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1 Supplementary Information An orally available, small-molecule interferon inhibits viral replication Hideyuki Konishi 1, Koichi Okamoto 1, Yusuke Ohmori 1, Hitoshi Yoshino 2, Hiroshi Ohmori 1, Motooki Ashihara 1, Yuichi Hirata 3, Atsunori Ohta 1, Hiroshi Sakamoto 1, Natsuko Hada 1, Asao Katsume 1, Michinori Kohara 3, Kazumi Morikawa 2, Takuo Tsukuda 1, Nobuo Shimma 1, Graham R Foster 4, William Alazawi 4, Yuko Aoki 1, Mikio Arisawa 1, & Masayuki Sudoh 1,* 1 Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd., 2 Kajiwara, Kamakura, Kanagawa , Japan, 2 Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd., 135, 1, Komakado, Gotemba, Shizuoka , Japan, 3 Department of Microbiology and Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo , Japan, 4 Queen Mary University of London, Blizard Institute of Cellular and Molecular Science, 4 Newark Street, London E1 4AT, UK. *Corresponding and requests for materials should be addressed to M.S. (sudomsy@chugai-pharm.co.jp) Supplementary Figures 1-14 Supplementary Tables 1-7 (separate file)

2 % of control % of control Replicon Cells that survived IFN-a RO8191 (mm) Supplementary Figure 1. After treatment with various concentrations of RO8191 for 24 h (upper graph) or 7 d (lower graph), the replication levels of HCV RNA (genotype 1b) were analyzed using a luciferase assay, and cell viability was determined using a WST-8 assay. The data are mean values, and the bars indicate the standard deviations based on triplicated results.

3 IFN-a (4 IU/mL) Fold change of non-treated cells RO8191 (2 mm) Supplementary Figure 2. The fold change in RO8191-treated or IFN-a-treated HCV replicon cells is plotted in comparison to untreated cells. All 54,675 probes (black circles) and 318 ISG 23 probes (red circles) are shown. The red lines to the left and below the center black line indicate.5-fold change, and red lines to the right and above the black line indicate 2.-fold change.

4 Fold change of control % of control a b h 2h 4h 8h 24h CXCL1 IP-1 TNF TNF IL-6 IL6 IL-1 IL1 IL-12A IL12A IL-12B IL12B CCL2 CCL2 CCL3 CCL3 CCL4 CCL4 (CXCL1) Post treatment 4h 1 8h 2 24h 3 4h 4 8h 5 24h 6 TNF-a RO8191 Supplementary Figure 3. (a) Gene expression levels of cytokines and chemokines at indicated time points in the RO8191-treated HCV replicon cells by DNA microarray analysis. (b) NF-kB reporter gene was transiently transfected into the Huh-7 cells, and at 48 h after transfection, the cells were treated with.3 ng/ml TNF-α or 5 mm RO8191 and a further 4, 8 or 24 h later, the luciferase activity was measured. The values indicate the mean of fold change.

5 Fold change of control Fold change of control Fold change of control 12 OAS1 mrna 25 MxA mrna 6 PKR mrna IFN-a RO8191 (mm) IFN-a RO8191 (mm) IFN-a RO8191 (mm) Supplementary Figure 4. ISG expression levels were measured using real-time RT-PCR. The values are listed relative to the mrna level of human b-actin and represent the mean fold change induction compared to untreated cells. Total RNA was extracted from Hc cells treated with the indicated concentrations of RO8191 or 1 IU/mL IFN-a for 8 h, and the mrna levels of OAS1, MxA, and PKR (means and SDs) were measured using real-time RT-PCR.

6 Fold change of control Fold change of control % of control Supplementary Figure 5 c a Imiquimod (mm) Oas1b mrna Replicon Cells that survived b STAT1 py-stat1 Control medium RO mm Murine IFN-aA 1, IU/mL MEF WT Tlr3 TLR3 KO TLR4 Tlr4 KO TLR7 Tlr7 KO TLR9 Tlr9 KO RO mm d ISRE-reporter Imiquimod 1 mm JAK inhibitor (-) JAK inhibitor (+) Control RO8191 (mm) Imiquimod (mm) IFN-a (IU/mL) h

7 Supplementary Figure 5. (a) After treatment with various concentrations of imiquimod for 72 h, the replication levels of the HCV replicon were analyzed using a luciferase assay, and cell viabilities were determined using a WST-8 assay. (b) Western blot analyses of the phosphorylated and total protein levels of STAT1 in HCV replicon cells cultured in a medium containing 1 mm RO8191 or 1 mm imiquimod at.5, 1, 2, 4 and 8 h. (c) Total RNA was extracted from TLR KO MEFs cultured in the presence of 5 mm RO8191 or 1, IU/mL murine IFN-aA for 8 h, and the Oas1b mrna level was analyzed using real-time RT-PCR. The data shown are the mean fold change induction compared to untreated cells, and the bars indicate the standard deviations based on triplicated results. (d) ISRE reporter gene activation by RO8191, imiquimod or IFN-a treatment. ISRE reporter gene was transiently transfected into Vero cells, and 48 h later the cells were treated with RO8191, imiquimod or IFN-a with or without a JAK inhibitor I. After an additional 8 h, luciferase activity was measured.

8 % of no transfection % of no transfection % of no transfection % of no transfection % of no transfection % of no transfection % of no transfection % of no transfection STAT1 mrna STAT2 mrna IRF9 mrna JAK1 mrna Ctrl STAT1-1 STAT1-2 sirna (1 nm) Ctrl STAT2-1 STAT2-2 sirna (1 nm) Ctrl IRF9-1 IRF9-2 sirna (1 nm) Ctrl JAK1-1 JAK1-2 sirna (1 nm) Ctrl Tyk2 mrna Tyk2-1 Tyk2-2 sirna (1 nm) ctrl JAK2 mrna JAK2-1 JAK2-2 sirna (1 nm) IFNAR1 mrna Ctrl IFNAR1-1 IFNAR1-2 sirna (1 nm) Ctrl IFNAR2 mrna IFNAR2-1 IFNAR2-2 sirna (1 nm) Supplementary Figure 6. HCV replicon cells transfected with the indicated sirnas (1 nm). Forty-eight hours after transfection, total RNA was extracted, and the mrna levels were analyzed using real-time RT-PCR analysis. The data shown are mean values and SDs relative to the mrna level of human b-actin in non-transfected cells.

9 Inhibition (%) Inhibition (%) RO8191 a si-ifnar b si-ifnar Ctrl sirna sirna-1 sirna-2 IFN-a sirna (nm) sirna (nm) Supplementary Figure 7. The anti-hcv replicon activity of RO8191 was attenuated by a knockdown of IFNAR2 (b) but not IFNAR1 (a). Serial dilution includes the data shown in Fig. 3a and b (5 nm sirna). The inhibition of HCV replicon replication by each agent is shown (the mean and SD from 3 experiments). The HCV replicon cells were transfected with various concentrations of the indicated sirnas (blue, red, and yellow bars). Twenty-four hours after treatment with each agent, the replication levels of HCV RNA were analyzed using a luciferase assay. Forty-eight hours after transfection, the HCV replicon cells were treated with 1.5 mm RO8191 (upper graph) or 3 IU/mL IFN-a (lower graph) for 24 h.

10 Fold change of control Oas1b mrna WT IFNAR1 KO MEF WT Ifnar1 KO Control medium RO mm Murine IFN-aA 1, IU/mL Supplementary Figure 8. Total RNA was extracted from Ifnar1 KO MEFs cultured in the presence of 5 mm RO8191 or 1, IU/mL murine IFN-aA for 8 h, and the Oas1b mrna level was analyzed using real-time RT-PCR. The data shown are the mean fold change induction compared to untreated cells, and the bars indicate the standard deviations based on triplicated results.

11 Inhibition (%) Inhibition (%) Inhibition (%) Inhibition (%) Inhibition (%) Inhibition (%) Inhibition (%) Supplementary Figure 9 RO8191 IFN-a IFN-g RO8191 IFN-a a si-jak sirna (nm) sirna (nm) si-tyk sirna (nm) d e f b sirna (nm) RO8191 si-stat1 si-stat2 si-irf-9 IFN-g c sirna (nm) si-jak sirna (nm) Ctrl sirna sirna-1 sirna-2

12 Supplementary Figure 9. The inhibition of HCV replicon replications by each agent is shown (means and SDs). Serial dilution includes the data shown in Fig. 4a e (5 nm sirna). The HCV replicon cells were transfected with various concentrations of the indicated sirnas (blue, red, and yellow bars). The anti-hcv replicon activity of RO8191 was reduced by a knockdown of JAK1 (a), but not Tyk2 (b) or JAK2 (c). Forty-eight hours after transfection, the HCV replicon cells were treated with 1.5 mm RO8191 (upper graph), 3 IU/mL IFN-a or 5 ng/ml IFN-g (lower graph). Twenty-four hours after treatment with each agent, the replication levels of HCV RNA were analyzed using a luciferase assay. The anti-hcv replicon activity of RO8191 was reduced by knockdowns of STAT2 (e) and IRF9 (f), but not STAT1 (d). Forty-eight hours after transfection, the HCV replicon cells were treated with 1.5 mm RO8191 (top graph), 3 IU/mL IFN-a (middle graph) or 5 ng/ml IFN-g (bottom graph). Twenty-four hours after treatment, the replication levels of HCV RNA were analyzed using a luciferase assay.

13 a Control sirna si-jak1-1 si-jak1-2 b Control sirna si-tyk2-1 si-tyk2-2 STAT1 NT 8191 IFN NT 8191 IFN NT 8191 IFN STAT1 NT 8191 IFN NT 8191 IFN NT 8191 IFN py-stat1 py-stat1 JAK1 Tyk2 Supplementary Figure 1. The phosphorylation of STAT1 was attenuated by knockdown of JAK1 (a) but not Tyk2 (b). The HCV replicon cells were transfected with the indicated sirnas (1 nm). Forty-eight hours after transfection, the cells were treated for 15 min with 1 mm RO8191 or 2 IU/mL IFN-a. Total lysates were subjected to western blot analysis in order to analyze the phosphorylated and total protein levels of STAT1.

14 Fold change of control OAS1 mrna 4 35 Control medium RO mm IFN-a 1 IU/mL fTGH (Tyk2-WT) 2fTGH U1A (Tyk2-null) U1A Supplementary Figure 11. Total RNA was extracted from 2fTGH and U1A cells cultured in the presence of 5 mm RO8191 or 1 IU/mL IFN-a for 8 h, and the OAS1 mrna level was analyzed using real-time RT-PCR. The data shown are the mean fold change induction compared to untreated cells, and the bars indicate the standard deviations based on triplicated results.

15 Fold change of control OAS1 mrna Ctrl sirna sistat1-1 sistat1-2 Supplementary Figure 12. Sixty-four hours after indicated 1 nm sirna transfection, we treated the cells with 1 mm RO8191 for 8h. Then, RNA was extracted from the cells and OAS1 expression was determined by RT-PCR. The data are mean values, and the bars indicate the standard deviations.

16 Fold change of HCV titer at day-1 Non-treatment 1 RO8191 (3 mg/kg) PEG-IFN-a2a (3 mg/kg).5 * P=.18.1 ** P=.7 day - 1 day 1 day 3 day 7 day 1 day 14 Supplementary Figure 13. The mice were treated for 14 days with RO mg/kg/day orally or PEG-IFN-a2a 3 mg/kg subcutaneously twice weekly. Time course of serum HCV RNA levels in mice treated with RO8191 (red line, n=5), PEG-IFN-a2a (blue line, n=4) or no treatment (green line, n=8) is shown. The data are mean values, and the bars indicate the standard deviations.

17 Cell viability after infection with EMCV (%) Cell viability after infection with EMCV (%) Ctrl sirna sistat1 sistat EMCV (-) (+) No drug RO8191 IFN-a Control medium RO8191 IFN-a + EMCV Supplementary Figure 14. We transfected 5 nm STAT1- or STAT2-siRNA to A549 cells, and after 72 h we infected EMCV to the cells and treated them with 1 mm RO8191 or 2 IU/mL IFN-a. After additional 48 h incubation, we evaluated the cell viability by staining with crystal violet.

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