Isothiafludine, a novel non-nucleoside compound, inhibits hepatitis B virus replication through blocking pregenomic RNA encapsidation.
OBJECTIVE
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, 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.


CONCLUSIONS
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.
Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Li
L
Yang
Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Li-ping
LP
Shi
Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Hai-jun
HJ
Chen
Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Xian-kun
XK
Tong
Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Gui-feng
GF
Wang
Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Yang-ming
YM
Zhang
Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Wen-long
WL
Wang
Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Chun-lan
CL
Feng
Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Pei-lan
PL
He
Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Feng-hua
FH
Zhu
Tongji Hospital of Tongji Medical College, Wuhan 430030, China.
You-hua
YH
Hao
Tongji Hospital of Tongji Medical College, Wuhan 430030, China.
Bao-ju
BJ
Wang
Tongji Hospital of Tongji Medical College, Wuhan 430030, China.
Dong-liang
DL
Yang
Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Wei
W
Tang
Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Fa-jun
FJ
Nan
Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Jian-ping
JP
Zuo
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D024921:Drug Resistance, Multiple, Viral / Q000235:genetics; D004372:Ducks; D056945:Hep G2 Cells; D018347:Hepadnaviridae Infections / Q000188:drug therapy* / Q000821:virology; D015611:Hepatitis B Virus, Duck / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D006524:Hepatitis, Viral, Animal / Q000188:drug therapy* / Q000821:virology; D006801:Humans; D009154:Mutation; D019251:Nucleocapsid / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000187:drug effects*; D013844:Thiazoles / Q000494:pharmacology*; D013997:Time Factors; D014162:Transfection; D014779:Virus Replication / Q000187:drug effects*
D000998:Antiviral Agents; D012367:RNA, Viral; D013844:Thiazoles; C000595585:isothiafludine