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    • The effect of microtubule inhibitors on HBV infection and

    2022-09-22

    The effect of microtubule inhibitors on HBV infection and replication were reported previously. In the study by Dr. Ai-Long Huang's group, microtubule inhibitors promoted HBV replication in Hep2.2.15 and HepG2-HBV1.1, in which HBV DNA is integrated into the chromosomal DNA (Xu et al., 2015). Integrated DNA may be affected by nearby genes or cis-elements and it is difficult to distinguish between HBV transcription from cccDNA and integrated DNA in these cell lines. However, we showed that KX2-391 inhibited HBV transcription in HBV-infected primary cells, such as PXB cells. Our experimental condition of HBV replication reflects a more natural condition than the use of Hep2.2.15 and HepG2-HBV1.1 cells. In a study by Dr. Michael Kann's group, microtubules were involved in transport of the intracytoplasmic HBV galanin receptor to the nucleus (Rabe et al., 2006). They used a lipid-based delivery of HBV capsid into HBV non-susceptible cells that do not express NTCP. Using this system, the authors observed an active microtubule-dependent capsid transfer to the nucleus. However, it is unclear whether NTCP-dependent HBV entry requires an active microtubule. In addition, we observed that KX2-391 did not affect the HBV entry step (Fig. 3). HBV transcription is initiated by the Precore, S1, S2 and X promoters (Moolla et al., 2002). KX2-391 specifically inhibited the HBV precore promoter but not the S1, S2 and X promoters. The HBV precore promoter is regulated by several transcription factors and nuclear receptors including C/EBP, HNF1, HNF3, HNF4, RXR, PPAR, COUP-TF1, and ARP1 (Gilbert et al., 2000, Quasdorff and Protzer, 2010). Of note, HNF4A expression stimulated Precore RNA and Core RNA from the precore promoter (Quasdorff and Protzer, 2010, Schrem et al., 2002, Yu and Mertz, 1997, Yu, 2003). In this study, treatment with KX2-391 or vincristine decreased HNF4A mRNA levels (Fig. 5F). In a previous report, HNF4A was suppressed at the transcription level by PXR, which was activated by the tubulin inhibitors vinblastine and paclitaxel (Kodama et al., 2015, Smith et al., 2010, Nallani et al., 2003). Therefore, the inhibitory effect of tubulin polymerization by KX2-391 may suppress HNF4A expression mediated by the activation of PXR, resulting in diminished precore promoter activity. In a phase I dose escalation study of KX2-391 in patients with solid tumors, the blood level of KX2-391 was less than 150 ng/ml at 4 h after treatment with 80 mg twice-daily (Naing et al., 2013). We calculated the concentration of KX2-391 required for use as an anti-HBV drug in vivo by using the model reported by Antonarakis et al. (2013). KX2-391 was 83% protein-bound in the serum, which correlates with an approximate 4-fold reduction in KX2-391 potency. The EC50 for HBV inhibition in PXB cells was 140 nM in the absence of human plasma and 560 nM in the presence of human plasma: 560 nM of KX2-391 corresponds to a plasma concentration of 241.92 ng/ml. KX2-391 partitioned into tumor tissue vs plasma at a ratio of 1.52. This reduced the required plasma concentration 1.52-fold. Therefore, 367 ng/mL of KX2-391 in plasma is required to inhibit HBV replication. However, KX2-391 plasma levels do not reach a level that can inhibit HBV replication in patients treated with oral KX2-391 at a dose of 80 mg twice daily. Therefore, further study is required before KX2-391 can be used as an anti-HBV drug in patients. In addition, KX2-391 suppressed cell growth under subconfluent conditions (Fig. 2D), and this suppression might be unfavorable for an anti-HBV treatment that requires administration over months to years. More studies of the mechanism of transcription inhibition by tubulin inhibitors are needed to discriminate between antiviral activity and cytotoxicity.
    Acknowledgments We thank Ms. Hiromi Yamamoto, Ritsuko Shiina and Atsuno Kaneto for technical assistance. This work was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Health, Labor, and Welfare of Japan (H24-006) and by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (22114004) and Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (15K19121) and by the Research Program on Hepatitis from Japan Agency for Medical Research and Development, AMED (17fk0310104h0001).