Mami Matsuda, Asuka Hirai-Yuki, Osamu Kotani, Michiyo Kataoka, Xin Zheng, Daisuke Yamane, Masaru Yokoyama,Koji Ishii, Masamichi Muramatsu, Ryosuke Suzuki
Abstract
No antiviral drugs currently are available for treatment of infection by hepatitis A virus (HAV), a causative agent of acute hepatitis, a potentially life-threatening disease. Chemical screening of a small-compound library using nanoluciferase-expressing HAV identified loxapine succinate, a selective dopamine receptor D2 antagonist, as a potent inhibitor of HAV propagation in vitro. Loxapine succinate did not inhibit viral entry nor internal ribosome entry site (IRES)-dependent translation, but exhibited strong inhibition of viral RNA replication. Blind passage of HAV in the presence of loxapine succinate resulted in the accumulation of viruses containing mutations in the 2C-encoding region, which contributed to resistance to loxapine succinate. Analysis of molecular dynamics simulations of the interaction between 2C and loxapine suggested that loxapine binds to the N-terminal region of 2C, and that resistant mutations impede these interactions.
Introduction
Hepatitis A virus (HAV) is an important causative agent of acute liver disease in humans; the virus is transmitted via the fecal-oral route through ingestion of contaminated food and water, or through person-to-person contact [1]. The World Health Organization (WHO) estimates that more than 100 million people world-wide are infected with HAV, resulting in more than 15,000–30,000 mortalities from hepatitis A annually. Recent hepatitis A outbreaks have occurred not only in developing countries but also in developed countries around the globe [2–5]. Although a vaccine consisting of inactivated virus is highly efficacious in preventing HAV infection, no HAV-specific antiviral drug is available for the treatment of those already infected. HAV, a member of the genus Hepatovirus within the family Picornaviridae, is a small, quasi-enveloped, positive-sense, single-stranded RNA virus.
Materials and methods
Ethics statement
All animal experiments were approved by the Animal Care and Use Committee of the National Institute of Infectious Diseases (approval no. 122002) and carried out in accordance with the approved guidelines.
Cell culture
Cells of the human hepatoma-derived Huh7.5.1 line, human embryonic kidney 293T line and the human rhabdomyosarcoma RD-A line were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with nonessential amino acids, penicillin at 100 U/mL, streptomycin at 100 μg/mL, and 10% fetal bovine serum (FBS). All cultures were grown at 37 °C in a 5% CO2 incubator.
Results
Identification of a novel small-molecule inhibitor of HAV replication
In an effort to identify small-molecule inhibitors of HAV infection, we employed a chemical screening assay using a NanoLuc luciferase (Nluc)-expressing HAV (HAV/NLuc) [7], as shown in Fig 1A. Huh7.5.1 cells were seeded into 96-well plates and infected with HAV/NLuc in the presence of compounds. Nluc activity was analyzed at 3 days post-infection (dpi). This assay allowed the detection of inhibitors of viral attachment/entry, translation, replication, and assembly/egress. In parallel, potential cytotoxic effects of the drugs were assessed by quantifying ATP levels (using the CellTiter-Glo Kit; Promega, Madison, WI, USA) in exposed cells.
Discussion
Despite the successful development and deployment of a vaccine for hepatitis A, HAV remains a common cause of enterically transmitted hepatitis throughout the world, and is responsible for epidemics in both developing and developed countries. This challenge reflects, in part, the current lack of a HAV-specific antiviral treatment for infected patients. Clearly, a better understanding of the potential molecular targets of such antivirals is critical for the development of novel therapies. In an effort to identify small-molecule inhibitors of HAV infection, we conducted a chemical screen using HAV/NLuc; exposure to the highest-potency compound identified in this screen, loxapine succinate, was found to decrease HAV propagation by blocking viral RNA replication in vitro. This result is the first demonstration, to our knowledge, that loxapine succinate inhibits HAV propagation. Loxapine is a US FDA-approved first-generation antipsychotic medication that is used clinically, primarily in the treatment of patients with schizophrenia.
Acknowledgments
We are grateful to Y. Hirama and M. Oizumi for administrative assistance; K. Tanno, M. Ushida, Y. Suzuki, N. Nishiyama, R. Yoshida, and S. Kobayashi for technical assistance; and M. Arita for helpful discussions. We thank S. M. Lemon for providing the HAV HM175/18f strain, the HM175/18f-NLuc reporter virus, and HAV replicon construct. We also thank H. Shimizu, Y. Nishimura, and T. Kiyohara for providing experimental materials, including EV-D68, pirodavir, and HAV strains KRM031 and TKM005. The MD simulations were performed, in part, on the TSUBAME3.0 supercomputer at the Tokyo Institute of Technology.
Citation: Matsuda M, Hirai-Yuki A, Kotani O, Kataoka M, Zheng X, Yamane D, et al. (2024) Loxapine inhibits replication of hepatitis A virus in vitro and in vivo by targeting viral protein 2C. PLoS Pathog 20(3): e1012091. https://doi.org/10.1371/journal.ppat.1012091
Editor: Jun Wang, Rutgers University: Rutgers The State University of New Jersey, UNITED STATES
Received: October 12, 2023; Accepted: March 2, 2024; Published: March 13, 2024
Copyright: © 2024 Matsuda et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data generated in this study have been included in the manuscript and its Supporting information files.
Funding: This research was supported, in part, by grants-in-aid from the Ministry of Health, Labour and Welfare: Grant Number 10KA1006 (RS); the Japan Society for the Promotion of Science (JSPS) of KAKENHI:Grant Numbers JP20K08852 (RS), JP21H02746 (AHY), and JP21K16330 (OK); and the Japan Agency for Medical Research and Development:Grant Numbers JP23fk0210109 (RS), JP23fk0210132 (RS), and JP23fk0108627 (OK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1012091#sec001
