Okumura Akinori, Muraki Yukiko, Arai Jun, Nishimura Sayaka, Inden Noriko, Sakamoto Kazumasa, Kitada Tomoya, Kimoto Satoshi, Kitano Rena, Inoue Tadahisa, Yamashita Yukiko, Iguchi Yusuke, Ohashi Hirofumi, Watashi Koichi, Wakita Takaji, Une Mizuho, Ito Kiyoaki
Department of Gastroenterology, Aichi Medical University, Nagakute, Aichi, Japan.
Faculty of Pharmaceutical Sciences, Hiroshima International University, Kure, Hiroshima, Japan.
Antiviral Res. 2025 Aug;240:106213. doi: 10.1016/j.antiviral.2025.106213. Epub 2025 Jun 11.
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.
乙型肝炎病毒（HBV）感染是全球肝脏疾病的主要病因，因此需要开发针对HBV生命周期各个步骤的新型抗病毒药物。HBV前S1区域介导病毒进入的关键步骤，包括与牛磺胆酸钠共转运多肽（NTCP）结合，使其成为抗HBV药物开发的潜在靶点。我们之前的研究表明，胆汁酸衍生物INT-767优先附着于肝细胞上的前S1而非NTCP，这表明INT-767与前S1的一个重要结构域结合可抑制HBV感染。为了阐明INT-767与前S1区域之间的特异性结合相互作用，我们使用荧光标记的前S1肽和表达NTCP的HepG2细胞进行了前S1结合试验，以评估INT-767的结合效力。此外，我们评估了前S1中氨基酸取代对INT-767结合和原代人肝细胞中HBV感染性的影响。前S1结合试验表明，INT-767与前S1的NTCP结合区域中的苯丙氨酸13、苯丙氨酸14和组氨酸17特异性相互作用。功能分析表明，用丙氨酸取代这些残基会显著降低HBV感染性，表明它们在病毒进入中起关键作用。INT-767与这些残基的结合有效地阻断了前S1与NTCP的相互作用，从而抑制了HBV感染。这些发现突出了一种新的抗病毒机制，即INT-767通过与前S1直接相互作用发挥其抗HBV作用，为靶向HBV进入提供了一种潜在的治疗策略。
