Inhibition of Hepatitis B Virus Replication by Drug-Induced Depletion of Nucleocapsids

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Science  07 Feb 2003:
Vol. 299, Issue 5608, pp. 893-896
DOI: 10.1126/science.1077215


Chronic hepatitis B virus (HBV) infection is a major cause of liver disease. Only interferon-α and the nucleosidic inhibitors of the viral polymerase, 3TC and adefovir, are approved for therapy. However, these therapies are limited by the side effects of interferon and the substantial resistance of the virus to nucleosidic inhibitors. Potent new antiviral compounds suitable for monotherapy or combination therapy are highly desired. We describe non-nucleosidic inhibitors of HBV nucleocapsid maturation that possess in vitro and in vivo antiviral activity. These inhibitors have potential for future therapeutic regimens to combat chronic HBV infection.

The development of novel combination-based therapies for HBV infections requires antivirals that block the viral life cycle by interference with functions other than those associated with the viral polymerase (1–3). Here, we present the drug profile and mechanism of Bay 41-4109 and the congeners Bay 38-7690 and Bay 39-5493. These compounds, also referred to as heteroaryldihydropyrimidines (HAP), were discovered as highly potent non-nucleosidic inhibitors of HBV replication in vitro and in vivo (4). Unlike the presently known HBV antivirals (3, 5), HAP prevented the proper formation of viral core particles (nucleocapsids), which are the site of viral DNA replication. Core particles are stable, high molecular weight aggregates assembled from HBV core protein subunits (6).

The chemical structures of Bay 41-4109 and congeners used in this study are shown in Fig. 1A. The inhibitory concentration of Bay 41-4109 needed to decrease HBV genome replication by 50% (IC50) in stably transfected HepG2.2.15 cells (HBV-producing hepatoma cells) (7) (supporting online text S1) was 0.05 μM, whereas the IC50 of 3TC was 0.3 μM (Fig. 1B). The antiviral activity in cell culture was enantio-selective and was observed for the (−) R-enantiomers only (supporting online text S2). In the endogenous-polymerase assay, Bay 41-4109 was inactive, suggesting that genome replication is not a direct target (supporting online text S2).

Figure 1

Bay 41-4109 blocks HBV replication in vitro. (A) Structure of Bay 41-4109 and congeners used in this study. The median inhibitory concentration (IC50) is the drug concentration required to decrease HBV replication in HepG2.2.15 cells by 50%. The median lethal concentration (TC50) is the drug concentration that impairs HepG2.2.15 cell viability by 50% compared with mock-treated cells. Toxicity of test compounds was assessed by the uptake and metabolic conversion of dye Alamar Blue (11) under conditions identical to those used in (B). (B) Block of HBV replication by treatment of HBV-producing HepG2.2.15 cells with Bay 41-4109 (•) or 3TC (▪). HepG2.2.15 cells were cultured in 96-well plates for 8 days under standard conditions in the presence or absence of drug. At day 4, culture medium was changed and new inhibitor added. At day 8, supernatant and cell lysate were transferred to nylon membranes. HBV DNA detected by Southern blotting with digoxygenin-labeled probes was quantified by chemiluminescence in a Lumi-Imager. The IC50of Bay 41-4109 was 0.05 μM, whereas the IC50 of 3TC was 0.3 μM.

As observed for the replication of viral DNA, Bay 41-4109 also reduced the steady-state amount of HBV core protein in HepG2.2.15 cells (Fig. 2). The amount of immunoreactive core protein (supporting online text S3) declined with increasing concentrations of Bay 41-4109, whereas 3TC did not show any effect on the viral core protein even at a concentration of 10 μM (Fig. 2A). Bay 41-4109 did not interfere with the efficiency of the immunoprecipitation and protein recovery (supporting online text S4, fig. S1). Testing the congener Bay 38-7690, we observed similar effects on newly synthesized core proteins and HBV DNA replication in transiently transfected hepatoma cells (supporting online text S5, fig. S2). When the effect of Bay 38-7690 on the formation of “empty core particles” induced by the core-protein expression vector pCS1aC1 (8) was studied, a substantial reduction was observed as well. Thus, interactions with viral components other than core protein were not essential for depletion (supporting online text S6, fig. S3).

Figure 2

Bay 41-4109 and Bay 38-7690 reduce HBV core protein levels in cell culture. (A) Dose-dependent reduction of core protein in the presence of Bay 41-4109 (lanes 4 to 7). 3TC showed no effect (lane 3). Lane 1, virus-free parent HepG2 cells; lane 2, mock-treated virus-producing HepG2.2.15 cells. At day 8 of culture, cell lysates were prepared and core protein was immunoprecipitated (11). Immunoprecipitates were resolved on SDS–polyacrylamide gel electrophoresis gels and visualized by immunoblotting. (B) Bay 38-7690–induced core particle (immunoblot I) and core protein (immunoblot II) depletion in cells of the Huh7 line transiently transfected with the wild-type HBV dimer DNA construct pd2 (9). (C) Cells of the HepG2 line transiently transfected with the preC minus dimer construct pd24 (9). Twenty-four hours after transfection, treatment was started (time point set at 0 hours) in five groups corresponding to lanes 1 to 5 of the respective immunoblots and RNA blots. Lane 1: control, harvest at 24 hours; lane 2: actinomycin (2 μg/ml), harvest at 24 hours; lane 3: Bay 38-7690 (5 μM), harvest at 24 hours; lane 4: Bay 38-7690 until 24 hours, removal of drug, harvest at 48 hours; lane 5: Bay 38-7690 until 24 hours, removal of drug, actinomycin until 48 hours, harvest.

To determine whether reduced core protein levels are the cause or consequence of the reduced capsid formation, we examined whether the decline of core particles coincided with the decline of core protein in the presence of Bay 38-7690. Transiently transfected hepatoma cells (9) were treated with Bay 38-7690 and, for comparison, with 3TC. Two portions of each sample were processed to visualize core particles and core protein separately (supporting online text S3). Core protein was readily detected, at concentrations between 0.4 and 2 μM Bay 38-7690, but no core particles were detected (Fig. 2B). In contrast, 3TC did not reduce core particle or core protein levels. Thus, Bay 38-7690 treatment interfered with the formation of core particles (assembly) without primarily affecting core protein levels. Consequently, no effect of the drug would be expected on transcript levels, as was indeed observed (Fig. 2C). Upon drug removal, efficient particle formation was resumed and the resumption was dependent on ongoing transcription, i.e., was actinomycin sensitive.

To study the interaction of HAP with isolated core protein, we investigated binding between tritium-labeled Bay 39-5493 and HBV core particles expressed in Escherichia coli by identifying drug-protein complexes with a molecular sieve column (Fig. 3). Only the active (−) enantiomer, and not the biologically inactive (+) enantiomer, bound to core particles. In the presence of core particles, a shift of radioactivity from late elution volumes to the void volume occurred (Fig. 3A). Binding was not only enantio-selective but also specific for the human virus. No interaction was observed for duck hepatitis B virus (DHBV) core particles (supporting online text S4 and S7, fig. S4).

Figure 3

HAP binding to HBV core particle is stereo-specific and reversible. (A) Bay 39-5493, a congener of Bay 41-4109, was used for radioactive labeling. Its biologically active (−) enantiomer eluted with HBV core particles (capsid) (▴) in the void volume of the size exclusion column (fractions 3 and 4) (supporting online text S7). No elution in the void volume was observed with the (+) enantiomer (◊) or biotinylated bovine serum albumin (BSA) plus (−) enantiomer (□). BSA plus core particle again showed elution of bound radioactivity in the void volume (•). Radioactivity contained in fractions 11 to 30 (unbound) is shown on the right. (B) Elution profile of (−) enantiomer [3H]Bay 39-5493 in the presence of HBV capsids and cold competitor (left). Molar excess of competitor is indicated as follows: (▪), no competitor; (▴), 1-fold molar excess; (▾), 10–fold molar excess; (⧫), 100–fold molar excess; (•), 1000–fold molar excess.

Binding of [3H]Bay 39-5493 to E. coli–expressed HBV core particles was reversible (Fig. 3B). This result was confirmed by isothermal titration calorimetry (10). The binding constant was calculated to be 3.32 × 107 ± 2.02 × 107 liter/mol, and the number of binding sites was ∼136 sites per capsid, which might indicate that [3H]Bay 39-5493 occupied as many binding sites as there are core protein–dimer subunits in the viral capsid. The dissociation constant (3 × 10−8 M) was in the range of the IC50 of Bay 39-5493 required to inhibit the viral replication by half in cell culture. Thus, reversible, enantio- selective, and species-specific binding of HAP to core particles in vitro was demonstrated. It is to be assumed that this binding also occurs in cells in which HBV replicates. However, HAP binding may not be confined to capsids but may also occur with core protein dimers and lower aggregation forms.

To define a possible binding site, we performed cross-linking experiments between azido-modified HAP and core particles. Amino acids assumed to participate in binding were mutated (substitution of His with Leu at amino acid residues 47, 51, and 52) to assess respective mutant viruses for drug resistance. Only one mutant (His-52) was replication competent, and it had an increased rather then a decreased sensitivity to Bay 39-5493 (supporting online text S8, fig. S5). It thus remains open whether it is possible to confer HAP resistance through mutation of HAP binding sites.

To monitor the fate of newly synthesized HBV core protein in the presence of drug (preceded by incubation for 48 hours) and to examine the ability of core proteins to aggregate into stable core particles, we performed pulse-chase experiments (10-min pulse) (Fig. 4A) (11). At zero and 1 hour of chase, cells synthesized similar amounts of core protein regardless of the presence or absence of the congener Bay 39-5493, indicating that there was no influence of the drug on core translation (Fig. 4A). However, at 24 hours of chase, the core protein signal, as determined by photostimulated luminescence (PSL), was reduced to ∼10% when compared with that of the untreated control (8 versus 81 PSL units). The apparent half-life of core protein was calculated to be 3 hours, whereas its half-life in the untreated sample exceeded 24 hours (Fig. 4A). When Bay 39-5493 was added to the cells at the beginning of the chase, no depletion of core protein was observed compared with untreated controls (supporting online text S9, fig. S6). This result was to be expected, assuming that within 10 min of labeling, most newly synthesized core protein was aggregated (supporting online text S3) and thereby rescued from the activity of the drug. Apparently, it was only when particle formation was inhibited by HAP that core protein did not become stabilized and was instead degraded. This process was proteasome mediated (Fig. 4B): The addition of the proteasome inhibitor lactacystin induced accumulation of HBV core protein at 4 hours (84 versus 34 PSL units) and even at 8 h of chase (58 versus 25 PSL units), whereas all of the core protein had virtually disappeared at this time point in the Bay 41-4109–treated samples that were devoid of lactacystin. In conclusion, our data provide strong evidence for an inhibition of particle formation as the primary event and an increased degradation of core protein as a consequence of this mode of HAP action.

Figure 4

Bay 41-4109 and Bay 38-7690 induce depletion of newly synthesized core proteins in HepG2.2.15 cells, apparently by way of the proteasome pathway. (A) Cells subjected to a pulse-chase procedure after 2 days in the presence or absence of Bay 39-5493 (0.2 μM) were extracted and immunoprecipitated for assay of core protein (11). Photostimulated luminescence (11) served as a quantitative measure for the amount of recovered labeled core protein. Newly synthesized core protein faded in the presence (+) of Bay 39-5493 (0.2 μM), but not in mock-treated cells (−). (B) Cells first treated with or without Bay 41-4109 (4109; 0.2 μM) and/or lactacystin (lacta; 10 μM) for 2 days were subjected to pulse chase (11) (top). Lactacystin delayed degradation of newly synthesized core protein by the proteasome pathway (see 4 and 8 hours of chase).

We present a substance class for the treatment of HBV infection that displays a highly specific antiviral principle, namely, inhibition of capsid formation, concomitant with a reduced half-life of the core protein. The candidate, Bay 41-4109, may become a valuable addition to future therapy (mono- or combination-therapy regimens) in light of its specific mechanism of action. It has a demonstrated efficacy in HBV transgenic mice (4) and a suitable preclinical pharmacokinetic and toxicology profile (supporting online text S10). The clinical efficacy of this treatment modality of HBV infection will now need to be demonstrated.

Supporting Online Material

Materials and Methods

SOM Text S1 to S10

Figs. S1 to S6


  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: karl.deres.kd1{at} (K.D.); c.schroeder{at}; arnold.paessens.ap{at} (A.P.);{at} (H.R.-W.)


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