Fig. 1

Discovery of rapavir as a novel inhibitor of NTCP with potent anti-HBV efficacy. a Identification of rapavir. Left-to-right: Construction of the rapafucin 3D microarrays and all of the compounds were spotted in duplicate. Chemical structures of HP07-C6 and JH-B10 (rapavir). Dose-dependent inhibition of TCA-d₄ uptake, anti-HBV activity against HBeAg and HBV 3.5-kb RNA in HepG2-NTCP cells by HP07-C6 and rapavir. Data represent the mean ± s.d.; n = 3 samples. b Rapavir interacts with NTCP with relatively high specificity and its activity is independent of FKBP12 protein. Left-to-right: Chemical structure of biotin-rapavir. Affinity pulldown of detergent-solubilized NTCP by a biotin-rapavir conjugate and competition by free rapavir. Volcano plot analysis reveals a significant reduction in the levels of conjugated bile acids after treatment of rapavir for 6 h. FK506 did not antagonize rapavir inhibition of NTCP-mediated TCA-d₄ uptake in HepG2-NTCP cells, and knockout of FKBP12 did not confer resistance to rapavir in HepG2-NTCP cells. c Rapavir blocks the HBV infection phase in HepG2-NTCP cells. Left-to-right: The intracellular HBV cccDNA levels were detected by Taq-man qPCR. The HBV 3.5-kb RNA levels were analyzed by RT-qPCR. HBeAg and HBsAg levels in the culture supernatant were detected by ELISA. Data represent the mean ± s.d.; n = 3 samples. d Rapavir resists HBV infection in humanized liver-chimeric mice. Left-to-right: HBV DNA in serum was measured by qPCR. Serum HBeAg was quantified by chemiluminescent immunoassay. HBV cccDNA and HBV 3.5-kb RNA in the liver tissues were determined by qPCR