In Vitro Inhibition Of HBV Replication By A Novel Compound And Its Efficacy To Adefovir-resistant HBV Mutations

Background and aims:Hepatitis B virus (HBV) infection continues to be a major cause of liver desease. Patients with chronic hepatitis B (CHB) can be successfully treated using nucleos(t)ide analogues (NA), but drug-resistant HBV mutants frequently arise, leading to treatment failure and progression to liver diseases. Therefore, future standard treatments of chronically persistent HBV infections should be comprised of a new HBV drug and/or an optimized combination of agents with different modes of antiviral actions. Recently, heteroaryldihydropyrimidines (HAPs) were discovered as highly potent non-nucleosidic inhibitors of HBV replication in vitro and in vivo. Unlike the presently known HBV antivirals, HAP prevented the proper formation of viral core particles (nucleocapsids). Here, we described the mechanism of a representative HAP compound, GH-1, which exhibits a novel and highly specific anti-HBV activity.Methods:(1) Cultured HepG2.2.15 cells and supernatants on day 8 following treatment of GH-1 and 3TC were harvested for analysis. The median toxic concentrations (TC50) were determined to investigate the toxicity of GH-1 by using MTS assays. (2) HBV replicative intermediates (RI) detected by Southern blotting were quantified and the median inhibitory concentration (IC50) of GH-1 and 3TC were estimated from dose-response curves. (3) HepG2.2.15 cells and HepG2 cells transfected transiently with a C-terminal flag-tagged HBcAg 1-149/pcDNA3.1 construct were treated with GH-1 and, for comparison, with 3TC. Two portions of each sample were processed to visualize core particles and core protein separately by immunoblotting. (4) Truncated hepatitis B core protein 149 (Cp149) was expressed in E coli and purified. T= 4 capsids were crystallized by the vapor-diffusion method. The particle size distribution was detected by dynamic light scattering, and the capsids assembled with Cp149 were observed by electron microscopy (EM). (5) To determine the antiviral activity of GH-1 to adefovir (ADV)-resistant HBV mutants, HepG2 cells were transiently transfected with PUC-HBV1.2 plasmids containing three major ADV-resistant HBV mutations (rtA181T, rtA181V and rtN236T, respectively) following GH-1 treatment. Intracellular HBV replicative intermediates were detected by Southern blotting. The ratios of mutant to wild-type IC50S for each drug (arbitrarily named "resistance factors") were determined. Drug sensitivity and resistance were defined on the basis of resistance factors.Results:(1) TC50S of GH-1 and 3TC were 73.5μM and>100μM, respectively. (2) The IC50 of GH-1 on HBV replication in HepG2.2.15 was 0.01μM, which was significantly lower than that of 3TC (0.3μM). (3) The amount of HBV capsids declined with increasing concentrations of GH-1 in HepG2.2.15 cells, whereas 3TC did not show any effect on the viral core particles even at a high concentration of 10μM. GH-1-induced core particle and core protein depletion in cells of the HepG2 cells transiently transfected with pHBc1-149. (4) With increasing concentrations of GH-1, the particle size increased and the observed proportion of intact capsids decreased, while anamorphic core protein masses increased. (5) GH-1 could efficiently inhibit the replication of ADV-resistant HBV mutants, with similar IC50 compared with that of wild-type HBV.Conclusions:GH-1 could efficiently inhibit replication of HBV in vitro. Inhibition of HBV core particle formation by GH-1 suggested its novel mechanism of action. GH-1 is also a highly active inhibitor of both wild-type and ADV-resistant HBV strains. The candidate, GH-1, may become a valuable addition to future therapy (mono- or combination-therapy regimens) in light of its specific mechanism of action.