Screening And Identification Of CDK2 Kinase Inhibitors Inhibiting HBV Replication

Objective: Hepatitis B virus(HBV)specifically infects liver cells,causing acute infections or chronic infections,of which chronic infections are the leading cause of cirrhosis and hepatocellular carcinoma.Among the current clinical drugs of treating chronic hepatitis B infection(CHB),long-term use of nucleoside analogs is likely to lead to drug resistance,and there is a rebound in drug withdrawal.Meanwhile interferon therapy has a low response,therefore,seeking for new anti-hepatitis B drugs is urgent.From virus-host perspective,finding direct antiviral drugs and host-based factors as targets can help enrich anti-hepatitis B drug development strategies.SAMHD1(Sterile alpha motif(SAM)and histidine/aspartic acid(HD)domain-containing protein 1)is an important host restriction factor inhibiting viral replication by its dNTPase activity.Recent research reports that SAMHD1 is regulated by cyclin-dependent kinases(CDKs)at the T592 site,and phosphorylated SAMHD1 loses its antiviral effect.However,it is still unclear which and how CDK kinase is involved in the regulation of phosphorylation of SAMHD1 and its antiviral effect during HBV infection.In addition,whether CDK kinase inhibitors can be used as potential drugs restricting HBV replication by limiting the phosphorylation of SAMHD1 has not been reported.Therefore,in this research,we identified CDK2 kinase is involved in the regulation of SAMHD1 phosphorylation in HBV-infected hepatocytes,and screened two kinds of CDK2 kinase inhibitors that effectively inhibit viral replication by reducing phosphorylation of SAMHD1 among the current CDK2 kinase inhibitors.Our results indicate that CDK2 can interact with SAMHD1 in the nucleus of hepatocytes,regulating SAMHD1 phosphorylation and viral replication;On the other hand,screening of the current anti-HBV inhibitors targeting CDK2 kinase,we identified CDK2 inhibitor II and CDK2 inhibitor III can block HBV replication by inhibiting SAMHD1 phosphorylation.These results help to elucidate the molecular mechanism that CDK2 regulated the phosphorylation of SAMHD1,antagonizing its antiviral function in HBV infecting hepatocytes.Furthermore,we found CDK2 inhibitor II and CDK2 inhibitor III can effectively block HBV replication by reducing the phosphorylation of SAMHD1.This would provid a new strategy for cure HBV infection.Methods: It is divided into two parts.Part I: Identification of the cell cycle kinase CDK2 that regulates phosphorylation of SAMHD1:(1)The exogenously expressed SAMHD1 and CDK1 or CDK2 were co-transfected in Huh7.0 cells,then the interaction of SAMHD1 with CDK1 or CDK2 was investigated by Co-IP.(2)The localization of SAMHD1 and CDK2 in Huh7.0 cells was studied by immunofluorescence;(3)The CDK1 or CDK2 were knocked down by siRNA in Huh7.0 cells,the viral replication parameters including HBV surface antigen and HBV DNA levels were measured by ELISA and Southern blot.The phosphorylation of SAMHD1 was detected by Western blot.Part II: Screening and identification of CDK2 kinase inhibitors that inhibit HBV replication:(1)The HepAD38 cells were treated with the current 13 specific inhibitors of cell cycle kinase CDK2,and the cytotoxicity effect of CDK2 inhibitors was detected by MTT assay,then four kinds of inhibitors with less toxicity were selected out;(2)The HepAD38 cells transfected with SAMHD1 were treated with the CDK2 inhibitors,the phosphorylation level of SAMHD1 was detected by Western blot,and the viral DNA or RNA levels were measured by qPCR;(3)The antiviral function dependent on reducing phosphorylation level of SAMHD1 of CDK2 inhibitor Ⅱor CDK2 inhibitor Ⅲ were also verified on Huh7.0 cells.Results: Part Ⅰ: We identified that CDK2 kinase is involved in the regulation of phosphorylation of SAMHD1 in HBV-infected hepatocytes.:(1)The CDK2,not CDK1 was found to interact with SAMHD1 by Co-IP;(2)SAMHD1 and CDK2 colocalize in the nucleus in the Huh7.0 cells detected by immunofluorescence;(3)The viral DNA in the core particles were extracted and measured by Southern blot and qPCR,the HBV DNA levels was significantly reduced after knockdown of CDK2,but there was no significant difference after knockdown of CDK1.In addition,HBV surface antigen was detected by ELISA,and the results indicated that silencing CDK1 and CDK2 had no significant difference on HBV surface antigen level.The phosphorylation level of SAMHD1 was significantly decreased after knockdown of CDK2,but no change was observed when knockdown of CDK1.In summary,we demonstrated that CDK2 can interact with SAMHD1 in hepatocytes,and CDK2 kinase is mostly involved in regulation of phosphorylation of SAMHD1 and replication of HBV in hepatocytes.Part II: Screening and identification of CDK2 kinase inhibitors that inhibit HBV replication.(1)The toxicity of current 13 specific inhibitors targeting CDK2 kinase were examined by MTT assay,and 4 kinds of inhibitors with less cytotoxicity were screened out;(2)The HepAD38 cells transfected with SAMHD1 were treated with CDK2 inhibitor II and CDK2 inhibitor III,and CDK2 inhibitor II and CDK2 inhibitor III had strong antiviral effects at a concentration of 5 μM.Western blot showed that the phosphorylation level of SAMHD1 gradually decreased as the concentration of CDK2 inhibitor II or CDK2 inhibitor III increased.In addition,HBV RNA levels were not changed under CDK2 inhibitor II or CDK2 inhibitor III treatment.The results indicate that CDK2 inhibitor II or CDK2 inhibitor III can inhibit viral replication and reduce the phosphorylation of SAMHD1;(3)The Huh7.0 cells transfected with SAMHD1 were treated with CDK2 inhibitor II and CDK2 inhibitor III,the results showed that the cells stayed in the G0/G1 phase by flow cytometry.The HBV DNA levels and the phosphorylation level of SAMHD1 were gradually reduced;(4)The Huh7.0 cells were treated with CDK2 inhibitor II or CDK2 inhibitor III after knockdown SAMHD1 by siRNA,the antiviral function of CDK2 inhibitor II or CDK2 inhibitor III on HBV disappeared after knockdown the endoexpression of SAMHD1.This suggested the restriction on HBV of CDK2 inhibitors is dependent on phosphorylation of SAMHD1.Conclusion: In hepatocytes that are infected by HBV,CDK2 kinase is involved in the regulation of viral replication and the phosphorylation of SAMHD1;CDK2 kinase interacts with SAMHD1 directly and both of them localize in the nucleus;CDK2 inhibitor II or CDK2 inhibitor III can block HBV replication and reduce the phosphorylation of SAMHD1 in hepatocytes.