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Antiviral Compounds Screening Targeting HBx Protein of the Hepatitis B Virus.

ABSTRACT: A functional cure of hepatitis B virus (HBV) infection or HB antigen loss is rarely achieved by nucleos(t)ide analogs which target viral polymerase. HBx protein is a regulatory protein associated with HBV replication. We thought to identify antiviral compounds targeting HBx protein by analyzing HBx binding activity. Recombinant GST-tagged HBx protein was applied on an FDA-approved drug library chip including 1018 compounds to determine binding affinity by surface plasmon resonance imaging (SPRi) using a PlexArray HT system. GST protein alone was used for control experiments. Candidate compounds were tested for anti-HBV activity as well as cell viability using HepG2.2.15.7 cells and HBV-infected human hepatocytes. Of the 1018 compounds screened, 24 compounds showed binding to HBx protein. Of the top 6 compounds with high affinity to HBx protein, tranilast was found to inhibit HBV replication without affecting cell viability using HepG2.2.15.7 cells. Tranilast also inhibited HBV infection using cultured human hepatocytes. Tranilast reduced HB antigen level dose-dependently. Overall, theSPRi screening assay identified novel drug candidates targeting HBx protein. Tranilast and its related compounds warrant further investigation for the treatment of HBV infection.

SUBMITTER: Ma Y

PROVIDER: S-EPMC9569680 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Antiviral Compounds Screening Targeting HBx Protein of the Hepatitis B Virus.

Ma Yaojia Y Nakamoto Shingo S Ao Junjie J Qiang Na N Kogure Tadayoshi T Ogawa Keita K Nakagawa Miyuki M Fujiwara Kisako K Iwanaga Terunao T Kojima Ryuta R Kanzaki Hiroaki H Koroki Keisuke K Kobayashi Kazufumi K Kanogawa Naoya N Kiyono Soichiro S Nakamura Masato M Kondo Takayuki T Nakagawa Ryo R Ogasawara Sadahisa S Muroyama Ryosuke R Chiba Tetsuhiro T Kato Jun J Kato Naoya N

International journal of molecular sciences 20221010 19

A functional cure of hepatitis B virus (HBV) infection or HB antigen loss is rarely achieved by nucleos(t)ide analogs which target viral polymerase. HBx protein is a regulatory protein associated with HBV replication. We thought to identify antiviral compounds targeting HBx protein by analyzing HBx binding activity. Recombinant GST-tagged HBx protein was applied on an FDA-approved drug library chip including 1018 compounds to determine binding affinity by surface plasmon resonance imaging (SPRi) ...[more]

PMID: 36233317

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Project description:BackgroundPreS1-binding protein (PreS1BP), recognized as a nucleolar protein and tumor suppressor, influences the replication of various viruses, including vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1). Its role in hepatitis B virus (HBV) replication and the underlying mechanisms, however, remain elusive.MethodsWe investigated PreS1BP expression levels in an HBV-replicating cell and animal model and analyzed the impact of its overexpression on viral replication metrics. HBV DNA, covalently closed circular DNA (cccDNA), hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), and HBV RNA levels were assessed in HBV-expressing stable cell lines under varying PreS1BP conditions. Furthermore, co-immunoprecipitation and ubiquitination assays were used to detect PreS1BP- hepatitis B virus X protein (HBx) interactions and HBx stability modulated by PreS1BP.ResultsOur study revealed a marked decrease in PreS1BP expression in the presence of active HBV replication. Functional assays showed that PreS1BP overexpression significantly inhibited HBV replication and transcription, evidenced by the reduction in HBV DNA, cccDNA, HBsAg, HBcAg, and HBV RNA levels. At the molecular level, PreS1BP facilitated the degradation of HBx in a dose-dependent fashion, whereas siRNA-mediated knockdown of PreS1BP led to an increase in HBx levels. Subsequent investigations uncovered that PreS1BP accelerated HBx protein degradation via K63-linked ubiquitination in a ubiquitin-proteasome system-dependent manner. Co-immunoprecipitation assays further established that PreS1BP enhances the recruitment of the proteasome 20S subunit alpha 3 (PSMA3) for interaction with HBx, thereby fostering its degradation.ConclusionsThese findings unveil a previously unidentified mechanism wherein PreS1BP mediates HBx protein degradation through the ubiquitin-proteasome system, consequentially inhibiting HBV replication. This insight positions PreS1BP as a promising therapeutic target for future HBV interventions. Further studies are warranted to explore the clinical applicability of modulating PreS1BP in HBV therapy.

Project description:Background & aimsCurrent antiviral drugs, including nucleoside analogs and interferon, fail to eliminate the HBV covalently closed circular DNA (cccDNA), which serves as a transcript template in infected hepatocytes. Silencing the HBV X protein, which plays a crucial role in cccDNA transcription, is a promising approach to inhibit HBV replication. Therefore, the identification of novel compounds that can inhibit HBx-mediated cccDNA transcription is critical.MethodsInitially, a compound library consisting of 715 monomers derived from traditional Chinese medicines known for their liver-protecting properties was established. Then, MTT assays were used to determine the cytotoxicity of each compound. The effect of candidates on Flag-HBx expression was examined by real-time PCR and western blotting in Flag-HBx transfected HepG2-NTCP cells. Ultimately, the antiviral effect of gambogic acid (GA) on HBV was observed in HBV-infected HepG2-NTCP cells. Mechanistically, the functional role of DTX1 in GA-induced HBV inhibition was examined using RNA-seq. Finally, the antiviral effect of GA was estimated in vivo.ResultsGambogic acid (GA), a natural bioactive compound with a myriad of biological activities, markedly reduced Flag-HBx expression. Potent and dose-dependent reductions in extracellular HBV RNAs, HBV DNA, HBsAg, HBeAg and HBc protein were discovered three days after GA treatment in HBV-infected cells, accompanied by the absence of significant cytotoxicity. Furthermore, our research revealed that GA exhibited a dose-dependent inhibition of HBx expression, which is a pleiotropic protein required for HBV infection in vivo. We explored the mechanisms underlying GA-mediated inhibition of HBV and confirmed that this inhibition is accomplished by upregulating the expression of the DTX1 gene and boosting the Notch signaling pathway. Finally, the inhibitory effect of GA on HBV replication was tested in vivo using a mouse model of hepatitis B virus recombinant cccDNA.ConclusionsHerein, we discovered GA, which is a natural bioactive compound that targets HBx to inhibit hepatitis B virus replication by activating the DTX1-Notch signaling pathway.

Project description:BackgroundOur previous report suggested that centrosomal P4.1-associated protein (CPAP) is required for Hepatitis B virus (HBV) encoded non-structure protein X (HBx)-mediated nuclear factor kappa light chain enhancer of activated B cells (NF-κB) activation. CPAP is overexpressed in HBV-associated hepatocellular carcinoma (HCC); however, the interaction between CPAP and HBx in HBV-HCC remains unclear.MethodsThe mRNA expression of CPAP and HBx was analyzed by quantitative-PCR (Q-PCR). NF-κB transcriptional activity and CPAP promoter activity were determined using a reporter assay in Huh7 and Hep3B cells. Immunoprecipitation (IP) and in situ proximal ligation assay (PLA) were performed to detect the interaction between CPAP and HBx. Chromatin-IP was used to detect the association of cAMP response element binding protein (CREB) and HBx with the CPAP promoter. Cell proliferation was measured using cell counting kit CCK-8, Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) incorporation, and clonogenic assays. The tumorigenic effects of CPAP were determined using xenograft animal models.ResultsHBx can transcriptionally up-regulate CPAP via interacting with CREB. Overexpressed CPAP directly interacted with HBx to promote HBx-mediated cell proliferation and migration; SUMO modification of CPAP was involved in interacting with HBx. Knocked-down expression of CPAP decreased the HBx-mediated tumorigenic effects, including cytokines secretion. Interestingly, overexpressed CPAP maintained the HBx protein stability in an NF-κB-dependent manner; and the expression levels of CPAP and HBx were positively correlated with the activation status of NF-κB in HCC. Increased expression of CPAP and CREB mRNAs existed in the high-risk group with a lower survival rate in HBV-HCC.ConclusionThe interaction between CPAP and HBx can provide a microenvironment to facilitate HCC development via enhancing NF-κB activation, inflammatory cytokine production, and cancer malignancies. This study not only sheds light on the role of CPAP in HBV-associated HCC, but also provides CPAP as a potential target for blocking the hyper-activated NF-κB in HCC.

Project description:ObjectivesHepatitis B virus X (HBx) is closely associated with HBV-related hepatocarcinogenesis via the inactivation of tumour suppressors. Protein phosphatase 2A (PP2A) regulatory subunit B56 gamma (B56γ), as a tumour suppressor, plays a critical role in regulating cellular phosphorylation signals via dephosphorylation of signalling proteins. However, the underlying mechanism that B56γ involved in regulating HBx-associated hepatocarcinogenesis phenotypes and mediating anti-HBx antibody-mediated tumour suppression remains unknown.Materials and methodsWe used bioinformatics analysis, paired HCC patient specimens, HBx transgenic (HBx-Tg) mice, xenograft nude mice, HBV stable replication in the HepG2.2.15 cells, and anti-HBx antibody intervention to systematically evaluate the biological function of protein kinase B (AKT) dephosphorylation through B56γ in HBx-associated hepatocarcinogenesis.ResultsBioinformatics analysis revealed that AKT, matrix metalloproteinase 2 (MMP2), and MMP9 were markedly upregulated, while cell migration and viral carcinogenesis pathways were activated in HBV-infected liver tissues and HBV-associated HCC tissues. Our results demonstrated that HBx-expression promotes AKT phosphorylation (p-AKTThr308/Ser473 ), mediating the migration and invasion phenotypes in vivo and in vitro. Importantly, in clinical samples, HBx and B56γ were downregulated in HBV-associated HCC tumour tissues compared with peritumor tissues. Moreover, intervention with site-directed mutagenesis (AKTT308A , AKTS473A ) of p-AKTThr308/Ser473 mimics dephosphorylation, genetics-based B56γ overexpression, and intracellular anti-HBx antibody inhibited cell growth, migration, and invasion in HBx-expressing HCC cells.ConclusionsOur results demonstrated that B56γ inhibited HBV/HBx-dependent hepatocarcinogenesis by regulating the dephosphorylation of p-AKTThr308/Ser473 in HCC cells. The intracellular anti-HBx antibody and the activator of B56γ may provide a multipattern chemopreventive strategy against HBV-related HCC.

Dataset Information

Antiviral Compounds Screening Targeting HBx Protein of the Hepatitis B Virus.

Publications

Antiviral Compounds Screening Targeting HBx Protein of the Hepatitis B Virus.

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