Inhibitory effect of CDK9 inhibitor FIT-039 on hepatitis B virus propagation
Introduction
Hepatitis B virus (HBV), which is the etiologic agent of hepatitis B in humans, persistently infects more than 350 million people worldwide (El-Serag, 2012) and causes acute and chronic hepatitis, leading to liver cirrhosis and hepatocellular carcinoma (Liang, 2009). Current therapies using interferon (IFN)-alpha and/or nucleos(t)ide analogs for chronic hepatitis B effectively decrease the viral load and ameliorate the clinical conditions (Gish et al., 2015), but the complete elimination of HBV has remained elusive due to the stability of the covalently closed circular (ccc) DNA and the integration of the viral DNA into the host genome.
HBV, which is classified into the genus Orthohepadnavirus of the family Hepadnaviridae, is the enveloped virus that carries the nucleocapsid consisting of a full-length negative and an incomplete positive DNA strand and core proteins (Seeger and Mason, 2015). HBV internalizes a hepatocyte via attachment to sodium taurocholate cotransporting polypeptide (NTCP) (Yan et al., 2012) and releases nucleocapsid into the cytoplasm. The nucleocapsid is delivered into the host nucleus and then is uncoated for its own genomic replication. The partially double-stranded DNA of HBV genome, which is termed relaxed circular (rc) DNA, is repaired by host and viral proteins, resulting in the formation of cccDNA. The synthesis of cccDNA requires viral polymerase and cellular DNA enzymes (Sohn et al., 2009), although the molecular mechanism is not known in detail. The viral genes are transcribed from cccDNA by host RNA polymerase II. The viral transcripts include pregenomic RNA (pgRNA) and three subgenomic mRNAs. The pgRNA is packaged with core proteins and then reverse-transcribed by the viral polymerase, resulting in the formation of nucleocapsid. The nucleocapsid is enveloped to form an infectious viral particle, or is imported again into the nucleus to form new cccDNA.
Several cyclin-dependent kinase (CDK) inhibitors are reported to suppress viral replication, as referred to in a review by Schang et al. (Schang et al., 2006). CDK family members exhibit serine/threonine kinase activity in a cyclin-dependent manner and are responsible for the regulation of cell division or gene transcription. CDK1, CDK4, and CDK5 are involved in the cell cycle, while CDK7, CDK8, CDK9, CDK11, and CDK20 are involved in transcription efficiency (Malumbres, 2014). CDK9 binds to T-type cyclins, resulting in the formation of positive transcription elongation factor-b (P-TEFb). RNA polymerase II C-terminal domain (CTD), which is composed of repeats of the heptapeptide sequence, is phosphorylated by active P-TEFb at the second residue Ser (Ser2) of the heptad repeat, leading to promotion of the transcription elongation of host and viral genes (Bowman and Kelly, 2014, Feichtinger et al., 2011, Ou and Sandri-Goldin, 2013). Development of an antiviral agent targeting a host factor is expected to contribute to a reduction in the risk of the emergence of drug-resistant viruses and establishment of antiviral therapies irrespectively of the viral genotype (Cholongitas et al., 2015). Recently, FIT-039, which is a novel CDK9-specific inhibitor, has been shown to selectively suppress replications of herpes simplex virus (HSV)-1, HSV-2, human cytomegalovirus and HIV irrespective of the host cell viability (Okamoto et al., 2015, Yamamoto et al., 2014). FIT-039 inhibits the kinase activity of CDK9/cyclin T1 complex with an IC50 value of 5.8 μM through binding to the ATP-binding pocket of CDK9, but does not affect the activity of other CDKs (Yamamoto et al., 2014). FIT-039 is being tested as a novel candidate antiviral agent for human papilloma virus under a Phase 1/2 clinical trial.
In this study, we employed HBV-replicating HepG2 cell lines and the HBV infectious cell culture system in order to investigate the antiviral activity of FIT-039. FIT-039 significantly impaired productions of intracellular HBV RNA, nucleocapsid DNA and cccDNA in a dose-dependent manner without remarkable cytotoxic effects. FIT-039 significantly reduced serum HBV DNA in combination with entecavir in vivo. These data suggest that FIT-039 is a promising anti-HBV drug candidate. The inhibitory effect of FIT-039 on HBV may provide an insight into the mechanism of HBV replication.
Section snippets
Reagents and antibodies
FIT-039 was synthesized as described previously (Yamamoto et al., 2014). Flavopiridol and roscovitine were purchased from Santa Cruz (Dallas, TX) and Sigma (St. Louis, MO), respectively. These compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50 mM and stored at −80 °C until use. Mouse monoclonal antibody to HBc antigen was reported previously (Watashi et al., 2014). Mouse monoclonal antibody to beta-actin was purchased from Sigma.
Cells
HepG2/NTCP cell line, which was
FIT-039 inhibited HBV infection
We tested the effect of the CDK9 inhibitor FIT-039 on HBV infection, compared with other CDK inhibitors, flavopiridol and roscovitine. FIT-039 specifically inhibited CDK9, but not other CDKs (Yamamoto et al., 2014). Fravopiridol is a pan-CDK inhibitor targeting CDK1, CDK2, CDK4, CDK5, CDK6, and CDK9 (Schang et al., 2006), while roscovitine is an oligo-specific CDK inhibitor targeting CDK1, CDK2, CDK5, and CDK7 (Khalil et al., 2015). HepG2/NTCP cells were incubated with HBV for 16 h in the
Discussion
The data in this study showed that the CDK9-specific inhibitor FIT-039 suppressed HBV replication at an IC50 value of 0.33 ± 0.16 μM in HBV-infected cells (Fig. 1, Fig. 2). FIT-039 treatment downregulated HBV production at an early infection step after virus attachment (Fig. 3, Fig. 4) and impaired viral replication in HepG2.2.15 and Hep38.7-Tet cell lines, although more than ten times the amount of FIT-039 would be required for suppression of HBV replication in both cell lines compared with
Financial supports
This work was supported by Research Programs on Hepatitis i and ii (KU, YT, MH and KM) and Project of Translational and Clinical Research Core Centers (MH) from the Japan Agency for Medical Research and Development, AMED.
Conflict of interest
MH is collaborating with and has stock in KinoPharma, Inc., which is a venture company developing FIT-039 as a clinical drug.
Acknowledgements
We gratefully thank M. Mori-Furugori for her secretarial work and K. Watashi, T. Wakita and T. Suzuki for kindly providing cell lines.
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The authors contributed equally to this work.