Cinnamic acid derivatives inhibit hepatitis C virus replication via the induction of oxidative stress
Introduction
Hepatitis C virus (HCV), which is the major causative agent of chronic liver diseases such as steatosis, cirrhosis and hepatocellular carcinoma, affects at least 71 million people worldwide (WHO, 2017). Over the past decade, combination therapy using pegylated interferon α and ribavirin has been employed for hepatitis C. Recently, tremendous efforts to develop efficient direct acting antivirals (DAAs) demonstrated the achievement of sustained viral response (SVR) in over 95% of cases with generally minor side effects after 12 to 24 weeks of treatment with DAAs (Asselah et al., 2016). However, several issues regarding DAA therapy remain (Pawlotsky, 2016). 1) Ribavirin, which exhibits moderate side effects, is still required for many groups of hepatitis C patients. 2) Treatment with DAA achieves a low SVR for genotype 3 patients, compared with other genotype patients. 3) DAA medication is extremely expensive in developed countries. 4) Treatment with DAA should be carefully applied to patients with underlying disorders including kidney disorders and patients co-infected with hepatitis B virus (HBV). Thus, further development of effective and safe anti-HCV agents remains necessary for the settlement of these issues.
Cinnamic acid is an organic chemical mainly isolated from cinnamon, and its derivatives are known as plant hormones correlated with the regulation of cell growth and differentiation. Natural and synthetic cinnamic acid derivatives were reported to exhibit multiple biological activities including anti-inflammatory, anti-microbial, anti-oncogenic, anti-oxidant and/or kinase-inhibitory effects (De et al., 2011, Mielecki and Lesyng, 2016, Sova, 2012). Several cinnamic acid derivatives have been known to suppress the propagations of several viruses. 3,5-Dicaffeoylquinic acid, 1-methoxyoxalyl-3,5-dicaffeoylquinic acid, and L-chicoric acid inhibited the enzymatic activity of human immunodeficiency virus type 1 integrase (Robinson et al., 1996). Synthetic cinnamyl derivatives of thieo[2,3-d]oxazinoes impaired the productions of herpes simplex virus type (HSV) 2, varicella zoster virus, and cytomegalovirus (Jarvest et al., 1999). Carboxylated lignins based on a cinnamic acid scaffold exhibited potent inhibitory effects on HSV-1 entry (Thakkar et al., 2010). The compound synthesized by attaching cinnamic acids to caudatin and its derivatives suppressed HBV promoters and enhancers, resulting in the inhibition of HBV replication (Wang et al., 2012). However, the effects of cinnamic acid on HCV infection have not yet been fully elucidated.
The activation of signal transducer and activator of transcription 3 (STAT3) is dependent on the phosphorylation of Tyr705 in response to several cytokines, including interleukin-6 (IL-6), leukemia inhibitory factor, epidermal growth factor and oncostatin M (Bromberg and Darnell, 2000, Wen et al., 1995, Zhong et al., 1994). The binding of IL-6 to its receptor leads to receptor dimerization and the activation of JAK1/2. Activated JAK1 and 2 can phosphorylate STAT3 Tyr705. Phosphorylated STAT3 proteins are homo-dimerized and then translocated into the nucleus for the promotion of targeted gene expression (Heinrich et al., 1998). STAT3 was reported to play a role in HCV replication (McCartney et al., 2013, Waris et al., 2005) and to associate with HCV-induced carcinogenesis (Yoshida et al., 2002). AG490, which is classified in the cinnamic acid group as a JAK2 inhibitor, specifically inhibits the phosphorylation of STAT3 Tyr705 (Mielecki and Lesyng, 2016) and suppresses HCV propagation (McCartney et al., 2013, Waris et al., 2005), suggesting that the phosphorylation of STAT3 Tyr705 is critical for the HCV life cycle.
In this study, we prepared cinnamic acid derivatives based on an AG490 scaffold and screened them using HCV replicon cells to identify novel antivirals against HCV. We also analyzed the mode of action of hit compounds.
Section snippets
Cell culture and luciferase reporter assay
The Huh7/Rep-Feo cell line harboring the subgenomic replicon RNA of the N strain (genotype 1b) was reported previously (Yokota et al., 2003). The HCV replicon cell line derived from strain JFH1 (genotype 2a) was described previously (Nishimura-Sakurai et al., 2010). The S52/SG-Feo (AI) cell line (genotype 3a) and ED43/SG-Feo (VYG) cell line (genotype 4a) were kindly provided by C. M. Rice (Saeed et al., 2012, Saeed et al., 2015). These replicon cell lines were maintained in Dulbecco's modified
Effect of cinnamic acid derivatives on HCV replication
To identify novel antivirals against HCV infection, we prepared 17 cinnamic acid derivatives based on an AG490 scaffold, and we screened them using genotype 1b replicon cells to identify more potent antiviral than AG490 (Table 1). AG490 could inhibit the replication of genotype 1b replicon with an EC50 value of 15.2 ± 0.8 μM (Supplementary Fig. 1). Each compound was added to the culture supernatant at a final concentration of 10 μM, which is the lowest concentration of AG490 among
Discussion
In this study, we screened 17 synthetic cinnamic acid derivatives to identify an inhibitor against HCV replication. Compound 6 was identified as an effective HCV antiviral by comparing its activity with activities of other compounds (Fig. 1, Table 2). Compound 6 inhibited the viral replication in replicon cell lines of genotypes 1b, 2a, 3a and 4a with EC50 values of 1.4–8.1 μM and SI values of 16.2–94.2 (Fig. 2, Table 3), but it did not affect ISRE promoter activity and expressions of ISGs in
Conclusion
In this study, our data suggest that a synthetic cinnamic acid derivative, compound 6, significantly inhibits HCV replication via the induction of oxidative stress. Further studies on the modification of this cinnamic acid derivative for the improvement of anti-HCV activity may lead to novel therapeutic strategies for chronic hepatitis C.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
We gratefully thank M. Mori-Furugori for her secretarial work and N. Sakamoto, T. Wakita, F. L. Cosset, R. Bartenschlager, and C. M. Rice for kindly providing the cell lines and plasmids. This work was supported by Research Programs from the Japan Agency for Medical Research and Development (16fk0210109h1301 and 16fk0210106h0001), from JSPS KAKENHI grants No. JP 15K08493, and from a scholarship donation from Yakult Co. Ltd.
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The authors contributed equally to this work.