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.

Mechanistic insights into the inhibition of NTCP by myrcludex B

“…3D), a dose also previously published. 14,18 Competition of non-fluorescently labeled Myrcludex B (up to a 5-fold excess) decreases the amount of bound Myrcludex B-FITC by~40% in a dose-dependent fashion within 60 min (Fig. 4).…”

“…Preparation of U2OS-HA-hNTCP cells for confocal microscopy was performed as described. 14 Live cell fluorescent imaging was performed using a Leica SP8X-SMD confocal microscope with LAS X software and fully enclosed incubation at 37°C. A 63x oil objective was used and images were captured while the cells were consecutively excited at 490 nm (FITC) and 350 nm (Hoechst).…”

Efficient delivery of HBV NLS siRNAs into HepG2.2.15 cells for HBV inhibition through novel recombinant preS1‑tP proteins

“…The treatment and eradication of HBV infections have been limited by the unavailability of effective therapeutic drugs and methods to completely remove the cccdNA in the infected hepatocytes. Due to the significance of the human NTcP gene during HBV infection and entry, the establishment of hepatocytes with stable NTcP gene expression has been widely applied as a cellular model for the investigation of cHB pathology, and for the screening of therapeutic agents and methods for the treatment of HBV (11,20,(24)(25)(26). Guided by the same rationale, the present study used HepG2.…”

Concept of Viral Inhibitors via NTCP

“…Other anti-HBV entry compounds with unknown modes of action include oxysterols, zafirlukast, genistein, and PA452. 64,83,84 Compounds Identified by Affinity-Based Screening Assays Using Recombinant NTCP Many of the above entry inhibitors identified from cell-based screenings were proven to target NTCP. NTCP is regarded as an attractive target for the development of anti-HBV agents, given a promising clinical data of the NTCP targeting.…”