LAUSR

Abstract Antiviral therapies for chronic hepatitis B virus (HBV) infection that are currently applicable for clinical use are limited to nucleos(t)ide analogs targeting HBV polymerase activity and pegylated interferon alpha (PEG‐IFN). Towards establishing an effective therapy for HBV related diseases, it is important to develop a new anti‐HBV agent that suppresses and eradicates HBV. This study used recombinant HBV encoding NanoLuc to screen anti‐HBV compounds from 1827 US Food and Drug Administration approved compounds and identified several compounds that suppressed HBV infection. Among them, KX2‐391, a non‐ATP‐competitive inhibitor of SRC kinase and tubulin polymerization, was identified as a lead candidate for an anti‐HBV drug. Treatment of sodium taurocholate cotransporting polypeptide (NTCP) transduced‐HepG2 (HepG2‐NTCP) or primary human hepatocytes with KX2‐391 suppressed HBV replication in a dose‐dependent manner. The anti‐HBV activity of KX2‐391 appeared not to depend on SRC kinase activity because siRNA for SRC mRNA did not impair the HBV infection/replication. The anti‐HBV activity of KX2‐391 depended on the inhibitory effect of tubulin polymerization similar to other tubulin polymerization inhibitors, some of which were shown to inhibit HBV replication. KX2‐391 inhibited HBV transcription driven by a HBV precore promoter in an HBV X protein‐independent manner but did not inhibit the activity of HBV‐S1, ‐S2, ‐X or cytomegalovirus promoters. Treatment with KX2‐391 reduced the expression of several various factors including hepatocyte nuclear factor‐4a. HighlightsTo identify new anti‐HBV agents, we screened a library of 1827 compounds using recombinant HBV encoding NanoLuc.The suppression of HBV replication by KX2‐391, a SRC kinase and tubulin polymerization inhibitor, was dose dependent.Anti‐HBV activity of KX2‐391 depends on the inhibitory effect of tubulin polymerization but not SRC kinase activity.KX2‐391 inhibits HBV RNA transcription by reducing the expression of HNF4A.