Discovery of bimodal hepatitis B virus ribonuclease H and capsid assembly inhibitors
Daniel P. Bradley, Caleb J. Valkner, Qilan Li, Makafui Gasonoo, Marvin J. Meyers, Georgia-Myrto Prifti, Dimitrios Moianos, Grigoris Zoidis, Adam Zlotnick, John E. Tavis
Abstract
Hepatitis B virus (HBV) ribonuclease H (RNaseH) inhibitors are a potent class of antivirals that prevent degradation of the viral pregenomic RNA during reverse transcription and block formation of mature HBV DNAs. Development of HBV RNaseH inhibitors is entering advanced preclinical analyses. To ensure the mechanism of action was fully understood, we defined the effects of RNaseH inhibitors on other steps of HBV replication. Some N-hydroxypyridinedione (HPD) HBV RNaseH inhibitors significantly reduced accumulation of capsids in HBV-replicating cells. A representative HPD 1466, with a 50% effective concentration against HBV replication of 0.25 µM, decreased capsid and core protein accumulation by 50–90% in HepDES19 and HepG2.2.15 cells.
Introduction
Hepatitis B virus (HBV) is a hepatotropic partially double-stranded DNA virus that chronically infects an estimated 254 million people worldwide and causes ~1 million deaths annually [1,2]. Current antiviral therapies primarily employ nucleos(t)ide analogs (NA) which block viral DNA synthesis and drive HBV titers to near or below clinical detection levels but are not curative [3,4]. Therefore, treatment is usually lifelong. The development of new anti-HBV therapies has focused on targeting multiple aspects of the viral replication cycle that will work in concert with current therapeutics to identify a functional cure that maintains undetectable viral levels after treatment termination [5].
Materials and method
Compounds
Structures of all compounds are in S1 Fig. The αHT compounds 110 and 390 were synthesized as described in [28,29]. The HNO 1073 was described in [55], compound 148 and 150 were reported in [60,61]. HNO 1562 was newly synthesized as described in S1 Data. The HPD 1133 (also called A23) was reported in [62]. The HPD 1736 was reported in [63]. HPDs 514, 1463, 1466, 1467, and 1740 were newly synthesized as described (S1 Data).
Results
RNaseH inhibitors can alter capsid accumulation
HBV RNaseH inhibitors block viral replication and produce large RNA:DNA heteroduplexes [7] that are stiffer than double-stranded DNA (dsDNA) [13,14]. Due to the capsid’s small size and metastable nature [22–24], we hypothesized that the RNA:DNA heteroduplex could exert a greater force than dsDNA on the nucleocapsid, potentially disrupting them. Therefore, we screened ten HBV RNaseH inhibitors from three chemotypes at 2x or 5x their 50% effective concentrations (EC50) against HBV replication for effects on capsid accumulation in replicating HepDES19 cells, a line carrying a tetracycline-repressible HBV pgRNA expression cassette [25].
Discussion
HPDs, including 1466, are potent HBV RNaseH inhibitors that preferentially inhibit synthesis of the HBV positive-polarity DNA strand (S1 Fig). As part of our preclinical development and evaluation of HBV RNaseH inhibitors, we aimed to define their effects on each step of HBV replication and found that HPD compounds suppress capsid accumulation in HepDES19 and HepG2.2.15 cells (Figs 1–2, and 5). Detailed analysis of the representative HPD compound 1466 revealed that it inhibits empty capsid accumulation in a dose-dependent manner independently of HBV DNA replication and when HBc is expressed by itself in cells (Fig 4).
Acknowledgments
We thank Ryan Murelli for synthesizing the αHT compounds, and Anna Pan, Suvajit Koley, and the Indiana University-Bloomington Electron Microscopy Center for technical assistance.
Citation: Bradley DP, Valkner CJ, Li Q, Gasonoo M, Meyers MJ, Prifti G-M, et al. (2025) Discovery of bimodal hepatitis B virus ribonuclease H and capsid assembly inhibitors. PLoS Pathog 21(2): e1012920. https://doi.org/10.1371/journal.ppat.1012920
Editor: Jianming Hu, Pennsylvania State University College of Medicine: Penn State College of Medicine, UNITED STATES OF AMERICA
Received: October 2, 2024; Accepted: January 17, 2025; Published: February 10, 2025
Copyright: © 2025 Bradley 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 relevant data are in the manuscript and its Supporting information files.
Funding: This work was supported by grants R01 AI148362 to JET and MJM, R01 AI118933 to AZ, both from the Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, US National Institutes of Health (https://www.nih.gov/), and also support from Gilead Sciences’ ‘ASKLEPIOS Grants Program’ (https://www.gilead.gr/en/our-purpose/corporate-giving/what-we-fund/) to GZ, and R01 AI156610 to JET from the Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, US National Institutes of Health (https://www.nih.gov/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: JET and MJM are inventors on patents covering HPD and HNO compounds as potential treatments for HBV. No other authors have a competing interest.
