Emetine inhibits Zika and Ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry

Abstract

The re-emergence of Zika virus (ZIKV) and Ebola virus (EBOV) poses serious and continued threats to the global public health. Effective therapeutics for these maladies is an unmet need. Here, we show that emetine, an anti-protozoal agent, potently inhibits ZIKV and EBOV infection with a low nanomolar half maximal inhibitory concentration (IC50) in vitro and potent activity in vivo. Two mechanisms of action for emetine are identified: the inhibition of ZIKV NS5 polymerase activity and disruption of lysosomal function. Emetine also inhibits EBOV entry. Cephaeline, a desmethyl analog of emetine, which may be better tolerated in patients than emetine, exhibits a similar efficacy against both ZIKV and EBOV infections. Hence, emetine and cephaeline offer pharmaceutical therapies against both ZIKV and EBOV infection.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1. Emetine is an inhibitor of ZIKV infection and replication.

a Chemical structures of emetine and cephaeline. b Dose-response curve showing the effect of emetine treatment on ZIKV NS1 protein expression (red) and cell viability (black) in HEK293 cells exposed to ZIKV MR766 strain. c Western blot of ZIKV NS1 protein after treatment of SNB-19 cells with varying concentrations of Emetine. SNB-19 cells were treated with emetine for 1 h before inoculation with ZIKV strains PRVABC59, FSS13025, or MR766 (MOI = 1), and cells were harvested 24 h after infection for Western Blot analysis. d Immunofluorescent images of astrocytoma cells stained for ZIKV PRVABC59 envelope (ENV) protein (green), nuclei (blue) and treated with emetine. Scale bar=100 µm. e Dose–response curves from cells in (d) showing the inhibition effect of emetine on ZIKV PRVABC59 infection measured by ENV+ SNB-19 cells (green), and cell number by nuclear counting (blue). f SNB-19 cells were treated with emetine at increasing concentrations for 1 h before infection with PRVABC59 at a MOI = 1 (n = 3 cultures). Cell culture supernatant was collected 24 h after infection, and infectious virions were quantified for focus-forming units (FFU) using Vero cells. All curves represent best fits for calculating the IC50 values (graph inset). All values represent mean ± SD (N = 3 replicates)

Fig. 2. Emetine inhibits ZIKV NS5 polymerase activity.

a Western blot showed the CETSA binding assay of ZIKV NS5 in the presence or absence of 50 μM emetine at different temperatures. b Temperature melting curves of ZIKV NS5 in (c). The relative chemiluminescent intensity of each sample at different temperatures was used to generate temperature-dependent melting curves and the apparent aggregation temperature (Tagg) was calculated by nonlinear regression. Values represent mean ± SEM (N = 3 replicates). c Dose–response curve showing the inhibition effect of emetine treatment on the RNA-dependent RNA polymerase (RdRp) activity of recombinant ZIKV NS5 enzyme. The curve represent best fits for calculating the IC50 values (graph inset). Values represent mean ± SD (N = 3 replicates). d Predicted binding model of emetine with ZIKV NS5 RNA-dependent RNA-polymerase (RdRp). The three domains of fingers (yellow), palm (blue), and thumb (green) are shown in ribbons, while the priming loop is shown in red color. The emetine bound at the active site is depicted as sticks. A close-up view of hydrogen-binding interactions of emetine at the active site of RdRp is shown

Fig. 3. Emetine suppresses ZIKV virus load in vivo.

a Three-month-old SJL male mice were infected retro-orbitally with ZIKVBR followed by IP administration of emetine (1 mg/kg/day) for the next 6 days (N = 4 mice per group). Two groups of SJL mice (N = 4) received the same volume of vehicle buffers. On the day 7 the blood samples were collected and ZIKV RNA was quantified by real-time qPCR. Statistical analysis by two tailed t-test. **p = 0.0014, ***p = 0.0005. b Ifnar1−/−−/− mice were dosed with emetine 1 mg/kg (E1, N = 6), 2 mg/kg (E2, N = 7), and PBS (VC, N = 8), respectively, 24 h prior to challenge with virus on day 0. Treatment with emetine/VC was done once daily intraperitoneally. On day 3, a blood sample was taken from tail vein and viral load in the serum is estimated by ZIKV NS1 ELISA kit. Values presented as mean ± SEM, one way ANOVA, followed by Dunnett’s test. **p = 0.007, ***p = 0.0008. c Ifnar1-/- mice (8–9 week old, male and female) were dosed with emetine 2 mg/kg/day IP starting at 24 h prior (N = 8) or 24 h after (N = 7) challenging with ZIKV on day 0. Drug was continued until day 3 and mice were killed. Blood was collected and the serum NS1 protein was measured using ZIKV NS1 ELISA kit. d Liver tissue sample was collected, total RNA was column-extracted and qPCR was done using ZIKV NS1 specific primers and GAPDH primers to calculate the relative expression of the NS1 gene. Statistical analysis was done using the Mann–Whitney ANOVA. *p < 0.05, ***p < 0.001

Fig. 4. Emetine inhibits EBOV infection in vitro and in vivo.

a Dose–response curve showing the inhibition effect of emetine treatment on Ebola VLP entry in HeLa cells. b Dose–response curve showing the inhibition effect of emetine treatment on infection of Ebola live virus in Vero E6 cells. c The survival curve of MA-EBOV infected mouse treated with 1 mg/kg emetine every day. Six to eight week-old female BALB/c mice were randomly assigned into groups (N = 6 animals). All the mice were challenged with a lethal dose of 1000 times the LD50 mouse adapted EBOV via IP treatments with either emetine (1 mg/kg/day) or PBS (same volume for the control group) were initiated at 3 h before the challenge and continued for up to 6 days post infection. Survival was monitored for 28 days post infection. d Top, dose–response curve showing the effect of emetine on cholesterol accumulation measured by filipin dye fluorescence intensity (IC50 = 9.03 µM). Bottom, fluorescence images of fibroblast cells treated with DMSO or emetine and stained with filipin (unesterified free cholesterol, green) and nuclear green (nuclei, blue). e Top, dose–response curve showing the effect of emetine on lipid accumulation measured by Nile red fluorescence intensity (IC50 = 551 nM). Bottom, fluorescence images of fibroblast cells treated with DMSO or emetine and stained with Nile red (lipids, yellow) and Hoechst 33342 (nuclei, blue). f Top, dose–response curve showing the effect of emetine on acidic organelle accumulation measured by LysoTracker dye fluorescence intensity (IC50 = 26.2 nM). Bottom, fluorescence images of fibroblast cells treated with DMSO or emetine and stained with LysoTracker dye (acidic organelles, red) and Hoechst 33342 (nuclei, blue). All curves represent best fits for calculating the IC50 values (graph inset). All values represent mean ± SD (N = 3 replicates). Scale bar=50 µm

Fig. 5. Emetine interrupts autophagy.

a Immunofluorescent images of astrocytoma SNB-19 cells treated with DMSO, emetine or chloroquine for 24 h, then stained with SQSTM1 protein (green), MAP1LC3B protein (red) and nuclei (blue). b Quantitative analysis of the SQSTM1 puncta staining per cell (average puncta signal intensity per cell × average total puncta area per cell, I × A) in (a), showing the dose-dependent increase after emetine or chloroquine treatment. c Quantitative analysis of the MAP1LC3B puncta staining per cell (I × A) in (a), showing the dose-dependent increase after emetine or chloroquine treatment. d Immunofluorescent images of astrocytoma SNB-19 cells treated with DMSO, emetine or chloroquine for 24 h, then stained with LAMP1 protein (green), MAP1LC3B protein (red) and nuclei (blue). e Quantitative analysis of the LAMP1 vesicle staining per cell (I × A) in (d), showing the dose-dependent decrease after emetine or chloroquine treatment. f Quantitative analysis of the MAP1LC3B puncta staining per cell (IxA) in d, showing the dose-dependent increase after emetine or chloroquine treatment. Error bars indicate mean ± S.D. All values represent mean ± SD (N ≥ 3 replicates)