High-Throughput Screening of Antiviral Compounds Using a Recombinant Hepatitis B Virus and Identification of a Possible Infection Inhibitor, Skimmianine
Mika Yoshita,
Masaya Funaki,
Tetsuro Shimakami,
Masaki Kakuya,
Kazuhisa Murai,
Saiho Sugimoto,
Shotaro Kawase,
Koji Matsumori,
Taro Kawane,
Tomoki Nishikawa,
Asuka Nakamura,
Reo Suzuki,
Atsuya Ishida,
Narumi Kawasaki,
Yuga Sato,
Ying-Yi Li,
Ariunaa Sumiyadorj,
Kouki Nio,
Hajime Takatori,
Kazunori Kawaguchi,
Kazuyuki Kuroki,
Takanobu Kato,
Masao Honda,
Taro Yamashita
Affiliations
Mika Yoshita
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
Masaya Funaki
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
Tetsuro Shimakami
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
Masaki Kakuya
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
Kazuhisa Murai
Department of Clinical Laboratory Medicine, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan
Saiho Sugimoto
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
Shotaro Kawase
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
Koji Matsumori
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
Taro Kawane
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
Tomoki Nishikawa
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
Asuka Nakamura
Department of Clinical Laboratory Medicine, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan
Reo Suzuki
Department of Clinical Laboratory Medicine, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan
Atsuya Ishida
Department of Clinical Laboratory Medicine, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan
Narumi Kawasaki
Department of Clinical Laboratory Medicine, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan
Yuga Sato
Department of Clinical Laboratory Medicine, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan
Ying-Yi Li
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
Ariunaa Sumiyadorj
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
Kouki Nio
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
Hajime Takatori
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
Kazunori Kawaguchi
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
Kazuyuki Kuroki
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
Takanobu Kato
Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
Masao Honda
Department of Clinical Laboratory Medicine, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan
Taro Yamashita
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan
We developed a novel hepatitis B virus (HBV) infection-monitoring system using a luminescent, 11-amino acid reporter (HiBiT). We performed high-throughput antiviral screening using this system to identify anti-HBV compounds. After the infection of primary human hepatocytes with the recombinant virus HiBiT-HBV, which contains HiBiT at its preS1, 1262 compounds were tested in a first screening using extracellular HiBiT activity as an indicator of viral infection. Following a second screening, we focused on the compound skimmianine, which showed a potent antiviral effect. When skimmianine was added at the same time as HiBiT-HBV infection, skimmianine inhibited HiBiT activity with EC50 of 0.36 pM, CC50 of 1.67 μM and a selectivity index (CC50:EC50 ratio) of 5,100,000. When skimmianine was added 72 h after HiBiT-HBV infection, the EC50, CC50 and selectivity index were 0.19 μM, 1.87 μM and 8.79, respectively. Time-lapse fluorescence imaging analysis using another recombinant virus, ReAsH-TC155HBV, with the insertion of tetra-cysteine within viral capsid, revealed that skimmianine inhibited the accumulation of the capsid into hepatocytes. Furthermore, skimmianine did not inhibit either attachment or internalization. These results imply that skimmianine inhibits the retrograde trafficking of the virus after internalization. This study demonstrates the usefulness of the recombinant virus, HiBiT-HBV, for high-throughput screening to identify anti-HBV compounds.
