Auranofin, identified by FDA-approved drug library screening, inhibits HBs antigen secretion via lysosomal damage

Abstract

Background and aim: Hepatitis B surface antigen (HBsAg) is associated with hepatocellular carcinoma risk and immune exhaustion and contributes to hepatitis B virus (HBV) persistence. Current anti-HBV treatments have a limited ability to reduce HBsAg levels. This study aimed to identify FDA-approved drugs capable of reducing HBsAg levels and explore the underlying mechanisms.

Methods: A total of 1134 FDA-approved compounds were screened at 9.9 μM for 7 days using HepG2.2.15.7 cells, which contain an integrated HBV genome. HBsAg in the supernatant and cell viability were assessed. Compounds whose viability was reduced by >1 SD were excluded. Compounds whose HBsAg concentration decreased by >1.5 SD were selected and validated at 0.5 and 5 μM. We evaluated the HBsAg-reducing effect of the final candidate compounds using HBV-infected primary human hepatocytes (HepaSH cells) derived from chimeric TK-NOG mice and investigated the underlying mechanism responsible for the reduction in HBsAg.

Results: After 126 cytotoxic compounds were excluded, the HBsAg levels of the 6 candidates decreased by >1.5 SD. Ethacridine and auranofin significantly reduced the level of HBsAg at both 5 and 0.5 μM. In HepaSH cells, only auranofin decreased the level of HBsAg. Auranofin did not affect the HBe antigen, HBV-DNA, or pregenomic RNA, nor did it reduce the level of intracellular HBsAg, as determined by Western blotting. Transmission electron microscopy revealed more vesicles in auranofin-treated HepaSH cells than in control cells. Immunofluorescence analysis of HepaSH cells treated with auranofin revealed increased Galectin-3 expression and colocalization of HBsAg with Galectin-3, which was consistent with lysosomal damage, compared with those of the untreated controls.

Conclusion: Auranofin, an FDA-approved antirheumatic agent, reduces HBsAg secretion via lysosomal damage.

Fig 1. FDA-Approved Drug Library Screening.

(A) Cell viability and HBsAg levels after treatment with the respective compounds. (B) HBsAg was treated with the remaining 1008 compounds. (C) Six compounds whose HBsAg levels decreased by more than 1.5 SD were identified as candidate compounds. (D) Cell viability and HBsAg levels after treatment with 6 compounds at concentrations of 0, 0.5, and 5 μM. (E) HBV-inoculated HepaSH cells (1000 GEq/cell) were treated with ethacridine or auranofin. (F) Viability and HBsAg levels of HBV-inoculated HepaSH cells treated with auranofin or ethacridine (n = 3. *: p < 0.05).

Fig 2. Auranofin Specifically Reduced HBsAg in the Supernatant of HepG2.2.15.7 and HBV-Inoculated HepaSH Cells.

HepG2.2.15.7 cells (n = 3) and HBV-inoculated HepaSH cells (1000 GEq/cell, n = 4) were treated with 1 μM auranofin. (A) HBsAg levels in the supernatant and cell viability of HepG2.2.15.7 cells treated with various concentrations of auranofin. (B) HBsAg, HBeAg, and HBV DNA levels in the supernatant and intracellular pgRNA and HBV DNA levels in HepG2.2.15.7 cells treated with or without auranofin. (*: p < 0.05). (C) Western blotting and band quantification of intracellular HBs proteins in HepG2.2.15.7 cells treated with or without auranofin. (D) HBsAg, HBeAg, and HBV DNA levels in the supernatant and intracellular pgRNA, HBV DNA and cccDNA levels in HBV-inoculated HepaSH cells treated with or without auranofin. (*: p < 0.05). (E) Western blotting and band quantification of intracellular HBs proteins in HBV-inoculated HepaSH cells treated with or without auranofin.

Fig 3. Auranofin did not affect proteasome activity, endoplasmic reticulum stress, or HBV promoter activity.

(A) HBV promoter activity with or without auranofin or with IFN (500 IU/mL) in HepG2.2.15.7 cells. (n = 3, *: p < 0.05). (B) Western blotting of ER stress-related proteins from 3 to 24 hours after auranofin treatment in HepG2.2.15.7 cells. (C) Proteasome activity in HepG2.2.15.7 cells was assessed by isolation of 20S proteasomes from cell lysates using an hHR23B ubiquitin-like domain resin (UbL-resin). The cell lysate of each sample was treated with/without UbL-Resin. (UbL-: samples prepared with UbL-Resin; Au: auranofin; MTX: mitoxantrone (positive control); Ctrl: control).

Fig 4. Auranofin Reduced the Extracellular Secretion of HBsAg by Inducing Lysosomal Damage.

(A) Transmission electron microscopy images of HBV-infected HepaSH cells treated with or without auranofin and noninfected HepaSH cells. (B) Number of vesicles in the cytoplasm of HBV-infected HepaSH cells treated with or without auranofin and noninfected HepaSH cells observed by electron microscopy. (C) Immunofluorescence staining of HBV-infected HepaSH cells treated with or without auranofin.