The human hepatoma cell lines HepG2.2.15 (stably transfected with HBV), Huh7, and Huh7D^hNTCP (stably expressing the HBV receptor NTCP) were kindly provided by the Wuhan Institute of Virology, Chinese Academy of Sciences. All cells were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum at 37 °C under 5% CO₂. KP and Entecavir (ETV) were purchased from Chengdu Alfa Biotechnology and Merck Life Science, respectively. Both compounds were dissolved in dimethyl sulfoxide as stock solutions, with the final dimethyl sulfoxide concentration in all treatments kept below 0.1%. The ERK inhibitor U0126 was obtained from MedChemExpress. The HBV promoter luciferase reporter plasmids, including pGL3-Cp, pGL3-Xp, pGL3-SpI, pGL3-SpII, were kindly provided by Prof. Mengji Lu from Institute of Virology, University Hospital of Essen, Germany and were constructed as previously described (Zhang et al., 2011). The pAAV-HBV1.2 HBV competent plasmid was kindly provided by Prof. Peijer Chen from National Taiwan University, and was constructed as previously described (Wu et al., 2017).

Commercial enzyme-linked immunosorbent assay kits for detecting HBsAg, HBeAg, and alanine aminotransferase (ALT), aspartate aminotransferase (AST) were acquired from Kehua Bio-Engineering and Beijing Solarbio Science & Technology, respectively. The PrimeScript™ RT Reagent Kit and TB Premix Ex Taq (Tli RNaseH Plus) were from Takara. The Cell Counting kit-8, Dual-Luciferase Reporter Assay Kit, and RNA-easy Isolation Reagent were from Vazyme Biotech. The QIAamp DNA Blood Mini Kit was from Qiagen.

Antibodies used for immunoblotting and immunofluorescence were as follows: anti-β-actin (#4967), anti-FOXO1(C29H4, #2880), anti-phosphorylated-FOXO1 (p-FOXO1, Ser256, #9461), anti-ERK (137F5, #4695), and anti-HNF4α (C11F12, #3113) from Cell Signaling Technology; anti-phosphorylated-ERK (p-ERK, T202/Y204, SAB5701896) from Sigma; anti-HBcAg (C1, EPR28251-34) from Abcam; anti-HBsAg (1023, sc-53299), anti-Pol (2C8, sc-81590), and anti-PGC-1α (D-5, sc-518025) from Santa Cruz Biotechnology.

Cell viability was assessed using the Cell Counting kit-8 assay. Briefly, cells were seeded in 96-well plates at a density of 5×10⁴ cells per well. After 24 h of adherence, cells were treated with KP at serial concentrations (6.25 to 400 μM) or an equivalent volume of vehicle dimethyl sulfoxide for 48 h. Subsequently, 10 μL of Cell Counting kit-8 regent was added to each well and incubated for 30 min at 37°C. The absorbance at 450 nm was measured using a Bio-Rad microplate reader. Cell viability was expressed as a percentage relative to the vehicle-treated control group.

Intracellular HBV DNA: Cells cultured in 6-well plates were lysed with the ice-cold lysis buffer. The lysate was treated with 10% SDS and proteinase K (0.5 mg/mL) at 56 °C for 2 h. Total DNA was then extracted via phenol/chloroform (1:1) extraction, precipitated with ethanol using glycogen as a carrier, and resuspended in nuclease-free water.

Extracellular HBV DNA: Viral DNA was extracted from 200μL of the cell culture supernatant using the QIAamp DNA Blood Mini Kit according to the manufacturer’s instructions.

The purified DNA was subjected to quantitative polymerase chain reaction using HBV-specific primers (Supplementary Table 1) and TB Green Premix Ex Taq (Tli RNaseH Plus) on a QuantStudio real-time PCR system. HBV DNA levels were quantified against a standard curve generated from known amounts of a plasmid containing the full-length HBV genome.

Total RNA was isolated from cells using the RNA-easy Isolation Reagent and reverse-transcribed into cDNA using the PrimeScript™ RT Reagent kit according to the manufacturer’s instructions. Quantitative polymerase chain reaction was performed with gene-specific primers listed in Supplementary Table 1 and TB Green Premix Ex Taq (Tli RNaseH Plus). The relative expression levels of target genes were calculated using the comparative threshold cycle (2^-△△Ct) method, with GAPDH serving as the endogenous control.

The levels of HBsAg, HBeAg and ALT, AST secreted into the cell culture supernatant or from mouse serum were quantified using commercial enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions. Absorbance was measured at 450 nm for HBsAg, HBeAg, and 505 nm for ALT, AST, respectively. The concentrations of ALT and AST were determined based on a standard curve.

HepG2.2.15 were seeded on coverslips in 6-well plates at a density of 2×10⁵ cells per well. After treatment, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin. Cells were then incubated overnight at 4 °C with primary antibodies, followed by incubation with a fluorophore-conjugated secondary antibody for 1 h at room temperature. Nuclei were counterstained with DAPI. Images were acquired using a fluorescence microscope.

Liver tissue samples from the right lobe of the mouse liver were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. After deparaffinization, rehydration, and antigen retrieval, sections were blocked for endogenous peroxidase activity and incubated with primary antibodies overnight at 4 °C. This was followed by incubation with a HRP-conjugated secondary antibody. The signal was developed with a DAB substrate, and nuclei were counterstained with hematoxylin. Slides were visualized under a bright-field microscope.

Cells or homogenized liver tissues were lysed in RIPA buffer containing protease and phosphatase inhibitors. Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking, membranes were probed with specific primary antibodies overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibodies. Protein bands were visualized using an enhanced chemiluminescence substrate on a Bio-Rad ChemiDoc imaging system and normalized to β-actin.

To assess HBV promoter activity, Huh7 cells were co-transfected in 24-well plates with a firefly luciferase reporter plasmid driven by the viral four promoters and a Renilla luciferase control plasmid. After 6 h, the medium was replaced with fresh medium containing KP or vehicle. 48 h post-transfection, cells were lysed, and luciferase activities were measured sequentially using the Dual-luciferase Reporter Assay Kit. Firefly luciferase activity was normalized to Renilla luciferase activity for each sample.

All animal procedures were approved by the Ethics Committee of China Pharmaceutical University (Approval NO. IACUC-2023-09-011) and conducted in accordance with institutional guidelines.

Male C57BL/6 mice (5–6 weeks old, 18–20 g) from the Comparative Medicine Center of Yangzhou University were housed under standard conditions. An HBV persistence infection model was established via hydrodynamic injection of 10 μg the pAAV-HBV1.2 plasmid in a volume equivalent to 10% of the mouse body weigh into the tail vein within 5–8 s. One day post-injection, mice were randomly assigned to six groups (n=6 per group): (1) Model group (vehicle:0.5% sodium carboxymethyl cellulose); (2) KP low-dose (10 mg/kg), (3) KP medium-dose (20 mg/kg); (4) KP high-dose (40 mg/kg); (5) Combination (KP 40 mg/kg and ETV 1 mg/kg); (6) Positive control (ETV, 1 mg/kg). KP was suspended in 0.5% sodium carboxymethyl cellulose solution. Administrations were performed via oral gavage every other day for a total of nine times. Mice in the model and combination groups received corresponding volumes of vehicles.

All data are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 8.0 software. Comparisons among multiple groups were analyzed by one-way analysis of variance followed by an appropriate post-hoc test. A p-value of less than 0.05 (p < 0.05) was considered statistically significant.

The molecular structure of KP was displayed in Figure 1, and we first evaluated the potential cytotoxicity of KP in relevant hepatoma cell lines. Treatment with KP at various concentrations for 48 h resulted in half-cytotoxic concentration (CC₅₀) values of 394.5 μM for Huh7D^hNTCP, 354.0 μM for HepG2.2.15, and 380.9 μM for Huh7 cells (Supplementary Figure 1), indicating low cytotoxicity. Based on these results, non-cytotoxic concentrations of 1, 5, and 25 μM KP were selected for all subsequent in vitro experiments.

To assess the anti-HBV activity of KP, Huh7D^hNTCP cells were infected with HBV for 24 h and then treated with KP according to the schedule outlined in Figure 2A. Myrcludex B (MyrB), an NTCP inhibitor, served as a negative control for HBV infection. KP treatment led to a potent, dose- and time-dependent reduction in the secretion of viral HBsAg, HBeAg, and extracellular HBV DNA into the supernatant (Figures 2B–D). Intracellular viral replication intermediates were similarly suppressed, with levels of intracellular HBV DNA, pgRNA, and total viral RNA significantly decreased in a dose- and time-dependent manner (Figures 2E–G). Additionally, Immunofluorescence analysis further confirmed that KP dose-dependently suppressed the expression of intracellular HBcAg in Huh7D^hNTCP cells (Figure 2H). While, the level of cccDNA was not affected by KP (Supplementary Figure 2).

The anti-HBV effect of KP was further validated in HepG2.2.15 cells, which stably support HBV replication. Cells were treated with KP every 48 h, and samples were collected at the indicated time points (Figure 3A). Consistent with the infection model, KP dose- and time-dependently inhibited the secretion of HBsAg and HBeAg in HepG2.2.15 cells (Figures 3B, C). Both extracellular and intracellular HBV DNA levels, as well as viral pgRNA and total RNA levels, were markedly downregulated by KP in a dose-dependent manner (Figures 3D–G). Notably, unlike the Pol inhibitor ETV, which primarily targets DNA synthesis, KP significantly reduced pgRNA levels (Figure 3F), suggesting an additional mechanism affecting viral transcription. Western blot analysis confirmed that KP dose-dependently reduced the expression of intracellular viral proteins, including Pol, HBsAg, and HBcAg in HepG2.2.15 cells (Figure 3H).

We next assessed the combined effect of KP and ETV. Co-treatment with 5 μM KP and 10 nM ETV reduced intracellular and extracellular HBV DNA levels by 87% and 84.73%, respectively. This inhibition was more pronounced than that achieved with 10 nM ETV alone (77% and 78.20% inhibition, respectively) (Supplementary Figure 3), suggesting a combined anti-HBV effect with ETV.

After established the in vitro activity of KP, we evaluated its efficacy in vivo using a hydrodynamic injection-based mouse model of chronic HBV infection. The experiment timeline is shown in Figure 4A. Serum levels of AST and ALT in model mice increased slightly in the early stage of infection, while remained within normal ranges, confirming that the hydrodynamic injection did not cause significant liver injury, and KP treatment did not induce hepatotoxicity (Supplementary Figure 4). Body weights were comparable across all group throughout the study (Supplementary Figure 5).

High serum HBsAg levels on day 0 confirmed successful model establishment (Figure 4B). KP administration induced a time- and dose-dependent decline in serum levels of HBsAg, HBeAg, and HBV DNA (Figures 4B–D). Consistent with the serum data, analysis of liver tissues revealed that KP dose-dependently reduced hepatic levels of HBV DNA, pgRNA, total viral RNA, and the viral proteins, including HBcAg, Pol, and HBsAg (Figures 4E–J). Notably, KP was more effective than ETV monotherapy in reducing viral pgRNA levels (Figure 4F). Furthermore, KP treatment dose-dependently attenuated the HBV-induced upregulation of hepatic pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, with levels in the high-dose KP group even falling below those of the normal control (Supplementary Figure 6), indicating combined antiviral and anti-inflammatory properties of KP.

The observed suppression of viral RNA, combination with ETV, and reduction in viral proteins suggested that KP may target HBV transcription. HBV transcription is governed by four viral promoters: the Cp, two surface promoters (SpI, SpII), and the X promoter (Xp), which regulates the transcription of viral X protein (HBx) (Figure 5A). Dual-luciferase reporter assays demonstrated that KP predominantly inhibited the activity of the Cp, with minimal effects on the other promoters (Figure 5B).

As the viral Cp is regulated by host factors, we examined key transcriptional regulators, including PGC-1α, HNF4α, FOXO1 (Shlomai and Shaul, 2009a; Zheng et al., 2004; Curtil et al., 2014). In HepG2.2.15 cells, KP treatment dose-dependently decreased FOXO1 protein levels while increasing its phosphorylated form (p-FOXO1) (Figure 5C). In contrast, the protein levels of PGC-1α and HNF4α were unchanged (Supplementary Figure 7). FOXO1 is a nuclear transcription factor known to enhance HBV transcription, and its phosphorylation induces cytoplasmic translocation and functional inactivation (Butt et al., 2011; Xing et al., 2018; Shlomai and Shaul, 2009b). Since ERK is a known upstream kinase regulating FOXO1 phosphorylation in hepatocytes (Jiao et al., 2013), we examined p-ERK as its activation status. KP treatment dose-dependently increased p-ERK levels (Figure 5D). Consistent with the in vitro findings, KP upregulated p-ERK and p-FOXO1 while downregulated FOXO1 in mouse liver tissues (Figure 5E). To directly link this pathway to the antiviral effect, we used the specific ERK inhibitor U0126. Pre-treatment with U0126 abolished KP-induced increases in p-ERK and p-FOXO1, and prevented the decrease in FOXO1 (Figure 5F). Crucially, U0126 also reversed the KP-mediated suppression of viral Cp activity by the luciferase assay (Figure 5G).

A final rescue experiment was performed to confirm the functional role of the ERK/FOXO1 pathway in the anti-HBV activity of KP. We assessed viral replication markers after co-treatment with KP and ERK inhibitor U0126. Although KP dose-dependently inhibited the levels of viral HBsAg, HBeAg, HBV RNA, and DNA, co-treatment with U0126 significantly attenuated this suppression across all viral markers (Figure 6). This reversal confirmed that the anti-HBV effect of KP was primarily mediated through activation of the ERK/FOXO1 signaling pathway.