Research ArticleEbola virus

A screen of approved drugs and molecular probes identifies therapeutics with anti–Ebola virus activity

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Science Translational Medicine  03 Jun 2015:
Vol. 7, Issue 290, pp. 290ra89
DOI: 10.1126/scitranslmed.aaa5597
  • Fig. 1. Mechanistic overview of compound library.

    Histogram showing the cellular compound mechanisms from the drug library across the horizontal axis. The vertical axis indicates the number of compounds with that particular mechanism in log scale. Blue bars indicate the number of all compounds that contain a particular mechanism (All). Red bars indicate the number of active compounds identified (Hits) per each mechanism. The screen identifies a number of compounds that are SERMs and antihistamines, and compounds that affect ion transport and protein processing or cell signaling pathways. GC, glucocorticoid nuclear receptor; HDAC, histone deacetylase; PPAR, peroxisome proliferator–activated receptor; GPCR, G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor.

  • Fig. 2. Confirmatory eight-point dose-response curves for select active compounds in Vero E6 cells.

    Compounds were evaluated using the eGFP-EBOV screening assay. The percent inhibition of the compound in the EBOV assay is shown in blue, and the percent cytotoxicity of the compounds on the host cell is shown in red. The maximum percent inhibition observed (Max response) of EBOV and IC50s are indicated. Error bars indicate the SEM. Results are from a minimum of two replicates. Data in Vero E6 cells for additional active compounds comprising a set of 30 prioritized compounds are shown in figs. S2 and S3.

  • Fig. 3. Confirmatory eight-point dose-response curves for select active compounds in HepG2 cells.

    Compounds were evaluated using the eGFP-EBOV screening assay. The percent inhibition of compounds in the EBOV antiviral assay is shown in blue, and the cytotoxic effect of the compounds on the host cell is shown in red. The maximum percent inhibition observed (Max response) of EBOV and IC50s are indicated. Error bars indicate the SEM. Results are from a minimum of two replicates. Data in HepG2 cells for additional active compounds comprising a set of 30 prioritized compounds are shown in figs. S4 and S5.

  • Fig. 4. Post-EBOV challenge effect of bepridil and sertraline on survival rate of mice.

    (A) Post-inoculation treatment with bepridil is 100% effective in protecting EBOV-infected animals. All mice in the vehicle control group succumbed to disease by day 9. n = 10 for both the vehicle and bepridil treatment groups. (B) Post-inoculation treatment with sertraline indicates that 70% of the EBOV-infected mice survived. Mice in the vehicle control group succumbed to disease by day 8. n = 10 for both the vehicle and sertraline treatment groups. The P value was determined using Fisher’s exact test.

  • Fig. 5. Effect of bepridil and sertraline on VLP entry.

    Bepridil and sertraline were tested for their ability to inhibit entry of VLPs displaying EBOV-GP1,2 glycoprotein (VLP-GP) into 293AD cells at the indicated concentrations. (A) Results show that both bepridil and sertraline inhibit VLP-GP entry in a dose-dependent fashion. The average normalized entry values for the two untreated (Mock) sets of samples were 19 and 24%. (B and C) Comparison of effects of bepridil (B) and sertraline (C) on entry of VLPs displaying EBOV-GP1,2, VSV-G, or LCMV-GP. In (B), the % entry values for untreated samples (0 μM drug) were 32, 51, and 52% for EBOV-GP1,2, VSV-G, or LCMV-GP, respectively. In (C), the % entry values for untreated samples (0 μM drug) were 10, 79, and 55% for EBOV-GP1,2, VSV-G, or LCMV-GP, respectively. The experiment in (C) was conducted two additional times with highly similar results, even when % entry values for untreated samples were as low as 6% for entry mediated by VSV-G and 4% for entry mediated by LCMV-GP. For all panels, triplicate samples were analyzed, and error bars represent SD of the mean. Results confirm that both bepridil and sertraline inhibit entry mediated by EBOV-GP1,2. Although these drugs have no effect on entry mediated by VSV-G, they inhibit entry mediated by LCMV-GP, albeit at somewhat higher doses than for EBOV-GP1,2. Entry values were normalized to that observed in untreated cells.

  • Fig. 6. Sertraline and bepridil do not inhibit EBOV-VLP–GP1,2 internalization, cathepsin processing, or traffic to NPC1+ endolysosomes.

    (A) Bepridil, sertraline, EIPA (50 μM), an inhibitor of macropinocytosis, and vehicle were tested for their ability to inhibit internalization of VLPs with the EBOV-GP1,2 glycoprotein. Samples were analyzed in duplicate or triplicate in four experiments, and the average internalization for mock-treated samples was 30.5%. (B) Bepridil, sertraline, positive control EST (10 μM), a cysteine protease inhibitor, and vehicle were evaluated for effects on the activities of cathepsin B (CatB) or cathepsin L (CatL) (singly and combined; some inhibition of CatL activity was seen with 10 μM of sertraline). Samples were analyzed in triplicate. (C) Bepredil, sertraline, positive control nocodazole (20 μM), a microtubule disruptor, and vehicle were tested for effects on trafficking of VLP-GP1,2 to NPC1+ endolysosomes (in 293AD cells). Eighteen microscope fields were analyzed to calculate Manders colocalization coefficients. Error bars indicate SE of average values. Asterisks indicate values that are statistically different from mock-treated samples at P < 0.02 as determined using a Student’s t test: **P < 1 × 10−4 and *P < 0.003 (A); **P < 3 × 10−5 and *P < 0.002 (B); and *P < 0.001 (C).

  • Fig. 7. In vitro dose-response curves for bepridil against native filovirus strains.

    The percent inhibition of bepridil against native filovirus strains [that is, EBOV/Kik (EBOV-95), SUDV, MARV, and RAVV] was evaluated by cell-based ELISA. The maximum percent inhibition (Max response) of these strains and IC50 values in μM are indicated. Bepridil is effective against all native virus strains evaluated. Results are from two or more replicates. Error bars represent SE. The cytotoxicity of bepridil is shown by inhibiting Vero E6 proliferation without virus exposure.

  • Fig. 8. In vitro dose-response curves for sertraline against native filovirus strains.

    The percent inhibition of bepridil against native filovirus strains [that is, EBOV/Kik (EBOV-95), EBOV/May (EBOV-76), SUDV, MARV, and RAVV] was evaluated by cell-based ELISA. The maximum percent inhibition (Max % response) of these strains and IC50 values in μM are indicated. Sertraline is effective across all native virus strains evaluated. Results are from two or more replicates. Error bars represent SE. The cytotoxicity of sertraline is shown by inhibiting Vero E6 proliferation without virus exposure.

  • Table 1. Anti-EBOV activity and cytotoxicity of active compounds.

    Parentheses indicate the SE for IC50 calculations. Table summarizes data shown in Fig. 2 and 3 and figs. S3 to S6. %I, maximum percent inhibition based on the curve fit; IC50, drug concentration in μM at which 50% inhibition of viral infection or host viability (without virus) was observed.

    Drug nameVero E6HepG2
    IC50 eGFP-EBOV
    μM (SE)
    %I eGFP-EBOVIC50 Host
    μM (SE)
    %I HostIC50 eGFP-EBOV
    μM (SE)
    %I eGFP-EBOVIC50 Host
    μM (SE)
    %I Host
    Aripiprazole8.1 (0.42)86.720.73.76 (0.18)94.719.4 (1.61)82
    Astemizole6.17 (1.34)96.537.91.37 (0.038)91.411.1 (0.055)99.6
    Atovaquone0.437 (0.016)73.935.45.713.62
    Azacitidine8.97 (2.6)74.727.910.3 (1.00)89.933.1
    Benztropine8.07 (0)89.121.02.82 (0.13)93.918.1
    Bepridil5.08 (0.38)9721.13.21 (0.15)91.249.3
    Clemastine5.44 (0.32)95.839.80.652 (0.037)93.343.7
    Clomiphene*2.42 (0.045)96.426.20.755 (0.046)92.015 (1.28)92
    Clomipramine11.4 (0.15)96.121.12.57 (0.16)89.923.5
    Dasatinib16.5 (4.6)64.235.64.23 (0.20)94.125.8 (0.80)71.3
    Efavirenz10.7 (2.0)63.97.3813.5 (2.07)869.64
    Flupentixol5.78 (0.20)95.941.91.59 (0.25)74.516.5
    Fluphenazine5.54 (0.19)96.846.43.05 (1.68)9230
    Hycanthone10.9 (0.85)96.842.95.96 (0.39)96.216.6 (0.82)62.9
    Lomerizine11.4 (1.8)89.919.72.42 (0.12)91.843.9
    Maprotiline9.63 (0.01)98.626.32.86 (0.15)92.641.3
    Mycophenolate mofetil43.83.270.29 (0.15)88.617.6
    Paroxetine7.45 (0.41)9639.91.38 (0.076)9223.6 (0.18)94.8
    Pimozide3.12 (0.11)96.820.31.67 (0.044)9118.9 (0.18)99.4
    Piperacetazine12.3 (0.85)96.223.13.3 (0.16)95.428.8
    Prochlorperazine5.96 (0.42)96.342.83.59 (0.19)94.727.3
    Quinacrine5.71 (0.61)92.323.6 (8.5)63.41.03 (0.052)86.212.9 (1.54)98.6
    Sertraline3.13 (0.24)96.229.41.44 (0.057)9518 (0.53)54.4
    Simvastatin44.6 (0.79)55.728.535.311.8
    Strophanthin0.0346 (0.020)74.328.80.0207 (0.0014)86.80.0569 (0.00047)68.4
    Teicoplanin7.28 (0.46)84.62.722.43 (0.16)772.33
    Terconazole8.26 (0.32)96.633.22.38 (0.068)91.836.5
    Thioridazine6.24 (0.79)95.410.2 (9.06)93.12.06 (0.12)93.421.6 (0.78)99.4
    Toremifene*0.162 (0.048)93.511.40.026 (0.0013)94.95.43
    Vinorelbine25.627.90.858 (0.034)78.832.9

    *EBOV antiviral activity also described by Johansen et al. (11).

    †EBOV antiviral activity also described by Shoemaker et al. (14).

    • Table 2. VLP entry inhibition results.

      Conc, concentration tested in initial VLP-GP1,2 entry inhibition assay in SNB19 cells. Yes indicates inhibition of >90%.

      Drug nameStructureConc (μM)Inhibit EBOV-VLP
      entry
      AripiprazoleAmphiphile10Yes
      AstemizoleAmphiphile5Yes
      AtovaquonePhenol0.8No
      AzacitidineCarbohydrate10No
      BenztropineCAD10Yes
      BepridilCAD10Yes
      ClemastineCAD4Yes
      Clomiphene*CAD5Yes
      ClomipramineCAD10Yes
      DasatinibCAD10Yes
      EfavirenzAnhydride10Yes
      FlupentixolCAD10Yes
      FluphenazineCAD10Yes
      HycanthoneCAD10Yes
      LomerizineAmphiphile10Yes
      MaprotilineCAD10Yes
      Mycophenolate mofetilAmphiphile1No
      ParoxetineAmphiphile10Yes
      PimozideAmphiphile10Yes
      PiperacetazineAmphiphile10Yes
      ProchlorperazineCAD10Yes
      QuinacrineCAD10Undetermined
      SertralineCAD10Yes
      SimvastatinEster10Yes
      StrophanthinCarbohydrate0.1No
      TeicoplaninCAD10Yes
      TerconazoleCAD12Yes
      ThioridazineCAD10Yes
      Toremifene*CAD0.8Yes
      VinorelbineAmphiphile5Yes

      *Entry inhibition activity also described by Johansen et al. (11).

      †Quinacrine is an autofluorescent compound, and its ability to inhibit EBOV-VLP entry could not be assessed in this assay.

      ‡Entry inhibition also described by Shoemaker et al. (14).

      Supplementary Materials

      • www.sciencetranslationalmedicine.org/cgi/content/full/7/290/290ra89/DC1

        Table S1. Results of a three-point dose antiviral screen of 2635 compounds against eGFP-EBOV infection (two Excel files).

        Table S2. Active compounds identified from preliminary three-point dose antiviral screen against eGFP-EBOV infection (Excel file).

        Table S3. Survival of ma-EBOV–infected C57BL/6 mice after treatment with additional priority active compounds identified in vitro.

        Fig. S1. Comparison of compound activity in the antiviral assay against eGFP-EBOV infection versus uninfected host cell proliferation.

        Fig. S2. Confirmatory in vitro eight-point dose-response curves for active compounds from EBOV antiviral screen in Vero E6 cells.

        Fig. S3. Additional confirmatory in vitro eight-point dose-response curves for active compounds from EBOV antiviral screen in Vero E6 cells.

        Fig. S4. Confirmatory in vitro eight-point dose-response curves for active compounds from EBOV antiviral screen in HepG2 cells.

        Fig. S5. Additional confirmatory in vitro eight-point dose-response curves for active compounds from EBOV antiviral screen in HepG2 cells.

        Fig. S6. Evaluation of sertraline and bepridil on endosome acidification.

        Fig. S7. Evaluation of sertraline and bepridil on EBOV-VLP–GP1,2 trafficking to NPC1+ endolysosomes.

        Fig. S8. Bepridil and sertraline, at the indicated concentrations, do not inhibit the viability of 293AD cells.

        Fig. S9. Assessment of bepridil and sertraline on VSV EBOV-GP pseudovirion entry into parental and NPC1-overexpressing CHO cell lines.

      • Supplementary Material for:

        A screen of approved drugs and molecular probes identifies therapeutics with anti–Ebola virus activity

        Lisa M. Johansen, Lisa Evans DeWald, Charles J. Shoemaker, Benjamin G. Hoffstrom, Calli M. Lear-Rooney, Andrea Stossel, Elizabeth Nelson, Sue E. Delos, James A. Simmons, Jill M. Grenier, Laura T. Pierce, Hassan Pajouhesh, Joseph Lehár, Lisa E. Hensley, Pamela J. Glass, Judith M. White, Gene G. Olinger*

        *Corresponding author. E-mail: gene.olinger{at}nih.gov

        Published 3 June 2015, Sci. Transl. Med. 7, 290ra89 (2015)
        DOI: 10.1126/scitranslmed.aaa5597

        This PDF file includes:

        • Table S3. Survival of ma-EBOV–infected C57BL/6 mice after treatment with additional priority active compounds identified in vitro.
        • Fig. S1. Comparison of compound activity in the antiviral assay against eGFPEBOV infection versus uninfected host cell proliferation.
        • Fig. S2. Confirmatory in vitro eight-point dose-response curves for active compounds from EBOV antiviral screen in Vero E6 cells.
        • Fig. S3. Additional confirmatory in vitro eight-point dose-response curves for active compounds from EBOV antiviral screen in Vero E6 cells.
        • Fig. S4. Confirmatory in vitro eight-point dose-response curves for active compounds from EBOV antiviral screen in HepG2 cells.
        • Fig. S5. Additional confirmatory in vitro eight-point dose-response curves for active compounds from EBOV antiviral screen in HepG2 cells.
        • Fig. S6. Evaluation of sertraline and bepridil on endosome acidification.
        • Fig. S7. Evaluation of sertraline and bepridil on EBOV-VLP–GP1,2 trafficking to NPC1+ endolysosomes.
        • Fig. S8. Bepridil and sertraline, at the indicated concentrations, do not inhibit the viability of 293AD cells.
        • Fig. S9. Assessment of bepridil and sertraline on VSV EBOV-GP pseudovirion entry into parental and NPC1-overexpressing CHO cell lines.

        [Download PDF]

        Other Supplementary Material for this manuscript includes the following:

        • Table S1. Results of a three-point dose antiviral screen of 2635 compounds against eGFP-EBOV infection (two Excel files).
        • Table S2. Active compounds identified from preliminary three-point dose antiviral screen against eGFP-EBOV infection (Excel file).

        [Download Tables S1 and S2]

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