AT-61 selectively inhibits HBV replication.
AT-61 belongs to a class of compounds that has an inhibitory effect on HBV replication in HepAD38 cells (22
). To extend our studies on the antiviral activity of AT-61, we have tested its ability to inhibit HBV replication in two additional systems, as well as its ability to inhibit a 3TC-resistant strain of HBV, WHBV, DHBV, HIV-1, HSV-1, NDV, and VSV.
To determine the antiviral effect of AT-61 in HepAD38 cells, we induced HBV replication in these cells by the removal of tetracycline from the medium and allowed the virus to replicate in the presence of various concentrations of drug. After 4 days, the medium was collected and assayed for the presence of HBV DNA by dot blot and phosphorimager analysis. We found that the media from cells treated with AT-61 at a concentration greater than 10 μM contained ≤10% of the HBV DNA found in the media of untreated cells (Fig. A; Table ). We found a similar result for AT-61 in HepG2 cells that were transiently transfected with a cDNA copy of the HBV pgRNA under the transcriptional control of the CMV IE promoter (Table ). However, in cells of the 2.2.15 cell line, a cell line that constitutively produces HBV, approximately fivefold more drug was required to inhibit HBV replication to the same extent (Table ). It is interesting that the 50% inhibitory concentration of AT-61 was similar (1.9 to 5.7 μM) in all three systems (Table ). In all three systems, the potency of 3TC was as expected.
FIG. 2 Inhibition of HBV replication by AT-61. HepAD38 cells were induced and treated with various concentrations of 3TC or AT-61. Three days after treatment commenced, the media were removed and replaced with fresh media containing compound. Twenty-four hours (more ...)
To determine whether AT-61 is inhibitory to the formation of immature core particles containing HBV DNA replicative intermediates, we isolated cytoplasmic core particles from induced HepAD38 cells that were treated with various concentrations of AT-61 or 3TC and analyzed them for the presence of HBV DNA replicative intermediates by Southern blot analysis. Replicative intermediates were quantified by phosphorimager analysis. AT-61 at 10 μM decreased the production of the DNA replicative intermediates by greater than 90% (Fig. B). In addition, all three replicative intermediates, relaxed circular DNA, linear DNA, and single-stranded DNA, were equally reduced (data not shown). As expected, 3TC also inhibited the production of the HBV replicative intermediates in a dose-responsive manner.
We have shown that a mutation in the HBV polymerase gene that caused an M539V substitution in the polymerase resulted in a virus that was resistant to 3TC, FTC, ddC, and zidovudine (16
). To determine if this mutation altered the sensitivity of the virus to the antiviral effects of AT-61, we induced HBV replication in HepAD79 cells by the removal of tetracycline from the medium and allowed the virus to replicate in the presence of various concentrations of AT-61. After 7 days, the medium was collected and assayed for the presence of HBV DNA by dot blot and phosphorimager analysis. The M539V polymerase variant of HBV was as sensitive as the wild type to the antiviral effects of AT-61 (Table ). In addition the EC50
of 3TC were similar to what we previously have reported (16
To determine the specificity of the antiviral activity of AT-61, we exposed replicating WHBV and DHBV for 7 days to various concentrations of AT-61 and found that AT-61 at a concentration of 81 μM did not affect the replication of either virus (Table ). Likewise, AT-61 (81 μM) had no effect on the replication of HIV-1, HSV-1, NDV, or VSV (Table ).
AT-61 does not affect the tetracycline-responsive promoter.
In HepAD38 and HepAD79 cells, transcription of HBV pgRNA is driven by a tetracycline-responsive CMV IE promoter. Since it was possible that some or all of the antiviral effect of AT-61 observed in these two cell lines may be caused by an inhibitory effect of the compound on this promoter, we created a cell line, HepAD43, that expresses the β-galactosidase gene under the transcriptional control of the tetracycline-responsive CMV IE promoter. HepAD43 cells were induced and maintained in various concentrations of AT-61 or tetracycline for 4 days. AT-61 had no effect on the production of β-galactosidase in HepAD43 cells, whereas tetracycline inhibited β-galactosidase expression in a dose-responsive manner (Fig. ).
FIG. 3 Susceptibility of the tetracycline-responsive promoter to AT-61. HepAD43 cells were induced and treated with various concentrations of tetracycline or AT-61. Three days after treatment commenced, the media were removed and replaced with fresh media containing (more ...) AT-61 has low toxicity in cells.
In the HepAD38 cell line, a compound that does not specifically inhibit HBV replication but that is cytotoxic or has an antiproliferative effect on the cell could also score as a positive hit in the antiviral assay. To rule out the possibility that the anti-HBV activity of AT-61 was due to an anticellular activity, HepAD38 and HepG2 cells, either in confluent monolayers or in active replication, were grown for 4 to 7 days in the presence of various concentrations of AT-61 and tested for viability and changes in cell number. AT-61 at concentrations as high as 81 μM had no effect on the cell viability or number of HepAD38 and HepG2 cells (Table ). In addition, we determined by microscopic examination that AT-61 at 81 μM had no cytopathic effect on HepAD43, 2.2.15, Vero, CA51, and MT-2 cells (Table ).
TABLE 2 Anticellular effects ofAT-61 AT-61 does not affect reverse transcriptase or DNA-dependent DNA polymerase activity.
Since the DNA replicative intermediates are produced from the pgRNA by the reverse transcriptase and DNA-dependent DNA polymerase activities of the HBV polymerase, we wanted to determine if the dose-dependent reduction of the replicative intermediates in HepAD38 cells treated with AT-61 was due to the inhibition of either one of these activities by AT-61. To measure the reverse transcriptase activity of the HBV polymerase, a [32P]TTP incorporation assay was performed with 0.1 μg of baculovirus-produced HBV polymerase in the presence of various concentrations of AT-61. We found that at 81 μM, AT-61 did not affect the ability of the HBV polymerase to incorporate radioactively labeled TTP into a newly synthesized strand of DNA; however, PFA at 1 mM inhibited reverse transcriptase activity, as expected (Table ).
TABLE 3 Effect of AT-61 on HBV reverse transcriptase and DNA-dependent DNAsynthesis
To assay for DNA-dependent DNA polymerase activity, HBV core particles were precipitated from the medium of induced HepAD38 cells. These core particles, which contain HBV polymerase, were incubated overnight with various concentrations of AT-61 and [32P]dCTP. The next day the viral DNA was separated by electrophoresis and analyzed by Southern blot and phosphorimager analysis for the ability of the HBV polymerase to incorporate radioactively labeled dCTP into the newly synthesized viral DNA. At 81 μM, AT-61 had no effect on the DNA-dependent DNA polymerase function of the HBV polymerase, whereas PFA at 1 mM completely inhibited polymerase activity (Table ).
AT-61 reduces the number of immature core particles containing pgRNA.
To determine if AT-61 affected the production of pgRNA or the packaging of pgRNA into immature core particles, we isolated both cytoplasmic and core-associated viral RNA from induced HepAD38 cells that were grown for 3 days in the presence of AT-61 or FTC at concentrations above their EC90s (27 and 1 μM, respectively). Viral RNA was quantified by RNase protection assay and phosphorimager analysis. We found that 27 μM AT-61 reduced the amount of cytoplasmic viral RNA by approximately 50%, whereas FTC did not affect the concentration of cytoplasmic HBV RNA (Fig. A). Moreover, AT-61 at a concentration of 27 μM reduced the level of core-associated viral RNA to below the level of detection (Fig. B). As expected, FTC had no effect on the amount of HBV RNA associated with immature core particles.
FIG. 4 Susceptibility of HBV RNA production and packaging to AT-61. (A) Production of HBV RNA. HepAD38 cells were induced and treated with AT-61 (27 μM) or FTC (1 μM) for 3 days. Cytoplasmic RNA was isolated as directed by the manufacturer (RNeasy (more ...)
Although the level of pgRNA, which serves as the mRNA template for the translation of core antigen, was reduced in the presence AT-61, Western blot analysis with antibodies specific for core and surface antigens (eAg and sAg, respectively) demonstrated that the levels of eAg and sAg in the cells were not affected by the presence of AT-61 (data not shown).
AT-61 inhibits the accumulation of cccDNA in the nucleus.
Since HBV may remain latent in hepatocytes in the form of cccDNA, it is important to determine how any potential inhibitor of HBV replication affects the level of cccDNA in the nucleus of the cell. Since the nucleus of HepAD38 cells accumulates cccDNA only after tetracycline has been removed from the medium, the treatment of HepAD38 cells with a potential inhibitor of HBV replication allows one to determine only the effect of the inhibitor on the accumulation of new cccDNA and not on the fate of existing cccDNA.
The nuclei of induced HepAD38 cells which had been treated for 3 days with AT-61 (27 μM) did not contain detectable levels of cccDNA, whereas cccDNA was abundant in the nuclei of induced cells that were not treated with AT-61 (Fig. ). As expected, FTC (1 μM) also prevented the accumulation of cccDNA in HepAD38 cells.
FIG. 5 Inhibition of cccDNA by AT-61. HepAD38 cells were induced and treated with AT-61 (27 μM) or FTC (1 μM). Three days after drug treatment started, the media were removed and replaced with fresh media containing compound. Twenty-four hours (more ...) Synergistic inhibition of HBV replication by AT-61 and 3TC.
To determine if AT-61 acts synergistically with 3TC to inhibit HBV replication, several concentrations of AT-61 and 3TC were assayed for their ability to inhibit the release of HBV DNA from HepAD38 cells. AT-61 was tested from 0.1 to 30 μM, whereas 3TC was tested from 0.003 to 1 μM. The raw data were analyzed in two ways. The first method was to create isobolograms of the inhibition data by using the Calcsyn software package (Biosoft). The shift of the curve in the isobologram to the left of the additivity line indicates that these drugs are acting synergistically at their EC50s and the EC90s (Fig. A).
FIG. 6 Synergistic inhibition of HBV replication by AT-61 and 3TC. HepAD38 cells were induced and treated with various concentrations of AT-61 and 3TC. Three days after treatment commenced, the media were removed and replaced with fresh media containing compounds. (more ...)
The second method for analyzing the data from the combination experiments was by generating CIs for several datum points at three molar ratios of compound (AT-61:3TC at 100:1, 10:1, and 1:1) by using the Calcsyn software package and plotting these in a median-effect plot. The CI values at 5, 15, 25, 35, 45, 55, 65, 75, 85, and 95% inhibition were used to determine if the drug combination was inhibiting HBV replication additively (CI = 1), synergistically (CI < 1), or antagonistically (CI > 1). For the 10:1 and 1:1 molar ratios of AT-61 to 3TC, the plots clearly show that these two drugs were acting synergistically to inhibit HBV replication; however, at a molar ratio of 100:1, the drugs appeared to be acting additively to slightly synergistically (Fig. B).