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1 Supplementary Materials 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* This PDF file includes: *Corresponding author. gene.olinger@nih.gov Published 3 June 2015, Sci. Transl. Med. 7, 290ra89 (2015) DOI: /scitranslmed.aaa5597 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 GP 1,2 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. Other Supplementary Material for this manuscript includes the following: (available at

2 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).

3 Fig. S1. Comparison of compound activity in the antiviral assay against egfp-ebov infection versus uninfected host cell proliferation. All compounds and their corresponding activities are displayed in gray. The maximum percent inhibition of Vero E6 proliferation is displayed along the horizontal axis versus the maximum percent inhibition of antiviral activity in the egfp-ebov assay along the vertical axis. Compounds selected as active inhibitors of egfp-ebov are displayed in red and yellow. Red represents actives identified in the 3-point screen but were not selected to advance to 8-point confirmation. The set of 30 active prioritized compounds is displayed in yellow. Compounds selected as active compounds have antiviral activity above 40% inhibition. In general, active compounds had a low impact on cell proliferation. In some cases, compounds selected as active displayed anti-proliferative activity at the highest concentration evaluated but demonstrated a therapeutic window at lower concentrations.

4 Fig. S2. Confirmatory in vitro eight-point dose-response curves for active compounds from EBOV antiviral screen in Vero E6 cells. The % inhibition in the antiviral EBOV assay by the

5 compounds is shown in blue, and the cytotoxic effect of the compounds on the host cells is shown in red. The maximum % inhibition observed (Max response) and IC 50 are indicated. Error bars indicate the SEM. Results are from a minimum of two replicates. Data for the dose response curves are from Table I.

6 Fig. S3. Additional confirmatory in vitro eight-point dose-response curves for active compounds from EBOV antiviral screen in Vero E6 cells. The % inhibition in the antiviral EBOV assay by the compounds is shown in blue, and the cytotoxic effect of the compounds on host cells is shown in red. The maximum % inhibition observed (Max response) and IC 50 are indicated. Error bars indicate the SEM. Results are from a minimum of two replicates. Data for the dose response curves are from Table I.

7 Fig. S4. Confirmatory in vitro eight-point dose-response curves for active compounds from EBOV antiviral screen in HepG2 cells. The % inhibition in the antiviral EBOV assay by the compounds is shown in blue, and the cytotoxic effect of the compounds on the host cells is shown in red. The maximum % inhibition observed (Max response) and IC 50 are indicated. Error bars indicate the SEM. Results are from a minimum of two replicates. Data for the dose response curves are from Table I.

8 Fig. S5. Additional confirmatory in vitro eight-point dose-response curves for active compounds from EBOV antiviral screen in HepG2 cells. The % inhibition in the antiviral EBOV assay by the compound is shown in blue, and the cytotoxic effect of the compounds on the host cell is shown in red. The maximum % inhibition observed (Max response) and IC 50 are indicated. Error bars indicate the SEM. Results are from a minimum of two replicates. Data for the dose response curves are from Table I.

9 Fig. S6. Evaluation of sertraline and bepridil on endosome acidification. 293AD cells were pre-incubated with DMSO, 10 mm NH 4 Cl (positive control), or the indicated concentrations of sertraline or bepridil for 1 hour at 37 C. At this time LysoTracker Red (+/- inhibitor, as indicated) was added, and the cells were incubated for an additional 60 minutes (at 37 o C). Cells were then imaged with a fluorescent microscope (within 30 minutes). A representative image of each sample is shown. At 5 µm, neither sertraline nor bepridil had a strong impact on endosome acidification. With 10 µm, inhibition was seen for both compounds. Scale bars= 40 µm

10 Fig. S7. Evaluation of sertraline and bepridil on EBOV-VLP GP 1,2 trafficking to NPC1 + endolysosomes. The figure shows representative confocal microscopic images from the experiment depicted in Fig. 6C. 293AD cells were pretreated with vehicle (DMSO, mock) or drugs prior to addition of EBOV VLP-GP 1,2 tagged with mcherry-vp40. Scale bars = 10 µm. Green, staining for NPC1; red, EBOV VLP-GP 1,2 (mcherry VP40); yellow (arrows), regions of overlap (colocalization). Scale bars = 10 µm.

11 Fig S8. Bepridil and sertraline, at the indicated concentrations, do not inhibit the viability of 293AD cells. Cells were pre-incubated and treated with DMSO (0 µm drug) or the indicated concentrations of sertraline or bepridil for the same times of incubations as for the VLP entry assay, but substituting DMEM for the courmarin cephalosporin fluorescein (CCF2) loading buffer. Cells were then lysed, and viability was measured using the Cell Titer-Glo luminescent cell assay kit (Promega) according to the manufacturer s instructions using a BioTek HT microplate reader. All samples were analyzed in triplicate and errors bars represent standard deviation of the mean.

12 Fig. S9. Assessment of bepridil and sertraline on VSV EBOV-GP pseudovirion entry into parental and NPC1-overexpressing CHO cells. Parental CHO cells (gray line, WT) and CHO cells stably overexpressing NPC1 (black line, NPC1) were pretreated with the indicated concentrations of (A) bepridil (2 experiments in triplicate), (B) sertraline (3 experiments in triplicate), (C) U18666A (positive control, 4-7 experiments in triplicate), or (D) E64d (negative control, 1 experiment in duplicate) or DMSO (mock) for 1 hour at 37 o C. Subsequently, cells were infected with VSV-pseudovirions encoding GFP with EBOV GP mucin for 18 hours in the continued presence of the compounds. At this time the cells were analyzed for GFP expression by flow cytometry. Dose shifts were observed for (A) bepridil and (B) sertraline, although the magnitude of shift appears lower than that observed with U18666A. Similar results for U18666A (dose shift) and E64d (no dose shift) were presented in (14). Entry values were normalized to DMSO-treated samples and averaged across experiments. Error bars represent standard error.

13 Table S3: Survival of ma-ebov infected C57BL/6 mice after treatment with additional priority active compounds identified in vitro. Compound Dose Regimen Survival at MTD (days) p-value day 28 Clomipramine 45 mg/kg QD 40% Lomerizine 22 mg/kg QD 30% Aripiprazole* 20 mg/kg QD 10% 8 N/A Teicoplanin 14 mg/kg QD 0% 8 N/A Paroxetine 15 mg/kg QD 0% 8 N/A Efavirenz 15 mg/kg QD 0% 8 N/A Animals were treated 1 hour following viral challenge and were treated for a total of 10 days. Animal survival was monitored out to 28 days. A low-to-modest percentage of animals survived following treatment with clomipramine and lomerizine, that reached significance with clomipramine. The p-value was determined using Fisher s exact test comparing vehicle- and compound-treated mice. *Extreme somnolence preventing eating and drinking was observed in mice treated with aripiprazole, and these side effects likely contributed to poor survival observed in these animals. Dosing optimization is recommended for these compounds to fully evaluate their in vivo efficacy. QD, once daily dosing; MTD, mean time to death.