Identification Of Compounds Inhibiting The Transcriptional Activity Of HBV Core Promoter

Objective: Although the existing therapies for the infection of chronic hepatitis B can effectively control the disease,it is still difficult to cure it.Therefore,it is necessary to develop new anti-hepatitis B drugs.Targeting the transcription process of hepatitis B virus(HBV)core promoter is an attractive but underdeveloped antiviral strategy.In this study,recombinant lentivirus was used to construct a Gaussia luciferase stable expression cell line(HepG2-Pcore-Gluc)driven by HBV core promoter on HepG2 cells.The cell line allowed the changes of core promoter activity to be reflected by luciferase activity,facilitating the screening of compounds modulating the transcriptional activity of core promoter.In this study,we used this model to screen candidate drugs that affect the transcriptional activity of HBV core promoter from a library of 1,450 small molecule compounds.Methods: A cell line(HepG2-Pcore-Gluc)stably expressing Gaussia luciferase driven by HBV core promoter was constructed by recombinant lentivirus,and a compound library containing 1,450 small molecules was screened by using this model.The compounds that could affect luciferase activity obtained from the first round of screening were tested for dose-effect relationship in the second round.Those compounds showing dose-dependent effects and no obvious toxicity were selected for further characterization.The effects of the candidate compounds on HBV RNA and HBV DNA in HBV replicating cell models were evaluated by Southern blot and quantitative fluorescence PCR.Western blot assay was used to evaluate the effect of candidate compounds on the translation of core protein.ELISA assay was used to evaluate the effects of the candidate compounds on the levels of HBV surface antigen and e antigen.Luciferase reporter assay was used to analyze the regions required for the effect of the candidate compounds.Results: 41 compounds that could affect luciferase activity more than2 times were screened out from a compound library containing 1,450 small molecules by using HepG2-Pcore-Gluc cell model.After a second round testing,2 compounds,Reversine and SCH58261,were identified for further verification.Subsequent testing showed that Reversine can effectively reduce the levels of HBV RNA,HBV DNA,HBe Ag and HBs Ag in HepG2 and HepG2.2.15 cells.Conclusion: A novel cell model was successfully established for the screening of drugs targeting the transcriptional activity of the core promoter of hepatitis b virus.Our results provided a proof-of-concept for the usage of this model in identifying new anti-HBV candidates,and demonstrated that Reversine might be used as a basis for further development of anti-HBV drugs.