Biology subjects

Isogawa, M.

Publications and source records attributed to Isogawa, M..

5 recordsLinked to original sources

bioRxiv2026

Vorapaxar and aripiprazole suppress hepatitis B virus replication through distinct host signaling pathways

Background & AimsCurrent nucleos(t)ide analogs efficiently suppress hepatitis B virus (HBV) replication but have limited effects on viral transcription from covalently closed circular DNA (cccDNA) and integrated HBV DNA. We aimed to identify clinically applicable compounds that directly inhibit HBV transcription by screening FDA-approved drugs. Approach & ResultsScreening of 1,470 FDA-approved compounds using an HBV enhancer I/X promoter reporter system identified vorapaxar and aripiprazole as potent inhibitors of viral promoter activity. Both compounds suppressed HBV replication in HBV-producing cells, HBV-infected HepG2-hNTCP cells, and primary human hepatocytes. Aripiprazole reduced hepatocyte nuclear factor 4 (HNF4) protein levels through an ERK/JNK-dependent pathway and inhibited HBV core promoter activity, whereas vorapaxar acted independently of HNF4. Both compounds suppressed enhancer I/X promoter activity through inhibition of STAT3 signaling. Vorapaxar inhibited PAR-1-mediated SRC, EGFR, and STAT3 activation, while aripiprazole suppressed SRC-STAT3 signaling independently of EGFR. PAR-1 activation enhanced HBV transcription, whereas PAR-1 knockdown reduced promoter activity and viral RNA expression. Both compounds also reduced HBV replication in human liver chimeric mice at clinically relevant exposure levels without apparent severe toxicity. ConclusionsVorapaxar and aripiprazole suppress HBV transcription and replication through distinct host signaling pathways. These findings identify PAR-1-STAT3 signaling as a previously unrecognized regulator of HBV transcription and suggest that host-targeting approaches may complement current therapies by suppressing viral gene expression from both cccDNA and integrated HBV DNA. Impact and implicationsCurrent nucleos(t)ide analogues effectively suppress HBV reverse transcription but have limited effects on viral transcription from cccDNA and integrated HBV DNA, highlighting the need for therapies targeting viral gene expression. We identify PAR-1- STAT3 signaling as a previously unrecognized regulator of HBV transcription and demonstrate that two clinically approved drugs, vorapaxar and aripiprazole, suppress HBV replication through distinct host signaling pathways. These findings are relevant to researchers developing host-targeting antivirals and to clinicians seeking complementary therapeutic strategies beyond current nucleos(t)ide analogue therapy. Although further clinical validation and combination studies are required, our results provide a rationale for repurposing approved drugs and for developing transcription-targeting therapies that may complement existing treatments for chronic hepatitis B. HighlightsO_LIVorapaxar and aripiprazole suppress HBV through distinct host pathways. C_LIO_LIBoth drugs inhibit HBV replication in vitro and in humanized liver mice. C_LIO_LIPAR-1 inhibition reduces HBV transcription by blocking SRC/EGFR/STAT3 signaling. C_LIO_LIPAR-1-STAT3 signaling is a novel regulator of HBV transcription. C_LIO_LIHost-targeting antiviral therapy complements current HBV treatment. C_LI

pharmacology and toxicology↗

bioRxiv2026

Identification of molecular determinants governing KIF4 cargo recognition of NTCP and their role in HBV/HDV infection

The surface availability of the sodium taurocholate co-transporting polypeptide (NTCP), which serves as entry receptor for Hepatitis B virus (HBV) and Hepatitis D virus (HDV), is a key determinant of HBV/HDV infectivity. We previously identified the Kinesin motor protein KIF4 as a key regulator of NTCP trafficking to the plasma membrane. However, the molecular mechanism by which NTCP is recognized as a KIF4 transport cargo has remained undefined. Here, we identified a leucine-rich motif, LLLI (aa 298-301), within the C-terminal transmembrane domain of NTCP as an essential determinant for KIF4 transport. Co-immunoprecipitation analyses revealed its importance for NTCP interaction with KIF4. This motif is conserved in another established KIF4 cargo, integrin {beta}1 (ITGB1), and mutation of the corresponding residues similarly disrupted ITGB1-KIF4 interaction, indicating a shared cargo recognition mechanism employed by KIF4. Functionally, disruption of the NTCP leucine-rich motif markedly impaired NTCP surface localization without affecting total protein expression, resulting in severe attenuation of HBV and HDV infection. Structural modeling and computational protein-protein interaction analysis indicated that the mutation does not alter NTCP structure or NTCP/preS1 binding affinity, supporting a trafficking-specific defect. Substitution mutations identified L298 and L299 as the minimal residues required for interaction with KIF4. Our findings define a recognition motif that regulates KIF4-dependent cargo identification and surface trafficking. It also underscores the potential of targeting KIF4-mediated NTCP recognition and transport to inhibit HBV and HDV entry. ImportanceHepatitis B and D viruses require the hepatocyte surface receptor NTCP for entry. We identified a conserved leucine-rich motif (LLLI, aa 298-301) in NTCP that is necessary for its recognition by the kinesin motor KIF4; disrupting this motif selectively impairs NTCP surface trafficking and markedly attenuates HBV/HDV infection without reducing total NTCP or intrinsic receptor function. Importantly, we identified a similar motif in integrin {beta}1 (ITGB1; aa 747-750) that is required for interaction with KIF4, revealing a similar cargo selection mechanism for different proteins. Scientifically, these findings define a motif-derived cargo-selection determinant implied by KIF4, advancing a general principle for kinesin-dependent cargo recognition. More broadly, this work highlights a possible host-directed antiviral approach that targets cargo-selection interfaces and provides a conceptual framework for manipulating kinesin-mediated trafficking of clinically relevant membrane proteins beyond NTCP, with implications for receptor regulation and membrane protein therapeutics.

microbiology↗

bioRxiv2025

RXR agonist S169 inhibits HBV/HDV entry in vitro by disrupting KIF4-dependent NTCP trafficking.

Chronic hepatitis B (HBV) and hepatitis D (HDV) viral infections pose serious global health challenges, and the lack of curative therapies calls for the development of new antiviral approaches. Recently, we have identified the Kinesin Family Member 4 (KIF4) as a crucial regulator of the surface transport for NTCP (HBV/HDV entry receptor). Our research has shown that the pan-RXR agonist, Bexarotene, suppresses KIF4 expression and inhibits HBV/HDV infections. However, the clinical use of Bexarotene is limited by poor physicochemical and pharmacokinetic (PK) properties and significant side effects. Here we investigated chemically diverse RXR modulators with tuned activity, and identified the RXR agonist S169 as a novel HBV/HDV entry inhibitor. S169 effectively blocks HBV and HDV infections in primary human hepatocyte (PXB) cultures without evident cytotoxicity. S169s suppressive effect on HBV infection was confirmed by reduction of secreted HBsAg and HBeAg, as well as inhibition of HBV cccDNA, pregenomic RNA, and intracellular HBV DNA. Mechanistically, S169 suppressed FOXM1-mediated KIF4 expression, leading to decreased NTCP surface levels and a marked decrease in HBV preS1 peptide binding to the hepatocyte cell surface. S169 is reported to exhibit enhanced oral bioavailability, favorable PK parameters, lower toxicity compared to Bexarotene. Interestingly, S169 induced a modest inhibition of NTCP-mediated bile acid transport up to 10 M, which remained unchanged at higher concentrations. Furthermore, S169 significantly impeded HBV spread in HBV infected long-time cultured PHHs (PXB). We speculate that S169 can be a promising seed for the development of novel oral HBV and HDV antiviral therapeutics.

microbiology↗

bioRxiv2021

Two doses of mRNA vaccine elicit cross-neutralizing memory B-cells against SARS-CoV-2 Omicron variant

SARS-CoV-2 Beta and Omicron variants have multiple mutations in the receptor-binding domain (RBD) allowing antibody evasion. Despite the resistance to circulating antibodies in those who received two doses of mRNA vaccine, the third dose prominently recalls cross-neutralizing antibodies with expanded breadth to these variants. Herein, we longitudinally profiled the cellular composition of persistent memory B-cell subsets and their antibody reactivity against these variants following the second vaccine dose. The vaccination elicited a memory B-cell subset with resting phenotype that dominated the other subsets at 4.9 months. Notably, most of the resting memory subset retained the ability to bind the Beta variant, and the memory-derived antibodies cross-neutralized the Beta and Omicron variants at frequencies of 59% and 29%, respectively. The preservation of cross-neutralizing antibody repertoires in the durable memory B-cell subset likely contributes to the prominent recall of cross-neutralizing antibodies following the third dose of the vaccine. One Sentence SummaryFully vaccinated individuals preserve cross-neutralizing memory B-cells against the SARS-CoV-2 Omicron variant.

immunology↗

bioRxiv2020

Interferon signaling suppresses the unfolded protein response and induces cell death in hepatocyte accumulating hepatitis B surface antigen

Virus infection, such as hepatitis B virus (HBV), often causes endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is counteractive machinery to ER stress, and the failure of UPR to cope with ER stress results in cell death. Mechanisms that regulate the balance between ER stress and UPR in HBV infection is poorly understood. Type 1 and type 2 interferons have been implicated in hepatic flares during chronic HBV infection. Here, we examined the interplay between ER stress, UPR, and IFNs using transgenic mice that express hepatitis B surface antigen (HBsAg) (HBs-Tg mice) and humanized-liver chimeric mice infected with HBV. IFN causes severe and moderate liver injury in HBs-Tg mice and HBV infected chimeric mice, respectively. The degree of liver injury is directly correlated with HBsAg levels in the liver, and reduction of HBsAg in the transgenic mice alleviates IFN mediated liver injury. Analyses of total gene expression and UPR biomarkers protein expression in the liver revealed that UPR is induced in HBs-Tg mice and HBV infected chimeric mice, indicating that HBsAg accumulation causes ER stress. Notably, IFN administration transiently suppressed UPR biomarkers before liver injury without affecting intrahepatic HBsAg levels. Furthermore, UPR upregulation by glucose-regulated protein 78 (GRP78) suppression or low dose tunicamycin alleviated IFN mediated liver injury. These results suggest that IFN induces ER stress-associated cell death by reducing UPR. IFN{gamma} uses the same mechanism to exert cytotoxicity to HBsAg accumulating hepatocytes. Collectively, our data reveal a previously unknown mechanism by which IFNs selectively induce cell death in virus-infected cells. This study also identifies UPR as a potential target for regulating ER stress-associated cell death. Author summaryHepatitis B virus (HBV) causes acute and chronic infections that kill over 600,000 people every year from severe hepatitis, liver cirrhosis, and cancer. Mechanisms of chronic liver injury remain largely unknown. Both type 1 and type 2 interferons (IFNs) have been implicated in hepatic flares during chronic HBV infection, although HBV per se is a poor IFN inducer. In addition, while IFN, a type 1 IFN, used to be the first-line treatment for chronic hepatitis B (CHB) patients, adverse side effects, including hepatic flares, severely limit their therapeutic effectiveness. These clinical observations suggest a pathogenic role of IFNs in HBV infection. Here, we demonstrate that IFN-1s cause severe and moderate hepatitis in transgenic mice expressing hepatitis B surface antigen (HBs-Tg mice) and human hepatocyte chimeric mice infected with HBV, respectively. HBsAg accumulation appears to cause ER stress because a counteractive response to ER stress, namely, unfolded protein response (UPR), was induced in both HBs-Tg mice and HBV infected chimeric mice. Our results indicate that IFN-1s suppress UPR before causing liver injury. UPR was also suppressed by IFN{gamma}. Induction of UPR in HBs-Tg mice before treatment with IFN and IFN{gamma} significantly alleviated liver injury. We suggest that IFNs exert cytotoxicity to ER stress accumulating cells by suppressing UPR.

microbiology↗