Preclinical Characterization of NVR 3-778, a First-in-Class Capsid Assembly Modulator against Hepatitis B Virus

Abstract

NVR 3-778 is the first capsid assembly modulator (CAM) that has demonstrated antiviral activity in hepatitis B virus (HBV)-infected patients. NVR 3-778 inhibited the generation of infectious HBV DNA-containing virus particles with a mean antiviral 50% effective concentration (EC₅₀) of 0.40 µM in HepG2.2.15 cells. The antiviral profile of NVR 3-778 indicates pan-genotypic antiviral activity and a lack of cross-resistance with nucleos(t)ide inhibitors of HBV replication. The combination of NVR 3-778 with nucleos(t)ide analogs in vitro resulted in additive or synergistic antiviral activity. Mutations within the hydrophobic pocket at the dimer-dimer interface of the core protein could confer resistance to NVR 3-778, which is consistent with the ability of the compound to bind to core and to induce capsid assembly. By targeting core, NVR 3-778 inhibits pregenomic RNA encapsidation, viral replication, and the production of HBV DNA- and HBV RNA-containing particles. NVR 3-778 also inhibited de novo infection and viral replication in primary human hepatocytes with EC₅₀ values of 0.81 µM against HBV DNA and between 3.7 and 4.8 µM against the production of HBV antigens and intracellular HBV RNA. NVR 3-778 showed favorable pharmacokinetics and safety in animal species, allowing serum levels in excess of 100 µM to be achieved in mice and, thus, enabling efficacy studies in vivo The overall preclinical profile of NVR 3-778 predicts antiviral activity in vivo and supports its further evaluation for safety, pharmacokinetics, and antiviral activity in HBV-infected patients.

Keywords: CHB; HBV inhibitors; capsid assembly modulator; chronic hepatitis B.

FIG 1

NVR 3-778 targets HBV core protein and inhibits viral replication. (A) Chemical structure of NVR 3-778. (B) Purified recombinant HBV core protein in the absence or presence of NVR 3-778. NVR 3-778 was incubated with recombinant core protein (1.2:1 compound-to-core monomer ratio) in buffer containing 0.15 M NaCl at room temperature overnight prior to staining and imaging by electron microscopy. (C, D) The effect of NVR 3-778 on intracellular rcDNA, extracellular HBV DNA, or cell viability (C) or on intracellular encapsidated pgRNA- or extracellular HBV RNA-containing particles (D) was determined upon treating HepG2.2.15 cells with increasing concentrations of NVR 3-778. (E, F) The effect of TFV on intracellular rcDNA, extracellular HBV DNA, or cell viability (E) or on intracellular encapsidated pgRNA- or extracellular HBV RNA-containing particles (F) was determined upon treating HepG2.2.15 cells with increasing concentrations of TFV. Secreted HBV DNA and secreted HBV RNA levels were determined from the supernatant of HepG2.2.15 cells. Intracellular encapsidated rcDNA and pgRNA levels were determined upon NP-40 lysis of cells and by using S7 nuclease to remove nonencapsidated nucleic acids. Cell viability was determined by measuring ATP levels using the CellTiter-Glo assay. Data points are mean values from at least three independent experiments, with standard deviations shown as error bars.

FIG 2

Transient-transfection studies using representative HBV strains from GT A to H. (A) Amino acid sequence alignment of HBV core encoded in the viral genomes from GT A to H isolates used in the transient-transfection studies. (B) Relative replication capacities of HBV of GT A to H. HepG2 cells were transfected with HBV-containing plasmids. HBV DNA replication was determined at 3 days posttransfection by measuring intracellular encapsidated HBV DNA signals. Data represent the mean values from at least three independent transfection experiments, and standard deviations are shown as error bars. (C) HepG2 cells transfected with HBV-containing plasmids were treated with increasing concentrations of NVR 3-778 for 3 days. Intracellular encapsidated HBV DNA levels were monitored and compared with those for untreated cells. Data points are EC₅₀ values from independent experiments. Means and standard deviations are shown.

FIG 3

Effect of NVR 3-778 in combination with nucleos(t)ide analogs. HepG2.2.15 cells were treated with increasing concentrations of NVR 3-778, LMV, TFV, or ETV for 6 days. (A to C) Synergy plots at 95% confidence calculated from MacSynergy II software. Three different assay plates of HepG2.2.15 cells treated with increasing concentrations of NVR 3-778 in combination with LMV (A), TFV (B), or ETV (C) in a checkerboard format were used. (D to G) The effect of combining NVR 3-778 and a nucleoside analog on secreted HBV RNA was further evaluated. HepG2.2.15 cells were treated for 6 days with NVR 3-778 (0.4 μM, 2 μM, and 4 μM) or LMV (0.15 μM, 0.75 μM and 1.5 μM) either alone or in combination. (D) The effect of NVR 3-778 or LMV on secreted HBV DNA, secreted HBV RNA, or cell viability in cells receiving treatment with a single drug. (E to G) Effect of combining NVR 3-778 and LMV on secreted HBV DNA (E), secreted HBV RNA (F), and cell viability (G). Each concentration of NVR 3-778 (0.4 μM, 2 μM, and 4 μM) was combined with LMV dosed at 0.15 μM, 0.75 μM, or 1.5 μM. Extracellular HBV DNA and HBV RNA levels were determined from supernatants and compared to those for untreated cells. Cell viability was determined by measuring ATP levels using the CellTiter-Glo assay. Data points represent mean values, and standard deviations are shown as error bars.

FIG 4

Susceptibility of HBV core variants to inhibition by NVR 3-778. HepG2 cells transiently transfected with wild-type HBV or the Y118F core variant (A), the I105L, I105T, or I105V core variant (B), or the T109S, T109M, or T109I core variant (C) were incubated with increasing concentrations of NVR 3-778 for 3 days. Intracellular encapsidated HBV DNA levels were monitored and compared with those for untreated cells. The dose-response curves for NVR 3-778 against HBV containing the wild-type genome are shown as dashed lines. Data points represent mean values from at least three independent antiviral studies, and standard deviations are shown as error bars.