HBV remains a major global health problem with approx. 240 million people chronically infected, despite the availability of prophylactic vaccines for more than 30 years. Patients chronically infected with HBV are at high risk of developing liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Standard of care includes (long-term) pegylated interferon and/or nucleo(t)ide analogs, which suppress viral replication but rarely control viral replication and achieve functional cure. This brings the risk of side effects and selection for drug-resistant variants with loss of drug susceptibility. The unmet medical need for chronic hepatitis B (CHB) patients is functional cure: a finite (combination) therapy that can control virus replication and maintain undetectable HBsAg and HBV DNA levels six months after cessation of treatment.

HBV is considered a “stealth” virus in early infection that evades recognition of innate immune sensors by multiple strategies. HBV polymerase was reported to inhibit RIG-I and TLR-3 mediated IFN-β induction (Yu, Chen et al. 2010) and disrupt the K63-linked ubiquitination of STING pathway (Liu, Li et al. 2015). HBx protein counteracts innate immunity by downregulating the RIG-I-MAVS signaling pathways (Wei et al., 2010, Wang et al., 2020). Activation of innate sensing pathways has been shown to enhance antiviral response against HBV (Lucifora et al., 2010, Luangsay et al., 2015, Zhang and Lu, 2015). Triphosphate-HBx-siRNAs have been shown to reverse HBV-induced immune tolerance in a RIG-I-dependent manner (Ebert et al., 2011, Han et al., 2011, Han et al., 2019).

Pattern recognition receptors (PRRs) like TLRs, STING, and RIG-I/MDA5 play important roles in the induction of innate immunity through sensing of pathogen-associated molecular patterns (PAMPs). RIG-I is a widely expressed cytosolic DExD/H box RNA helicase and preferentially binds to short blunt dsRNA sequences with 5′-phosphates (Schlee et al., 2009, Schmidt et al., 2009). Activation of RIG-I results in the oligomerization of the signaling adaptor MAVS and triggers the TBK1-IRF3 signaling cascade that stimulates the production of IFN-β. IFN-β binds to the cognate receptor and induces the expression of interferon stimulated genes (ISGs). Small molecules targeting the RIG-I signaling pathway have been shown to elicit a potent antiviral response both in vitro and in vivo. SB9200 (inarigivir), an oral small molecule nucleic acid hybrid RIG-I agonist exerts anti-HBV activity in the woodchuck model with a dose-response reduction of HBsAg and HBV DNA (Suresh, Korolowicz et al. 2017). However, the clinical development was discontinued due to the occurrence of unexpected serious adverse events (Smolders, Burger et al. 2020). Small molecule IRF3 agonists that can activate RIG-I-like receptor (RLR) pathway and induce innate immune gene expression displayed highly potent antiviral activity against a broad range of RNA viruses including West Nile virus, dengue virus, hepatitis C virus (HCV), and influenza virus. In addition, one of the benzothiazole derivatives, F7, suppresses HBV cccDNA in HBV-infected cells by directing a specific RIG-I-dependent innate immune response (Lee S 2021). These observations support the therapeutic potential of small molecules targeting RIG-I signaling pathways as antiviral drugs. However, the balance between therapeutic effectiveness and toxicity remains a challenge.

Here, we established an HBV specific-IFN-β luciferase reporter assay to screen for small molecules that stimulate the RIG-I signaling pathway as an HBV antiviral. A high throughput screen was performed with a small molecule diversity library and the identified leading compound JJ#1 induced high levels of IFN-β promoter-driven luciferase signal without cytotoxicity up to the maximum concentration tested. Western blot analysis demonstrated JJ#1 activated the RIG-I signaling pathway by inducing the phosphorylation of TBK1 and STAT1. Moreover, JJ#1 induced the expression of ISGs and decreased HBsAg level in HBV minicircle DNA transfected HepaRG cells, supporting its potential as an antiviral therapeutic.