Inhibition of hepatitis B virus by a novel L-nucleoside, beta-L-D4A and related analogues

Abstract

Aim: To explore the inhibition of beta-L-D4A on hepatitis B virus (HBV) in 2.2.15 cells derived from HepG2 cells transfected with HBV genome.

Methods: 2.2.15 cells were plated at a density of 5X104 per well in 12-well tissue culture plates, and treated with various concentrations of beta-L-D4A for 6 days. In the end, 5 microl of medium was used for the estimation of HBsAg and HBeAg, the other medium was processed to obtain virions by a polyethylene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with HindIII. Both DNAs were subjected to Southern blot, hybridized with a (32)P-labeled HBV probe and autoradiographed. Intensity of the autoradiographic bands was quantitated by densitometric scans of computer and ED(50) was calculated. Then Hybond-N membrane was washed and rehybridized with a (32)P-labeled mtDNA-specific probe, and effect of beta-L-D4A on mitochondrial DNA was studied. 2.2.15 cells were also seeded in 24-well tissue culture plates, and cytotoxicity with different concentrations was examined by MTT method. ID(50) was calculated. Structure-activity relationships between D2A and D4A were also studied as above.

Results: Autoradiographic bands were similar between supernatant and intracellular HBV DNA. Episomal HBV DNA was inhibited in a dose-dependent manner. ED(50) was 0.2 microM. HBsAg or HBeAg was not apparently decreased, and inhibition of mitochondrial DNA was not obvious. The experiment of cytotoxicity gained ID(50) at 200 microM.

Conclusion: beta-L-D4A possesses potent inhibitory effects on the replication of HBV in vitro with little cytotoxicity and mitochondrial toxicity, TI value is 1000. It is expected to be developed as a new clinically anti-HBV drug.

Figure 1

Structures of β-L-D4A and analogues.

Figure 2

Inhibition of isomers of D4A and D2A on HBV DNA replication in supernatant. Lane 1 as negative control; lanes 2, 3, 4 as D-D2A at 2 μM, 4 μM and 8 μM, respectively; lanes 5, 6, 7 as L-D2A at 2 μM, 4 μM and 8 μM, respectively; lanes 8, 9, 10 as D-D4A at 2 μM, 4 μM and 8 μM, respectively; lanes 11, 12, 13 as L-D4A at 2 μM, 4 μM and 8 μM, respectively. RC: relaxed circular HBV DNA; SS: single-stranded HBV DNA.

Figure 3

Inhibition on replication of extracellular HBV DNA by β-L-D4A. lanes 1, 2 as negative control; lanes 3, 4 as lamivudine at 1 μM; lanes 5, 6 as β-L-D4A at 10 μM; lanes 7, 8 as β-L-D4A at 2 μM; lanes 9, 10 as β-L-D4A at 0.4 μM; lanes 11, 12 as β-L-D4A at 0.08 μM; lanes 13, 14 as blank control (HepaG₂). RC: relaxed circular HBV DNA; SS: single-stranded HBV DNA.

Figure 4

Inhibition on replication of extracellular HBV DNA by β-L-D4A.

Figure 5

Inhibition on replication of intracellular HBV DNA by β-L-D4A. lane 1 as negative control; lane 2 as lamivudine at 1 μM; lane 3 as β-L-D4A at 10 μM; lane 4 as β-L-D4A at 2 μM; lane 5 as β-L-D4A at 0.4 μM; lane 6 as β-L-D4A at 0.08; lane 7 as blank control (HepaG2). RC: relaxed circular HBV DNA; SS: single-stranded HBV DNA.

Figure 6

Toxicity of mitochondrial DNA. lane 1 as blank control; lane 2 as β-L-D4A at 0.4 μM; lane 3 as β-L-D4A at 10 μM; lane 4 as ddC at 0.4 μM.

Figure 7

Effect of β-L-D4A on cell growth curve.