Reduced hepatitis B and D viral entry using clinically applied drugs as novel inhibitors of the bile acid transporter NTCP

Abstract

The sodium taurocholate co-transporting polypeptide (NTCP, SLC10A1) is the main hepatic transporter of conjugated bile acids, and the entry receptor for hepatitis B virus (HBV) and hepatitis delta virus (HDV). Myrcludex B, a synthetic peptide mimicking the NTCP-binding domain of HBV, effectively blocks HBV and HDV infection. In addition, Myrcludex B inhibits NTCP-mediated bile acid uptake, suggesting that also other NTCP inhibitors could potentially be a novel treatment of HBV/HDV infection. This study aims to identify clinically-applied compounds intervening with NTCP-mediated bile acid transport and HBV/HDV infection. 1280 FDA/EMA-approved drugs were screened to identify compounds that reduce uptake of taurocholic acid and lower Myrcludex B-binding in U2OS cells stably expressing human NTCP. HBV/HDV viral entry inhibition was studied in HepaRG cells. The four most potent inhibitors of human NTCP were rosiglitazone (IC₅₀ 5.1 µM), zafirlukast (IC₅₀ 6.5 µM), TRIAC (IC₅₀ 6.9 µM), and sulfasalazine (IC₅₀ 9.6 µM). Chicago sky blue 6B (IC₅₀ 7.1 µM) inhibited both NTCP and ASBT, a distinct though related bile acid transporter. Rosiglitazone, zafirlukast, TRIAC, sulfasalazine, and chicago sky blue 6B reduced HBV/HDV infection in HepaRG cells in a dose-dependent manner. Five out of 1280 clinically approved drugs were identified that inhibit NTCP-mediated bile acid uptake and HBV/HDV infection in vitro.

Figure 1

Screening approach and results. (A) Flow diagram of the approach to identify drugs that inhibit human NTCP. (B) B-scores for each compound of the two screening assays are depicted with inhibition of uptake present in the lower quadrants and inhibition of Myrcludex B binding in the left quadrants. Each dot represents a compound, with bile acids in green, previously known NTCP-inhibitors in blue, and the compounds selected for further analysis in red.

Figure 2

Inhibition of human NTCP by zafirlukast. (A) Taurocholate uptake into U2OS-HA-hNTCP cells was reduced in the presence of zafirlukast in a concentration-dependent fashion. (B) After normalization to maximal NTCP inhibition, the IC₅₀ value was calculated. N = 2–4 wells/condition, experiment was repeated twice. (C) Representative confocal microscopy pictures of reduced Myrcludex B-FITC fluorescence upon co-administration with increasing amounts of zafirlukast. (D) Quantification of C, n = 3 per condition. All data are presented as mean ± SD.

Figure 3

Inhibition of human NTCP by chicago sky blue 6B, rosiglitazone, sulfasalazine, and TRIAC. Taurocholate uptake and Myrcludex B binding was reduced to U2OS-HA-hNTCP cells in a concentration-dependent manner by chicago sky blue 6B (A,B), rosiglitazone (C,D), sulfasalazine (E,F), and TRIAC (G,H). Data are presented as mean ± SD, n = 2–4 wells/condition, experiment was repeated twice. From the confocal studies, representative pictures are shown, n = 3 per condition.

Figure 4

Myrcludex B inhibits uptake of tauro-ursodeoxycholic acid. Sodium dependent tauro-ursodeoxycholic acid uptake into U2OS-HA-hNTCP cells was reduced in the presence of Myrcludex B. N = 6 wells/condition, data are depicted as mean ± SD.

Figure 5

Inhibition of HBV and HDV infection in HepaRG cells. Rosiglitazone, sulfasalazine, TRIAC, zafirlukast, and irbesartan reduce both HBV (A–D) as well as HDV (E,F) infection in HepaRG cells. In a concentration-dependent fashion, hepatitis B extracellulair (A) and surface (B) antigen production was reduced by all compounds. Also, all compounds were effective in decreasing the amount of cells positively stained for HBcAg (C,D) and HDAg (E,F) in a concentration-dependent manner. Myrcludex B (1 µM) was included as positive control in all assays. Data are presented as mean ± SD, n = 3 × 2 wells/condition.

Figure 6

Compounds also inhibit mouse NTCP. (A–G) Taurocholate uptake into U2OS cells transiently transfected with mouse NTCP is inhibited similarly as human NTCP by sulfasalazine (D), tolfenamic acid (E) and zafirlukast (G), while chicago sky blue 6B (A), flufenamic acid (B), and rosiglitazone (C) are more potent in inhibiting mouse NTCP. TRIAC on the other hand inhibits human NTCP more strongly (F). Data are presented as mean ± SD, n = 2–4 wells/condition.

Figure 7

Some NTCP inhibitors also reduce ASBT-mediated bile acid uptake. (A–E) Sulfasalazine (C) is largely ineffective in inhibiting taurocholate uptake into MDCK-ASBT cells, while chicago sky blue 6B (A), rosiglitazone (B), TRIAC (D), and zafirlukast (E) inhibit ASBT in a concentration-dependent fashion. Data are presented as mean ± SD, n = 2–3 wells/condition.