Molecular mechanisms of hepatitis B virus entry inhibition by a bile acid derivative INT-767 binding to the preS1 region

Hepatitis B virus (HBV) infection is a leading cause of liver disease worldwide, necessitating the development of novel antiviral agents targeting various steps in the HBV life cycle. The HBV preS1 region mediates critical steps in viral entry, including binding to sodium taurocholate co-transporting polypeptide (NTCP), making it a potential target for anti-HBV drug development. Our previous study suggested that the bile acid derivative INT-767 preferentially attaches to preS1 rather than to NTCP on hepatocytes, indicating that HBV infection is inhibited by INT-767 binding to an important domain of preS1. To elucidate the specific binding interactions between INT-767 and the preS1 region, we performed a preS1 binding assay using fluorescently labeled preS1 peptides and NTCP-expressing HepG2 cells to assess INT-767 binding efficacy. Furthermore, we evaluated the effect of amino acid substitutions in preS1 on both INT-767 binding and HBV infectivity in primary human hepatocytes. The preS1 binding assay revealed that INT-767 interacts specifically with Phe13, Phe14, and His17 in the NTCP-binding region of preS1. Functional analysis demonstrated that substitution of these residues with alanine significantly reduced HBV infectivity, indicating their crucial role in viral entry. INT-767 binding to these residues effectively blocked preS1 interaction with NTCP, thereby inhibiting HBV infection. These findings highlight a novel antiviral mechanism by which INT-767 exerts its anti-HBV effects through direct interaction with preS1, providing a potential therapeutic strategy targeting HBV entry.