AIM: To explore the inhibition of β-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 5×10^4 per well in 12-w...AIM: To explore the inhibition of β-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 5×10^4 per well in 12-well tissue culture plates, and treated with various concentrations of β-L-D4A for 6 days. In the end,5μl of medium was used for the estimation of HBsAg and HBeAg, the other medium was processed to obtain virions by a polyethlene 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 ^32P-labeled HBV probe and autoradiophed.Intensity of the autoradiographic bands was quantitated by densitometric scans of computer and ED50 was calculated. Then Hybond-N membrane was washed and rehybridized with a ^32P-labeled mtDNA-specific probe, and effect of β-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. ID50 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 DNAwas inhibited in a dose-dependent manner. ED50 was 0.2μM. HBsAg or HBeAg was not apparently decreased, and inhibition of mitochondrial DNA was not obvious. The experiment of cytotoxicity gained ID50 at 200 μM.CONCLUSION: β-L-D4A possesses potent inhibitory effects on the replication of HBV in vitro with little cytotoxidty and mitochondrial toxicity, TI value is 1000. It is expected to be developed as a new clinically anti-HBV drug.展开更多
AIM:To investigate the safety of β-L-D4A on DNA polymeraseα. METHODS:Ion exchange chromatography was used to separate DNA polymeraseαfrom crude extract of human Hela cells.Detailed kinetic parameters were determine...AIM:To investigate the safety of β-L-D4A on DNA polymeraseα. METHODS:Ion exchange chromatography was used to separate DNA polymeraseαfrom crude extract of human Hela cells.Detailed kinetic parameters were determined for β-L-D4A against DNA polymeraseα. RESULTS:DNA polymeraseαwas purified with 4% yield and 31000 units/rag specific activity.The Michaelis constant (Km=3.22μmol/L),50% inhibition concentration (IC50=178.49μmol/L) and inhibition constant (Ki=126μmol/L) of β-L-D4A were determined by kinetic analysis. CONCLUSION:β-L-D4A is a more safe nucleoside for hepatitis B virus (HBV) infection with a lower host toxicity.展开更多
基金National Natural Science Foundation of China,No.39970858
文摘AIM: To explore the inhibition of β-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 5×10^4 per well in 12-well tissue culture plates, and treated with various concentrations of β-L-D4A for 6 days. In the end,5μl of medium was used for the estimation of HBsAg and HBeAg, the other medium was processed to obtain virions by a polyethlene 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 ^32P-labeled HBV probe and autoradiophed.Intensity of the autoradiographic bands was quantitated by densitometric scans of computer and ED50 was calculated. Then Hybond-N membrane was washed and rehybridized with a ^32P-labeled mtDNA-specific probe, and effect of β-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. ID50 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 DNAwas inhibited in a dose-dependent manner. ED50 was 0.2μM. HBsAg or HBeAg was not apparently decreased, and inhibition of mitochondrial DNA was not obvious. The experiment of cytotoxicity gained ID50 at 200 μM.CONCLUSION: β-L-D4A possesses potent inhibitory effects on the replication of HBV in vitro with little cytotoxidty and mitochondrial toxicity, TI value is 1000. It is expected to be developed as a new clinically anti-HBV drug.
基金National Natural Science Foundation of China,No.30330680
文摘AIM:To investigate the safety of β-L-D4A on DNA polymeraseα. METHODS:Ion exchange chromatography was used to separate DNA polymeraseαfrom crude extract of human Hela cells.Detailed kinetic parameters were determined for β-L-D4A against DNA polymeraseα. RESULTS:DNA polymeraseαwas purified with 4% yield and 31000 units/rag specific activity.The Michaelis constant (Km=3.22μmol/L),50% inhibition concentration (IC50=178.49μmol/L) and inhibition constant (Ki=126μmol/L) of β-L-D4A were determined by kinetic analysis. CONCLUSION:β-L-D4A is a more safe nucleoside for hepatitis B virus (HBV) infection with a lower host toxicity.