An orally available, small-molecule interferon inhibits viral replication

Abstract

Most acute hepatitis C virus (HCV) infections become chronic and some progress to liver cirrhosis or hepatocellular carcinoma. Standard therapy involves an interferon (IFN)-α-based regimen, and efficacy of therapy has been significantly improved by the development of protease inhibitors. However, several issues remain concerning the injectable form and the side effects of IFN. Here, we report an orally available, small-molecule type I IFN receptor agonist that directly transduces the IFN signal cascade and stimulates antiviral gene expression. Like type I IFN, the small-molecule compound induces IFN-stimulated gene (ISG) expression for antiviral activity in vitro and in vivo in mice, and the ISG induction mechanism is attributed to a direct interaction between the compound and IFN-α receptor 2, a key molecule of IFN-signaling on the cell surface. Our study highlights the importance of an orally active IFN-like agent, both as a therapy for antiviral infections and as a potential IFN substitute.

Figure 1. Identification of a small molecule that inhibits HCV replication.

(a) The chemical structure of RO8191. (b) After treatment with various concentrations of RO8191 or 100 IU/mL IFN-α for 72 h, HCV replication levels were examined using a luciferase assay (left graph), and cell viabilities were determined using a WST-8 assay (right graph). The mean values and their SDs were recorded for treated cells as a percentage of the values for untreated cells, and the values represent the means of 3 independent experiments. (c) Total RNA was extracted from HCV replicon cells cultured with the indicated concentration of RO8191 or 100 IU/mL IFN-α for 72 h; HCV RNA levels were analyzed using real-time RT-PCR. The mean values and their SDs were recorded for treated cells relative to the mRNA levels of β-actin, and are shown as a percentage of untreated cells. The values represent the means of 3 independent experiments. (d) HCV replicon cells were treated with control medium (left panels) or 10 μM RO8191 (right panels) for 24 h and immunostained with Hoechst 33452 (blue), anti-NS3 antibody (green), and anti-NS4A antibody (red). The results were then merged (yellow). (e) HCV replicon cells were treated with the indicated concentrations of RO8191 or 100 IU/mL IFN-α for 72 h. Whole cell lysates were immunoblotted with antibodies specific to the indicated HCV NS proteins. (f) After infection with the HCV JFH1 strain, Huh-7/K4 cells were treated with the indicated concentrations of RO8191 for 72 h. Total RNA was extracted, and the HCV RNA levels were analyzed using quantitative real-time RT-PCR.

Figure 2. RO8191 activates JAK/STAT and induces IFN-like signals.

(a) The cytopathic effect of EMCV infection was inhibited by the indicated concentrations of RO8191 or IFN-α. Viable cells were stained with crystal violet. The data shown are the mean values and SDs based on experiments performed in quadruplicate. (b) ISG expression levels were measured using real-time RT-PCR. Total RNA was extracted from HCV replicon cells cultured in the presence of 50 μM RO8191 or 100 IU/mL IFN-α for 2 or 8 h and known ISGs were analyzed. Asterisk indicates 2-h treatment with agents. The values shown are the mean fold change induction compared to the mRNA level of human β-actin and the fold change induction compared to untreated cells. (c) HCV replicon cells were treated with various concentrations of the indicated agents for 15 min. Total lysates were immunoblotted with antibodies to STATs or JAK kinases. The HCV replicon IC₅₀ of IFN-α was 0.4 IU/mL, that of IFN-β was 3 IU/mL, and that of IFN-γ was 0.3 ng/mL.

Figure 3. RO8191 requires and binds IFNAR2.

(a, b) The anti-HCV replicon activity of RO8191 was attenuated by knockdown of IFNAR2 (b), but not IFNAR1 (a). Inhibition of HCV replicon replication by each agent is shown (the mean and SD from 3 experiments). The HCV replicon cells were transfected with 50 nM of the indicated siRNAs (blue, red, and yellow bars). Forty-eight hours after transfection, the HCV replicon cells were treated with 1.5 μM RO8191 or 3 IU/mL IFN-α for 24 h. Twenty-four hours after treatment with each agent, the replication levels of HCV RNA were analyzed using a luciferase assay. (c, d) U5A cells that lack IFNAR2 were transfected with either an empty vector or a vector expressing the IFNAR2 gene. (c) Forty-eight hours after transfection, the cells were treated with 50 μM RO8191 (red bars) or 100 IU/mL IFN-α (yellow bars). After an additional 8 h of incubation, total RNA was extracted from the U5A cells, and the OAS1 mRNA level was measured using real-time RT-PCR. The values shown are relative to the mRNA level of human β-actin. (d) Forty-eight hours after transfection, the cells were lysed, and the whole cell lysates were immunoblotted with the indicated antibodies. (e) Real-time kinetic SPR analysis of the binding of RO8191 to the IFNAR2 ECD (red and blue lines). The results are consistent with 1:1 binding. PEG-IFN-α2a was also injected as a positive interacting control for IFNAR2 (black line, K_D: 30 nM). (f, g) The phosphorylation of STAT1 was attenuated by a knockdown of IFNAR2 (g) but not IFNAR1 (f). The HCV replicon cells were transfected with the indicated siRNAs (10 nM). Forty-eight hours after transfection, the cells were treated for 15 min with 10 μM RO8191 or 200 IU/mL IFN-α. The total lysates were subjected to western blot analysis to analyze the phosphorylated and total protein levels of STAT1. The data were statistically analyzed using Student's t-test.

Figure 4. RO8191 activates a novel IFN-like pathway.

(a–e) The inhibition of HCV replicon replications by each agent is shown (mean and SD). The HCV replicon cells were transfected with 50 nM of the indicated siRNAs (blue, red, and yellow bars). (a, b) The anti-HCV replicon activity of RO8191 was reduced by knockdown of JAK1 (a) but not Tyk2 (b). Forty-eight hours after transfection, the HCV replicon cells were treated with 1.5 μM RO8191 or 3 IU/mL IFN-α. Twenty-four hours after treatment with each agent, the replication levels of HCV RNA were analyzed using a luciferase assay. (c–e) The anti-HCV replicon activity of RO8191 was reduced by knockdowns of STAT2 (d) and IRF9 (e), but not STAT1 (c). Forty-eight hours after transfection, the HCV replicon cells were treated with 1.5 μM RO8191, 3 IU/mL IFN-α, or 50 ng/mL IFN-γ. Twenty-four hours after treatment, the replication levels of HCV RNA were analyzed using a luciferase assay. (f) A schematic showing the pathways of RO8191 and IFN-α. The data were statistically analyzed using Student's t-test, and differences were considered significant at p values < 0.05.