br Introduction Hepatitis B virus HBV infects about billion

Introduction Hepatitis B virus (HBV) infects about 2 billion people worldwide and is a leading cause of liver disease including liver fibrosis, cirrhosis and hepatocellular carcinoma (Trepo et al., 2014). Most current HBV therapies are based on nucleos(t)ide analogs and pegylated interferon alpha (PEG-IFN) plus ribavirin. Although these therapies effectively suppress virus proliferation they do not completely remove virus covalently closed circular DNA (cccDNA) from infected cells, allowing them to exist as reservoirs that contribute to viral re-emergence after treatment interruption or emerging immuno-suppressive conditions (Yuen and Lai, 2001, Raimondo et al., 2007). Therefore, the development of anti-HBV drugs that target the different steps of virus replication is needed to eradicate HBV. To identify host factors as well as low molecular weight agents that regulate HBV replication, pUC1.2xHBV/NL, a recombinant virus producing plasmid, was constructed by deleting a section of the first codon of the HBV precore coding frame and inserting the NanoLuc gene. Recombinant HBV/NL was prepared by transfecting HepG2 AZD 3965 with pUC1.2xHBV/NL and pUC1.2xHBV-D plasmids that produce all HBV proteins but cannot produce self-replicating virus. NL activity in HBV/NL infected primary hepatocytes or NTCP-transduced human hepatocyte derived cell lines increased linearly for several days after infection and correlated with cellular HBV RNA levels. Treatment with HBV inhibitors such as heparin and cyclosporine A during the period of infection reduced NL activity in a dose-dependent manner. HBV/NL can be used to monitor HBV replication between the virus entry and transcription steps. We demonstrated that this system provides a unique opportunity for studying the mechanisms of HBV replication and for the high-throughput, cost-effective screening of anti-HBV compounds (Nishitsuji et al., 2015). Taking advantage of this feature, we examined the effect of 1827 US Food and Drug Administration approved compounds that affect HBV replication using HBV/NL. We identified several agents that affected the early stage of HBV infection/replication. Among them, KX2-391, a known non-ATP-competitive inhibitor of SRC kinase and tubulin polymerization (Antonarakis et al., 2013, Anbalagan et al., 2012, Fallah-Tafti et al., 2011), had a suppressive effect on HBV infection. A time of addition assay revealed that KX2-391 suppressed HBV transcription. To delineate the steps involved in its suppressive function, an in vitro promoter assay was conducted. KX2-391 inhibited the HBV precore promoter containing enhancer II. Suppression of HBV transcription by KX2-391 was dependent on the inhibitory effect of tubulin polymerization but not SRC kinase activity. Because some tubulin polymerase inhibitors also suppress transcription from the HBV precore promoter, KX2-391 might exert a similar mechanism. Enhancing our understanding of the detailed mechanisms involved in the anti-HBV suppressive function of KX2-391 may aid the development of a new therapeutic strategy for HBV.
Materials and methods
Results
Discussion To identify novel anti-HBV agents, we screened 1827 commercially available bioactive compounds using recombinant HBV/NL that mimics the early events of the HBV life cycle. Of these compounds, 218 suppressed NL activity in HBV/NL infected HepG2/NTCP cells. Moreover, 31 compounds significantly inhibited HBV replication in PXB cells derived from primary human hepatocytes propagated in mice. Cyclosporin A and Irbesartan that were previously shown to have anti-HBV functions (Watashi et al., 2014, Wang et al., 2015), were also identified as suppressors of NL activity using this assay, indicating this system is applicable for screening candidate anti-HBV agents. Among the 31 compounds, KX2-391, a known SRC kinase and tubulin polymerization inhibitor, was identified as a new candidate HBV inhibitor. The current study showed that the inhibition of tubulin polymerization by KX2-391 suppressed HBV by inhibiting the transcription of the gene encoding a precore protein. However, the specific knockdown of mRNA for SRC kinase, a target of KX2-391, did not affect HBV replication (Fig. 4B and C). Previous studies reported that HBx protein enhanced viral DNA replication by stimulating SRC kinase, which in turn activated Pyk2, a cytoplasmic calcium-activated kinase, thus enhancing mitochondrial calcium signaling (Bouchard et al., 2001, Wang et al., 2009, Wu et al., 2010). The anti-HBV function of cyclophilin A correlated with the disruption of mitochondrial calcium signaling by binding to various cyclophilins including mitochondrial cyclophilins and preventing Pyk2 activation, which is a target of HBx (Bouchard et al., 2001). However, the time of drug-addition experiment revealed cyclosporin A inhibited HBV infection at the entry step but not during DNA replication (data not shown). Moreover, other SRC kinase inhibitors (Saracatinib and PP2) did not affect wild type HBV (Fig. 4D), indicating that SRC signaling is not required for the HBV life cycle under these cell conditions.