Phenylpropenamide Derivatives AT-61 and AT-130 Inhibit Replication of Wild-Type and Lamivudine-Resistant Strains of Hepatitis B Virus In Vitro

The phenylpropenamide derivatives AT-61 and AT-130 are nonnucleoside analogue inhibitors of hepatitis B virus (HBV) replication. They inhibited the replication of wild-type HBV with 50% inhibitory concentrations of 21.2 ± 9.5 and 2.40 ± 0.92 μM, respectively, compared to 0.064 ± 0.020 μM lamivudine. There were no significant differences in sensitivity between wild-type and nucleoside analogue-resistant (rtL180M, rtM204I, and rtL180M + rtM204V) HBV

In Vitro Study of the Effects of Precore and Lamivudine-Resistant Mutations on Hepatitis B Virus Replication

Understanding the consequences of mutation in the hepatitis B virus (HBV) genome on HBV replication is critical for treating chronic HBV infection. In this study, HBV replication in HepG2 cells initiated by transduction with precore (PC), rtM204I, and wild-type (wt) HBV recombinant baculoviruses was compared. The pattern and magnitude of HBV replication initiated by the PC HBV recombinant baculovirus were similar to those observed for wt HBV throughout the time course examined. In contrast, when the rtM204I mutation was introduced into wt HBV, by day 10 postinfection the levels of intra- and extracellular HBV DNA were markedly reduced compared to those for wt HBV. Although the rtM204I mutation reduced the production of HBV replicative intermediates, no effect on the level of covalently closed circular DNA or HBV transcripts was observed at late time points. Coinfection studies with different ratios of wt and rtM204I baculoviruses showed that the rtM204I variant did not produce a product that inhibited HBV replication. However, the combination of the wt and rtM204I baculoviruses yielded HBV DNA levels at late time points that were greater than those for the wt alone, suggesting that wt polymerase may function in trans to boost rtM204I replication. We concluded that the rtM204I mutation generates a polymerase that is not only resistant to lamivudine but also replicates nucleic acids to lower levels in vitro

Intracellular Metabolism and In Vitro Activity of Tenofovir against Hepatitis B Virus

Tenofovir is an acyclic nucleotide analog with activity against human immunodeficiency virus (HIV) and hepatitis B virus (HBV). Tenofovir disoproxil fumarate (tenofovir DF), a bis-alkoxyester prodrug of tenofovir, is approved for the treatment of HIV and is currently being developed to treat chronic hepatitis B. In this report, we further characterize the in vitro activity of tenofovir against HBV as well as its metabolism in hepatic cells. We show that tenofovir is efficiently phosphorylated to tenofovir diphosphate (TFV-DP) in both HepG2 cells and primary human hepatocytes. TFV-DP has a long intracellular half-life (95 h) and is a potent and competitive inhibitor of HBV polymerase (K(i) = 0.18 μM). Tenofovir has a 50% effective concentration of 1.1 μM against HBV in cell-based assays, and potency is improved >50-fold by the addition of bis-isoproxil progroups. Tenofovir has previously demonstrated full activity against lamivudine-resistant HBV in vitro and clinically. Here we show that the major adefovir resistance mutation, rtN236T, confers three- to fourfold-reduced susceptibility to tenofovir in cell culture; the clinical significance of this susceptibility shift has not yet been determined. The rtA194T HBV polymerase mutation recently identified in tenofovir DF-treated HIV/HBV-coinfected patients did not confer in vitro resistance to tenofovir as a single mutation or in a lamivudine-resistant viral background. Overall, the antiviral and metabolic profile of tenofovir supports its development for the treatment of chronic hepatitis B

Summary of HCV(1b/2a) Levels After 21 Days of Treatment +/− Anti-CD81 Ab.

a<p>The percentage of infected cells was estimated after anti-NS5A Ab staining (see Materials and Methods).</p

Summary of Antiviral Assay Results for the HCV Inhibitors Used in this Work.

a<p>All values are reported in nM unless otherwise indicated.</p>b<p>The HCV(1b/2a) chimeric virus expresses HCV(1b) envelope proteins and HCV(2a) replicative proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065273#pone.0065273-Chan1" target="_blank">[26]</a>.</p>c<p>Not determined.</p

Characterization of HCV persistently-infected cultures.

<p>(A) Examples of the level of HCV(1b/2a)-infected cells <i>vs</i>. uninfected cells after 21 days with or without replication inhibitor/entry inhibitor combination therapy. The yellow cells are infected (<i>i.e.</i> stained with anti-NS5A Ab as described in the Materials and Methods) and the blue cells are uninfected. The pluses signify relative quantifications of the percentage of infected cells in each culture (see Materials and Methods). (B) Bar graph depicting quantification of infected <i>vs</i>. uninfected cells in the untreated HCV persistently-infected culture shown in Figure. 1A (see Materials and Methods). (C) Viability of HCV persistently-infected cells after 25 days of incubation (see Materials and Methods). (D) Extracellular HCV levels (log₁₀ RNA copies/ml) during an 18-day time course initiated 7 days post infection (average of 3 assays) (HCV(2a) levels (solid circles), HCV(1b/2a) levels (solid squares, dashed line)).</p

Reduction of HCV(1b/2a) levels by anti-CD81 Ab alone and in combination with BILN-2061 or BMS-790052.

<p>HCV(1b/2a) persistently-infected cell cultures were treated with 5×EC₅₀ concentrations of the indicated HCV inhibitors for 21 days (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065273#pone-0065273-t001" target="_blank">Table 1</a> and Materials and Methods). HCV levels were normalized relative to the level of the DMSO control at each time point. This data is the average of 3 assays. Error bars represent standard deviation. Asterisks indicate statistically significant differences at day 21 from the DMSO day 21 time point (<i>t</i> test P≤0.05). (A) DMSO (solid circles and solid line), anti-CD81 Ab (pierced circles and dashed line), BILN-2061 (solid squares and dashed line), BMS-790052 (pierced squares and solid line), BILN-2061/anti-CD81 Ab (solid diamonds and solid line), BMS-790052/anti-CD81 Ab (solid hexagons and solid line), and BILN 2061/BMS-790052 (pierced diamonds and dashed line).</p

Reduction of HCV(1b/2a) levels by EI-1 alone and in combination with BILN-2061 or BMS-790052.

<p>HCV(1b/2a) persistently-infected cell cultures were treated with 5×EC₅₀ concentrations of the indicated HCV inhibitors for 20 days (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065273#pone-0065273-t001" target="_blank">Table 1</a> and Materials and Methods). HCV levels were normalized relative to the level of the DMSO control at each time point. This data is the average of 3 assays. Error bars represent standard deviation. Asterisks indicate statistically significant differences at day 20 from the DMSO day 20 time point (<i>t</i> test P≤0.05). DMSO (solid circles and solid line), EI-1 (pierced diamonds and dashed line), BILN-2061 (solid squares and dashed line), BMS-790052 (pierced squares and solid line), BILN-2061/EI-1 (diamonds and solid line),BMS-790052/EI-1 (solid hexagons and solid line), and BILN 2061/BMS-790052 (pierced diamonds and dotted line).</p

Summary of HCV(2a) Levels After 21 Days of Treatment +/− Anti-CD81 Ab.

a<p>The percentage of infected cells was estimated after anti-NS5A Ab staining (see Materials and Methods).</p