Isothiafludine, a novel non-nucleoside compound, inhibits hepatitis B virus replication through blocking pregenomic RNA encapsidation
Abstract
Li YANG1, #, Li-ping SHI1, #, Hai-jun CHEN2, #, Xian-kun TONG1, Gui-feng WANG1, Yang-ming ZHANG2, Wen-long WANG2, Chun-lan FENG1, Pei-lan HE1, Feng-hua ZHU1, You-hua HAO3, Bao-ju WANG3, Dong-liang YANG3, Wei TANG1, Fa-jun NAN2, *, Jian-ping ZUO1, *
1Laboratory of Immunopharmacology, 2Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 3Tongji Hospital of Tongji Medical College, Wuhan 430030, China
Aim: To investigate the action of isothiafludine (NZ-4), a derivative of bis-heterocycle tandem pairs from the natural product leucamide A, on the replication cycle of hepatitis B virus (HBV) in vitro and in vivo.
Methods: HBV replication cycle was monitored in HepG2.2.15 cells using qPCR, qRT-PCR, and Southern and Northern blotting. HBV protein expression and capsid assembly were detected using Western blotting and native agarose gel electrophoresis analysis. The interaction of pregenomic RNA (pgRNA) and the core protein was investigated by RNA immunoprecipitation. To evaluate the anti-HBV effect of NZ-4 in vivo, DHBV-infected ducks were orally administered NZ-4 (25, 50 or 100 mg·kg-1·d-1) for 15 d.
Results: NZ-4 suppressed intracellular HBV replication in HepG2.2.15 cells with an IC50 value of 1.33 μmol/L, whereas the compound inhibited the cell viability with an IC50 value of 50.4 μmol/L. Furthermore, NZ-4 was active against the replication of various drug-resistant HBV mutants, including 3TC/ETV-dual-resistant and ADV-resistant HBV mutants. NZ-4 (5, 10, and 20 μmol/L) concentration-dependently reduced the encapsidated HBV pgRNA, resulting in the assembly of replication-deficient capsids in HepG2.2.15 cells. Oral administration of NZ-4 dose-dependently inhibited DHBV DNA replication in the DHBV-infected ducks.
Conclusion: NZ-4 inhibits HBV replication by interfering with the interaction between pgRNA and HBcAg in the capsid assembly process, thus increasing the replication-deficient HBV capsids. Such mechanism of action might provide a new therapeutic strategy to combat HBV infection.
Keywords: hepatitis B; virus replication; capsid assembly; pregenomic RNA; non-nucleoside compound; isothiafludine; lamivudine; adefovir; entecavir
We thank Dr Meng-ji LU (Institute of Virology, University Hospital of Essen, Essen, Germany) for data analysis and reading of the manuscript.
The work was supported by the Chinese Academy of Sciences (CAS) Knowledge Innovation Project KSCX1-YW-10-03, National 863 Program Fund 2008AA02Z431, National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” 2009ZX09102-024 and 2012ZX09101-113.
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail jpzuo@mail.shcnc.ac.cn (Jian-ping ZUO); fjnan@mail.shcnc.ac.cn (Fa-jun NAN)
Received 2013-09-27 Accepted 2013-11-11
Keywords:
1Laboratory of Immunopharmacology, 2Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 3Tongji Hospital of Tongji Medical College, Wuhan 430030, China
Aim: To investigate the action of isothiafludine (NZ-4), a derivative of bis-heterocycle tandem pairs from the natural product leucamide A, on the replication cycle of hepatitis B virus (HBV) in vitro and in vivo.
Methods: HBV replication cycle was monitored in HepG2.2.15 cells using qPCR, qRT-PCR, and Southern and Northern blotting. HBV protein expression and capsid assembly were detected using Western blotting and native agarose gel electrophoresis analysis. The interaction of pregenomic RNA (pgRNA) and the core protein was investigated by RNA immunoprecipitation. To evaluate the anti-HBV effect of NZ-4 in vivo, DHBV-infected ducks were orally administered NZ-4 (25, 50 or 100 mg·kg-1·d-1) for 15 d.
Results: NZ-4 suppressed intracellular HBV replication in HepG2.2.15 cells with an IC50 value of 1.33 μmol/L, whereas the compound inhibited the cell viability with an IC50 value of 50.4 μmol/L. Furthermore, NZ-4 was active against the replication of various drug-resistant HBV mutants, including 3TC/ETV-dual-resistant and ADV-resistant HBV mutants. NZ-4 (5, 10, and 20 μmol/L) concentration-dependently reduced the encapsidated HBV pgRNA, resulting in the assembly of replication-deficient capsids in HepG2.2.15 cells. Oral administration of NZ-4 dose-dependently inhibited DHBV DNA replication in the DHBV-infected ducks.
Conclusion: NZ-4 inhibits HBV replication by interfering with the interaction between pgRNA and HBcAg in the capsid assembly process, thus increasing the replication-deficient HBV capsids. Such mechanism of action might provide a new therapeutic strategy to combat HBV infection.
Keywords: hepatitis B; virus replication; capsid assembly; pregenomic RNA; non-nucleoside compound; isothiafludine; lamivudine; adefovir; entecavir
We thank Dr Meng-ji LU (Institute of Virology, University Hospital of Essen, Essen, Germany) for data analysis and reading of the manuscript.
The work was supported by the Chinese Academy of Sciences (CAS) Knowledge Innovation Project KSCX1-YW-10-03, National 863 Program Fund 2008AA02Z431, National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” 2009ZX09102-024 and 2012ZX09101-113.
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail jpzuo@mail.shcnc.ac.cn (Jian-ping ZUO); fjnan@mail.shcnc.ac.cn (Fa-jun NAN)
Received 2013-09-27 Accepted 2013-11-11