Research ArticleEbola

FDA-Approved Selective Estrogen Receptor Modulators Inhibit Ebola Virus Infection

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Science Translational Medicine  19 Jun 2013:
Vol. 5, Issue 190, pp. 190ra79
DOI: 10.1126/scitranslmed.3005471
  • Fig. 1 In vitro eight-point dose-response curves for clomiphene and toremifene.

    Compounds were evaluated in both the Vero E6 and HepG2 cell lines. The percent inhibition of the compound in the EBOV assay is shown in green, and the cytotoxic effect of the compounds on the host cell is shown in red. The maximum percent inhibition observed (Max Effect) and IC50 are indicated. Error bars indicate SEM. Results are from two replicates.

  • Fig. 2 In vitro eight-point dose-response curves for clomiphene evaluated against native filovirus strains, EBOV/Kik (EBOV-95), EBOV/May (EBOV-76), SUDV, MARV, and RAVV.

    Results are from ELISA as described in Materials and Methods. Shown is the Max Effect (in % inhibition) along with the IC50 values (in μM). Vero E6 proliferation shows toxicity of clomiphene on uninfected cells. Results indicate that clomiphene is effective across all native virus strains evaluated. Results are from two or more replicates.

  • Fig. 3 In vitro eight-point dose-response curves for toremifene evaluated against native filovirus strains, EBOV/Kik, EBOV/May, SUDV, MARV, and RAVV.

    Results are from ELISA as described in Materials and Methods. Shown is the Max Effect (in % inhibition) along with the IC50 values (in μM). Vero E6 shows the toxicity of toremifene on uninfected cells. Results indicate that toremifene is effective across all native virus strains evaluated. Results are from two or more replicates.

  • Fig. 4 Survival plots for clomiphene and toremifene in a mouse model of EBOV infection.

    (A) A survival plot for clomiphene shows 90% of treated animals survived EBOV infection. All animals in the vehicle control group succumbed to disease by day 9. n = 10 for both the vehicle and clomiphene treatment groups. (B) A survival plot for toremifene indicating that 50% of the treated animals survived infection by EBOV. Animals in the vehicle control group succumbed to disease by day 7. n = 7 in the vehicle control treatment group and n = 10 in the toremifene treatment group. The P value for each study was determined by Fisher’s exact test. **P < 0.0001; *P < 0.0441.

  • Fig. 5 ER expression in EBOV permissive cell lines.

    (A) Western blot analysis of Vero E6 and HepG2 cells treated with clomiphene, dimethyl sulfoxide (DMSO) (vehicle control), or no treatment (NT) and probed for either ER-α or ER-β expression. MCF-7 and SK-BR-3 cell lysates were used as controls for ER-α and ER-β, respectively. (B) eGFP-EBOV was used to infect cell lines both positive and negative for ER-α and/or ER-β expression (as shown in fig. S4 and summarized in Table 2) that were treated with clomiphene. (C) Clomiphene and toremifene treatment of eGFP-EBOV–infected HUVECs that do not express ER-α or ER-β (fig. S5). Results indicate that both compounds inhibit virus infection similarly to cells that express ER receptors.

  • Fig. 6 Effect of clomiphene and toremifene on VLP entry.

    (A and B) Clomiphene (A) and toremifene (B) were tested for their ability to inhibit VLPs with the EBOV GP1,2 glycoprotein (VLP-GP), the VSV-G glycoprotein (VLP-G), and LCMV GP (VLP-LCMV). Results indicate that both clomiphene and toremifene exhibit greater specificity to VLPs bearing the EBOV GP1,2 glycoprotein compared to VLPs bearing VSV-G or the LCMV GP. For each compound, assays with the different VLPs were performed in parallel. Error bars represent the SE for two or more replicates.

  • Fig. 7 Evaluation of clomiphene and toremifene on Ebola virus VLP-GP1,2 internalization and cathepsin processing.

    (A) Clomiphene and toremifene were evaluated at 5 and 0.8 μM, respectively. EIPA is a known inhibitor of EBOV internalization. Results indicate that neither clomiphene nor toremifene inhibits EBOV internalization. (B) Clomiphene (5 μM) and toremifene (0.8 μM) were evaluated, as described in Materials and Methods, for their effects on cathepsin B (CatB) and cathepsin L (CatL) activity (singly and combined) in SNB19 cells. EST is a cysteine protease inhibitor that was included as a positive control for the assay. Data in the main plot are from the 1.5-hour time point. Data for CatL at 18 hours are shown in fig. S8.

  • Fig. 8 Endosomal pH upon exposure to clomiphene and toremifene.

    (A to D) SNB19 cells were preincubated in the presence of (A) DMSO vehicle, (B) 10 mM NH4Cl as a positive control for inhibition of acidification, (C) 5 μM clomiphene, or (D) 0.8 μM toremifene for 1 hour at 37°C. At this time, LysoTracker Red (± inhibitor, as indicated) was added, and the cells were incubated for an additional 30 min at 37°C. At this time, the cells were fixed, viewed, and photographed with a confocal microscope. Images are representative of 10 fields observed per condition.

  • Fig. 9 VLP-GP trafficking to late endosomes/lysosomes after clomiphene or toremifene treatment.

    SNB19 cells were pretreated with either DMSO, 2.5 μM clomiphene, or 1 μM toremifene for 1 hour, followed by the addition of VLP-GP1,2 as described in Materials and Methods. The impact of clomiphene or toremifene treatment on trafficking was assessed with confocal microscopy to determine the localization of VLP-GP in relation to LAMP1. (A) Ten random image fields per sample were analyzed with the JACoP plugin on ImageJ. Auto-thresholds were subtracted for each component 8-bit channel. Colocalization of VLPs (red, marked by mCheery-VP40) with LAMP1 (green) is reported as the average Mander’s overlap coefficient. Error bars represent SD. (B) Representative images (enlarged regions from an image field) from each treatment are shown with the green channel thresholds raised to 255 to better reveal areas of colocalization. This adjustment was made uniformly in all three images. White arrows indicate examples of colocalization.

  • Table 1 Activity of clomiphene and toremifene across native filovirus strains.

    IC50, the concentration at which 50% inhibition of viral infection was observed; Max effect, the maximum percent inhibition observed.

    EBOV/KikEBOV/MaySUDVMARVRAVV
    CompoundIC50 (μM)Max effectIC50 (μM)Max effectIC50 (μM)Max effectIC50 (μM)Max effectIC50 (μM)Max effect
    Clomiphene11.189.43.8397.84.9697.65.8297.07.1896.0
    Toremifene1.7396.60.9733.833.9697.85.7395.76.1795.6
  • Table 2 Estrogen receptor expression and infection status in cell lines.

    −, no expression; +, positive expression based on analysis by Western blotting; Y, cells that could be infected with eGFP-EBOV; N, cells resistant to infection with eGFP-EBOV; Low, a low level of eGFP-EBOV infection noted.

    Cell lineER-αER-βInfection
    Vero E6+Y
    HepG2+Y
    ZR-75-1++N
    MDA-MB-231+Low
    MCF-7++Y
    SK-BR-3+Low
    A549+Y
    H460+Y
    H322+N
    H1650+Y

Additional Files

  • Supplementary Material for:

    FDA-Approved Selective Estrogen Receptor Modulators Inhibit Ebola Virus Infection

    Lisa M. Johansen,* Jennifer M. Brannan, Sue E. Delos, Charles J. Shoemaker, Andrea Stossel, Calli Lear, Benjamin G. Hoffstrom, Lisa Evans DeWald, Kathryn L. Schornberg, Corinne Scully, Joseph Lehár, Lisa E. Hensley, Judith M. White, Gene G. Olinger*

    *Corresponding author. E-mail: gene.olinger@us.army.mil (G.G.O.); ljohansen@zalicus.com (L.M.J.)

    Published 19 June 2013, Sci. Transl. Med. 5, 190ra79 (2013)
    DOI: 10.1126/scitranslmed.3005471

    This PDF file includes:

    • Fig. S1. SERMS inhibit EBOV infection in vitro.
    • Fig. S2. The activity of clomiphene and toremifene against native strains of filoviruses using qRT-PCR.
    • Fig. S3. Kaplan-Meier survival curves for clomiphene in both male and female mice infected with a mouse-adapted Ebola virus.
    • Fig. S4. Western blot analysis showing ER-α and ER-β expression in cell line panel.
    • Fig. S5. Activity of clomiphene and toremifene in HUVECs.
    • Fig. S6. Representative flow cytometry plots from experiments conducted for Fig. 6.
    • Fig. S7. Effect of clomiphene and toremifene treatment on entry of VLPs bearing GP from SUDV.
    • Fig. S8. Cathepsin L processing at 18 hours.

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