Senko Tsukuda

Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter, sodium taurocholate cotransporting polypeptide (NTCP).

Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. Although nucleos(t)ide analogs inhibiting viral reverse transcriptase are clinically available as anti‐HBV agents, emergence of drug‐resistant viruses highlights the need for new anti‐HBV agents interfering with other targets. Here we report that cyclosporin A (CsA) can inhibit HBV entry into cultured hepatocytes. The anti‐HBV effect of CsA was independent of binding to cyclophilin and calcineurin. Rather, blockade of HBV infection correlated with the ability to inhibit the transporter activity of sodium taurocholate cotransporting polypeptide (NTCP). We also found that HBV infection‐susceptible cells, differentiated HepaRG cells and primary human hepatocytes expressed NTCP, while nonsusceptible cell lines did not. A series of compounds targeting NTCP could inhibit HBV infection. CsA inhibited the binding between NTCP and large envelope protein in vitro. Evaluation of CsA analogs identified a compound with higher anti‐HBV potency, having a median inhibitory concentration <0.2 μM. Conclusion: This study provides a proof of concept for the novel strategy to identify anti‐HBV agents by targeting the candidate HBV receptor, NTCP, using CsA as a structural platform. (Hepatology 2014;59:1726–1737)

Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP.

Hepatitis B virus (HBV) entry has been analyzed using infection-susceptible cells, including primary human hepatocytes, primary tupaia hepatocytes, and HepaRG cells. Recently, the sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor. In this study, we established a strain of HepG2 cells engineered to overexpress the human NTCP gene (HepG2-hNTCP-C4 cells). HepG2-hNTCP-C4 cells were shown to be susceptible to infection by blood-borne and cell culture-derived HBV. HBV infection was facilitated by pretreating cells with 3% dimethyl sulfoxide permitting nearly 50% of the cells to be infected with HBV. Knockdown analysis suggested that HBV infection of HepG2-hNTCP-C4 cells was mediated by NTCP. HBV infection was blocked by an anti-HBV surface protein neutralizing antibody, by compounds known to inhibit NTCP transporter activity, and by cyclosporin A and its derivatives. The infection assay suggested that cyclosporin B was a more potent inhibitor of HBV entry than was cyclosporin A. Further chemical screening identified oxysterols, oxidized derivatives of cholesterol, as inhibitors of HBV infection. Thus, the HepG2-hNTCP-C4 cell line established in this study is a useful tool for the identification of inhibitors of HBV infection as well as for the analysis of the molecular mechanisms of HBV infection.

A Novel Tricyclic Polyketide, Vanitaracin A, Specifically Inhibits the Entry of Hepatitis B and D Viruses by Targeting Sodium Taurocholate Cotransporting Polypeptide

ABSTRACT Anti-hepatitis B virus (HBV) drugs are currently limited to nucleos(t)ide analogs (NAs) and interferons. A challenge of drug development is the identification of small molecules that suppress HBV infection from new chemical sources. Here, from a fungus-derived secondary metabolite library, we identify a structurally novel tricyclic polyketide, named vanitaracin A, which specifically inhibits HBV infection. Vanitaracin A inhibited the viral entry process with a submicromolar 50% inhibitory concentration (IC50) (IC50 = 0.61 ± 0.23 μM), without evident cytotoxicity (50% cytotoxic concentration of >256 μM; selectivity index value of >419) in primary human hepatocytes. Vanitaracin A did not affect the HBV replication process. This compound was found to directly interact with the HBV entry receptor sodium taurocholate cotransporting polypeptide (NTCP) and impaired its bile acid transport activity. Consistent with this NTCP targeting, antiviral activity of vanitaracin A was observed with hepatitis D virus (HDV) but not hepatitis C virus. Importantly, vanitaracin A inhibited infection by all HBV genotypes tested (genotypes A to D) and clinically relevant NA-resistant HBV isolate. Thus, we identified a fungal metabolite, vanitaracin A, which was a potent, well-tolerated, and broadly active inhibitor of HBV and HDV entry. This compound, or its related analogs, could be part of an antiviral strategy for preventing reinfection with HBV, including clinically relevant nucleos(t)ide analog-resistant virus. IMPORTANCE For achieving better treatment and prevention of hepatitis B virus (HBV) infection, anti-HBV agents targeting a new molecule are in great demand. Although sodium taurocholate cotransporting polypeptide (NTCP) has recently been reported to be an essential host factor for HBV entry, there is a limited number of reports that identify new compounds targeting NTCP and inhibiting HBV entry. Here, from an uncharacterized chemical library, we isolated a structurally new compound, named vanitaracin A, which inhibited the process of entry of HBV and hepatitis D virus (HDV). This compound was suggested to directly interact with NTCP and inhibit its transporter activity. Importantly, vanitaracin A inhibited the entry of all HBV genotypes examined and of a clinically relevant nucleos(t)ide analog-resistant HBV isolate.

Dysregulation of Retinoic Acid Receptor Diminishes Hepatocyte Permissiveness to Hepatitis B Virus Infection through Modulation of Sodium Taurocholate Cotransporting Polypeptide (NTCP) Expression

Background: Host factors regulating hepatitis B virus (HBV) entry receptors are not well defined. Results: Chemical screening identified that retinoic acid receptor (RAR) regulates sodium taurocholate cotransporting polypeptide (NTCP) expression and supports HBV infection. Conclusion: RAR regulates NTCP expression and thereby supports HBV infection. Significance: RAR regulation of NTCP can be a target for preventing HBV infection. Sodium taurocholate cotransporting polypeptide (NTCP) is an entry receptor for hepatitis B virus (HBV) and is regarded as one of the determinants that confer HBV permissiveness to host cells. However, how host factors regulate the ability of NTCP to support HBV infection is largely unknown. We aimed to identify the host signaling that regulated NTCP expression and thereby permissiveness to HBV. Here, a cell-based chemical screening method identified that Ro41-5253 decreased host susceptibility to HBV infection. Pretreatment with Ro41-5253 inhibited the viral entry process without affecting HBV replication. Intriguingly, Ro41-5253 reduced expression of both NTCP mRNA and protein. We found that retinoic acid receptor (RAR) regulated the promoter activity of the human NTCP (hNTCP) gene and that Ro41-5253 repressed the hNTCP promoter by antagonizing RAR. RAR recruited to the hNTCP promoter region, and nucleotides −112 to −96 of the hNTCP was suggested to be critical for RAR-mediated transcriptional activation. HBV susceptibility was decreased in pharmacologically RAR-inactivated cells. CD2665 showed a stronger anti-HBV potential and disrupted the spread of HBV infection that was achieved by continuous reproduction of the whole HBV life cycle. In addition, this mechanism was significant for drug development, as antagonization of RAR blocked infection of multiple HBV genotypes and also a clinically relevant HBV mutant that was resistant to nucleoside analogs. Thus, RAR is crucial for regulating NTCP expression that determines permissiveness to HBV infection. This is the first demonstration showing host regulation of NTCP to support HBV infection.

Cyclosporin derivatives inhibit hepatitis B virus entry without interfering with NTCP transporter activity

Background & Aims The sodium taurocholate co-transporting polypeptide (NTCP) is the main target of most hepatitis B virus (HBV) specific entry inhibitors. Unfortunately, these agents also block NTCP transport of bile acids into hepatocytes, and thus have the potential to cause adverse effects. We aimed to identify small molecules that inhibit HBV entry while maintaining NTCP transporter function. Methods We characterized a series of cyclosporine (CsA) derivatives for their anti-HBV activity and NTCP binding specificity using HepG2 cells overexpressing NTCP and primary human hepatocytes. The four most potent derivatives were tested for their capacity to prevent HBV entry, but maintain NTCP transporter function. Their antiviral activity against different HBV genotypes was analysed. Results We identified several CsA derivatives that inhibited HBV infection with a sub-micromolar IC50. Among them, SCY446 and SCY450 showed low activity against calcineurin (CN) and cyclophilins (CyPs), two major CsA cellular targets. This suggested that instead, these compounds interacted directly with NTCP to inhibit viral attachment to host cells, and have no immunosuppressive function. Importantly, we found that SCY450 and SCY995 did not impair the NTCP-dependent uptake of bile acids, and inhibited multiple HBV genotypes including a clinically relevant nucleoside analog-resistant HBV isolate. Conclusions This is the first example of small molecule selective inhibition of HBV entry with no decrease in NTCP transporter activity. It suggests that the anti-HBV activity can be functionally separated from bile acid transport. These broadly active anti-HBV molecules are potential candidates for developing new drugs with fewer adverse effects. Lay summary In this study, we identified new compounds that selectively inhibited hepatitis B virus (HBV) entry, and did not impair bile acid uptake. Our evidence offers a new strategy for developing anti-HBV drugs with fewer side effects.

Camptothecin (CPT) directly binds to human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and inhibits the hnRNP A1/topoisomerase I interaction.

Camptothecin (CPT) is an anti-tumor natural product that forms a ternary complex with topoisomerase I (top I) and DNA (CPT-top I-DNA). In this study, we identified the direct interaction between CPT and human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) using the T7 phage display technology. On an avidin-agarose bead pull down assay, hnRNP A1 protein was selectively pulled down in the presence of C20-biotinylated CPT derivative (CPT-20-B) both in vitro and in vivo. The interaction was also confirmed by an analysis on a quartz-crystal microbalance (QCM) device, yielding a K(D) value of 82.7 nM. A surface plasmon resonance (SPR) analysis revealed that CPT inhibits the binding of hnRNP A1 to top I (K(D): 260 nM) in a non-competitive manner. Moreover, an in vivo drug evaluation assay using Drosophila melanogaster showed that the knockout of the hnRNP A1 homolog Hrb87F gene showed high susceptibility against 5-50 μM of CPT as compared to a wild-type strain. Such susceptibility was specific for CPT and not observed after treatment with other cytotoxic drugs. Collectively, our data suggests that CPT directly binds to hnRNP A1 and non-competitively inhibits the hnRNP A1/top I interaction in vivo. The knockout strain loses the hnRNP A1 homolog as a both CPT-binding partner and naïve brakes of top I, which enhances the formation of the CPT-top I-DNA ternary complexes and subsequently sensitizes the growth inhibitory effect of CPT in D. melanogaster.

A new class of hepatitis B and D virus entry inhibitors, proanthocyanidin and its analogs, that directly act on the viral large surface proteins

Introduction of direct‐acting antivirals against hepatitis C virus (HCV) has provided a revolutionary improvement in the treatment outcome. In contrast to HCV, however, the strategy for developing new antiviral agents against hepatitis B virus (HBV), especially viral‐targeting compounds, is limited because HBV requires only four viral genes for its efficient replication/infection. Here, we identify an oligomeric flavonoid, proanthocyanidin (PAC) and its analogs, which inhibit HBV entry into host cells by targeting the HBV large surface protein (LHBs). Through cell‐based chemical screening, PAC was identified to inhibit HBV infection with little cytotoxic effect. PAC prevented the attachment of the preS1 region in the LHBs to its cellular receptor, sodium taurocholate cotransporting polypeptide (NTCP). PAC was shown to target HBV particles and impair their infectivity, whereas it did not affect the NTCP‐mediated bile acid transport activity. Chemical biological techniques demonstrated that PAC directly interacted with the region essential for receptor binding in the preS1 region in the LHBs protein. Importantly, PAC had a pan‐genotypic anti‐HBV activity and was also effective against a clinically relevant nucleoside analog‐resistant HBV isolate. We further showed that PAC augmented the ability of a nucleoside analog, tenofovir, to interrupt HBV spread over time in primary human hepatocytes by cotreatment. Moreover, derivative analysis could identify small molecules that demonstrated more‐potent anti‐HBV activity over PAC. Conclusion: PAC and its analogs represent a new class of anti‐HBV agents that directly target the preS1 region of the HBV large surface protein. These agents could contribute to the development of a potent, well‐tolerated, and broadly active inhibitor of HBV infection. (Hepatology 2017;65:1104‐1116).

Specific inhibition of hepatitis C virus entry into host hepatocytes by fungi-derived sulochrin and its derivatives.

Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma. Although various classes of anti-HCV agents have been under clinical development, most of these agents target RNA replication in the HCV life cycle. To achieve a more effective multidrug treatment, the development of new, less expensive anti-HCV agents that target a different step in the HCV life cycle is needed. We prepared an in-house natural product library consisting of compounds derived from fungal strains isolated from seaweeds, mosses, and other plants. A cell-based functional screening of the library identified sulochrin as a compound that decreased HCV infectivity in a multi-round HCV infection assay. Sulochrin inhibited HCV infection in a dose-dependent manner without any apparent cytotoxicity up to 50 μM. HCV pseudoparticle and trans-complemented particle assays suggested that this compound inhibited the entry step in the HCV life cycle. Sulochrin showed anti-HCV activities to multiple HCV genotypes 1a, 1b, and 2a. Co-treatment of sulochrin with interferon or a protease inhibitor telaprevir synergistically augmented their anti-HCV effects. Derivative analysis revealed anti-HCV compounds with higher potencies (IC50<5 μM). This is the first report showing an antiviral activity of methoxybenzoate derivatives. Thus, sulochrin derivatives are anti-HCV lead compounds with a new mode of action.

Anti-hepatitis C Virus Natural Product from a Fungus, Penicillium herquei

New diazabicyclo[2.2.2]octane derivatives, peniciherquamides A-C (1-3), and a novel herqueinone derivative, neoherqueinone (5), were isolated from a fungal culture broth of Penicillium herquei. The structures of these novel compounds were determined by interpretation of spectroscopic data (1D/2D NMR, MS, and IR). Four known compounds, preparaherquamide (4), peniciherqueinone (6), and herqueinone/isoherqueinone (7/7a), were also obtained. The isolated compounds were tested for anti-hepatitis C virus (HCV) activity, and peniciherquamide C (3) was found to display an IC50 value of 5.1 μM. To our knowledge, this is the first report of a diazabicyclo[2.2.2]octane derivative with anti-HCV activity.

Ridaifen B, a tamoxifen derivative, directly binds to Grb10 interacting GYF protein 2.

Ridaifen B (RID-B) is a tamoxifen derivative that potently inhibits breast tumor growth. RID-B was reported to show anti-proliferating activity for a variety of estrogen receptor (ER)-positive human cancer cells. Interestingly, RID-B was also reported to possess higher potency than that of tamoxifen even for some ER-negative cells, suggesting an ER-independent mechanism of action. In this study, a T7 phage display screen and subsequent binding analyses have identified Grb10 interacting GYF protein 2 (GIGYF2) as a RID-B-binding protein. Using a cell-based assay, the Akt phosphorylation level mediated by GIGYF2 was found to have decreased in the presence of RID-B.