Identification Of Compounds Targeting Hepatitis B Virus Capsid Formation

Objective: Targeting the formation of capsid is an attractive strategy for the treatment of chronic hepatitis B.Currently available candidates targeting capsid formation mainly interfere with the process of multimerization of HBV core protein dimers,and compounds targeting the formation of dimers of HBV core protein monomers were not extensively screened.We previously established a cell model,SRluc-HBc6,which reflects the formation of HBC dimer by split luciferase complementation.In this study,we aim to screen a commercial library containing 3884 compounds to identify candidates targeting HBV capsid formation using this cell model.Methods: SRluc-HBc6 cells were used to drug screening model constructed in the early stage of the research group,the drug screening of HBV core protein dimers in the drug library was conducted.Using SRluc-HBc6 cell model,dose-dependent drugs with significant effect were selected as the drugs to be selected.Southern blotting was used to identify the effect of the candidate drugs on HBV DNA replication.Plasmids Rluc N-L-C was transfected into HEK 293 cells,and dose-response experiments were carried out to candidate drugs with no trend of luciferase activity inorder to eliminate false-positive results.The effect of candidate compounds on the formation of HBV nucleocapsid was identified by Particle Gel electrophoresis.The effect of candidate drugs on HBV core protein was identified by Western blotting.The effect of candidate drugs on protein stability was analyzed by thermal shift analysis.The binding affinity of drugs and core protein were detected directly in cells by cellular thermal transformation analysis(CETSA).Results: In the first round of screening,3884 compounds were screened by using SRluc-HBc6 cell model,among which 223 compounds showed double or more double reduction of Rluc activity.In addition to the compounds causing cell death,22 compounds showed significant dose-dependent effects.Six compounds showed significant inhibitory effects on HBV DNA replication in the third round of screening.In further tests for the candidate drugs,Chlorambucil and CCT128930 were found to reduce the formation of HBV core proteins and nucleocapsid,except for false positive results.In further tests,CCT128930 did not affect the stability of intracellular proteins.Conclusion: Chlorambucil and CCT128930 target the formation of core protein dimers and inhibit the replication of HBV DNA in a dose dependent manner.These two compounds also deserve further study to dissect theirmechanisms of action and develop effective drugs to help cure HBV infection.