical cells were mock-infected or ZIKV-infected at MOI 10 and treated with 6, 12, and 25 μM of C-Sds or 6 and 12 μM of mefloquine for 48 h. The Vero cells were mock-infected or ZIKV-infected at MOI 1 and treated with 12 μM of C-Sds or mefloquine for 48 h. (A) The cultures’ supernatant was collected and diluted (1:1000) for the plaque assay. The graphs represent the mean ± standard deviation of (B) the ZIKV titer (PFU/mL) and (C) the dose-response curve of PFR%. (D) The table summarizes the parameters of antiviral activity evaluation in cervical and Vero cells. The data are representative of 3–5 experiments run in duplicate. The statistical significance was determined by one-way ANOVA, followed by Dunnett’s multiple-comparisons test. **** p < 0.0001.