HCV Inhibitors And Methods Of Using Them
a technology of hcv and inhibitors, which is applied in the direction of heterocyclic compound active ingredients, biocides, drug compositions, etc., can solve the problems of long-term improvement of hepatitis c, and achieve the effect of increasing the sustained response ra
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example 1
Assay Example 1
HCV Replicon Assay
[0123] Actively dividing 5-2Luc replicon cells were seeded at the density of 5000-7500 cells / well in the volume of 90 μl / well into 96 well plate(s). The cells were then incubated at 37° C. and 5% CO2 for 24 hours The 5-2 cells are replicon cells licensed from Ralf Bartenschlager (Germany) and have a self-replicating RNA molecule in the Huh7 cell; the RNA contains HCV non-structural proteins that make the self-replication possible.
[0124] Various concentrations of compounds (in the volume of 10 μl) were added into each well 24 hours after seeding the cells. The cells were incubated for another 24 hours before luciferase assay.
[0125] After incubating the 5-2Luc replicon cells with the compounds for 24 hours, media were aspirated from each well and Bright-Glo (Pharmacia) luciferase assay reagents were added to each well according to the manufacturer's manual. Briefly, the BrightGlo reagent was diluted with equal volume of PBS and an aliquote (100 μl) ...
example 2
Assay Example 2
Luciferase Counter Assay
[0126] Actively dividing CMV-Luc cells (Luc cells in which DNA construct (CMV promoter followed by Luciferase gene) is permanently integrated into the chromosome of Huh7 cells) were seeded at the density of 5000-7500 cells / well in the volume of 90 μl / well into 96 well plate(s). The cells were then incubated at 37° C. and 5% CO2 for 24 hours.
[0127] Various concentrations of compounds (in the volume of 10 μl) were added into each well 24 hours after seeding the cells. The cells were incubated with the compounds for another 24 hours before luciferase assay.
[0128] After incubating the CMV-Luc cells with the compounds for 24 hours, media were aspirated from each well and Bright-Glo (Pharmacia) luciferase assay reagents were added to each well according to the manufacturer's manual. Luciferase counts were taken using a luminometer.
[0129] The activity of a number of compounds according to the invention measured by the luciferase assay is displayed...
example 3
Assay Example 3
Immunoblotting Assay
[0130] Actively dividing 9-13 replicon cells (Huh7 cells comprising an HCV replicon) were seeded at the density of 1×105 cells / well in the volume of 2 ml / well into 6 well plate(s), The cells were then incubated at 37° C. and 5% CO2 for 24 hours.
[0131] Various concentrations of compounds (in the volume of 10 μl) were added into each well 24 hours after seeding the cells. The cells were incubated with the compounds for another 48 hours.
[0132] Protein samples were prepared from the cultured cells and resolved on a SDS-PAGE gel.
[0133] After electrophoresis, the protein samples on the SDS-PAGE gel were transferred to a nitrocellulose membrane.
[0134] The membrane was blocked with 5% non-fat milk in PBS for 1 hr at room temperature.
[0135] Primary antibody incubation was performed for 1 hour at room temperature before the membrane was washed for 3 times with PBST (PBS plus 0.1% Tween 20), 15 minutes each.
[0136] Horse Radish Peroxidase conjugated sec...
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