AbstractThe 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.