Novel Hepatitis B Virus Capsid-Targeting Antiviral That Aggregates Core Particles and Inhibits Nuclear Entry of Viral Cores

Abstract

An estimated 240 million are chronically infected with hepatitis B virus (HBV), which can lead to liver disease, cirrhosis, and hepatocellular carcinoma. Currently, HBV treatment options include only nucleoside reverse transcriptase inhibitors and the immunomodulatory agent interferon alpha, and these treatments are generally not curative. New treatments with novel mechanisms of action, therefore, are highly desired for HBV therapy. The viral core protein (Cp) has gained attention as a possible therapeutic target because of its vital roles in the HBV life cycle. Several classes of capsid assembly effectors (CAEs) have been described in detail, and these compounds all increase capsid assembly rate but inhibit HBV replication by different mechanisms. In this study, we have developed a thermal shift-based screening method for CAE discovery and characterization, filling a much-needed gap in high-throughput screening methods for capsid-targeting molecules. Using this approach followed by cell-based screening, we identified the compound HF9C6 as a CAE with low micromolar potency against HBV replication. HF9C6 caused large multicapsid aggregates when capsids were assembled in vitro and analyzed by transmission electron microscopy. Interestingly, when HBV-expressing cells were treated with HF9C6, Cp was excluded from cell nuclei, suggesting that this compound may inhibit nuclear entry of Cp and capsids. Furthermore, mutational scanning of Cp suggested that HF9C6 binds the known CAE binding pocket, indicating that key Cp-compound interactions within this pocket have a role in determining the CAE mechanism of action.

Keywords: antiviral; capsid; hepatitis B virus.

Figure 1.. Development and validation of thermal shift assay for Cp binders.

(A) Schematic of thermal shift assay (TSA). (B) TSA was performed under non-assembly (50 mM sodium bicarbonate, pH 9.6, no NaCl, no pre-incubation) and full assembly (50 mM HEPES, pH 7.5, 0.5 M NaCl, 30 min pre-incubation at 37°C) conditions for Cp (7.5 μM dimer). (C-D) TSA was performed for Cp (7.5 μM dimer) in the presence or absence of 20 μM (C) Bay 38-7690 or (D) DVR-56

Figure 2.. Screening for Cp-binding antivirals.

Cp thermal shift profiles for DMSO (solid lines) and selected hit compounds (dashed lines).

Figure 3.. Antiviral screening.

(A) HepAD38 cells were treated with hit compounds (10 μM), ETV (2.5 nM), or Bay 38-7690 (500 nM) and assessed for HBV DNA production. Dashed lines indicate no change and 50% reduction compared to the DMSO control. (B) The chemical structures for the NRTIs 3TC and ETV and the CAEs HF9C6 and Bay 38-7690 are shown.

Figure 4.. HF9C6 promotes aggressive aggregation of HBV capsid particles.

TEM analysis of capsids assembled in the presence and absence of 20 μM HF9C6 or 5 μM Bay 38-7690; scale bar, 50 nm. Images are representative of at least 3 independent experiments.

Figure 5.. HF9C6 promotes cytoplasmic localization of Cp without significant changes in Cp levels.

(A) HepAD38 cells were treated with ETV (25 nM), Bay 38-7690 (3 μM), or the indicated concentrations of HF9C6 and assessed for Cp content by western blot. (B) HepAD38 cells were induced in the presence of DMSO, HF9C6 (20 μM), orBay 38-7690 (5 μM), and Cp was stained 4 days later; scale bar, 20 μm. Images are representative of at least 2 independent experiments.

Figure 6.. HF9C6 likely binds Cp in the HAP binding pocket.

Huh7 cells were transfected with HBV-expressing plasmids harboring the indicated mutations in Cp, treated with (A) 500 nM Bay 38-7690 or (B) 4 μM HF9C6, and assessed for HBV core-associated DNA by qPCR. (C-F) Purified WT or V124W Cp was subjected to TSA in the presence of DMSO (solid lines), 20 μM Bay 38-7690 (dashed lines), or 20 μM HF9C6 (dotted lines) with 500 mM or 50 mM NaCl.