HCV Protease

The toxicity of BMS-790052 (BMS) and Telaprevir (TPV) was measured by seeding 96-well plates to 70% confluence and exposing the cells to up to 50 μM of compound for 72 hours. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added to each well at a final concentration of 500 μg/ml 4 h before dissolving crystals in 100 μl of DMSO and measuring at 550 nm UV wavelengths. The 50% cytotoxic concentration (CC50) was then calculation using the OD550 value and the following formula: log CC50=log concentration of HPP-[(HPP-50)/(HPP-LPP)×log d HPP: highest protective percentage closest to 50% LPP: Lowest protective percentage closest to 50% d: dilution factor

HCV replicon genotype 1b cells were passaged in the presence of G418 alone (medium 694 control) or G418 with miravirsen, SPC4729 (oligonucleotide negative control) or telaprevir for 28 days in fixed concentrations at a multiple (X) of the EC₅₀ of miravirsen or telaprevir. Colony formation was assessed by staining surviving cells with crystal violet.

Genetic barriers of JTK-853, NNI-A, PF-868554, VX-222, PSI-6130, BMS-790052, and TMC435 for Con1. VX-222 and the other DAAs were treated at 1/3xEC90 for 17 days.

Western blot analysis of HCV NS3 protease cleavage of MAVS. The catalytic efficiency of four proteases (H, H-A156T, 41 and 41-A156T) from individuals H and 41 were tested in the absence or presence of an NS3 protease inhibitor (danoprevir). Expression of the HCV NS3 proteases resulted in cleavage of the lambda cI repressor with MAVS cleavage site. Expression of the protease was induced with IPTG for 3 h. The lambda cI repressor with MAVS cleavage site was not cleaved by an NS3 protease that included a substitution in catalytic residue S139A. Similarly, different catalytic efficiencies were observed with different proteases.

Representative confocal microscope image showing phosphorylated Drp1 translocation to mitochondria and mitochondrial fission in the presence of Sofosbuvir. At 24 hours after treatment with Sofosbuvir (100 nM), Huh7 cells were immunostained with antibodies against TOM20 (red) and p-Drp1 (S616) (green). Nuclei are demarcated with white dotted circles. Treated (+) and untreated (–) cells are marked. In the zoomed images, the arrows indicate the colocalization of TOM20 and p-Drp1 (S616) in Sofosbuvir-treated cells (yellow spots).

(C) Impact of HCV clearance by DAA treatment on the Nrf2 nuclear translocation. HCV infected Huh-7.5 cells day 12 were given two rounds of antiviral treatment with IFN-α, sofosbuvir, ledipasvir or combination of sofosbuvir plus ledipasvir. After 72 hours, cells were split, and then treated again with the same antiviral agent. After two rounds of antiviral treatment, the expressions of HCV core and pNrf2 were measured by immunostaining.

The toxicity of BMS-790052 (BMS) and Telaprevir (TPV) was measured by seeding 96-well plates to 70% confluence and exposing the cells to up to 50 μM of compound for 72 hours. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added to each well at a final concentration of 500 μg/ml 4 h before dissolving crystals in 100 μl of DMSO and measuring at 550 nm UV wavelengths. The 50% cytotoxic concentration (CC50) was then calculation using the OD550 value and the following formula: log CC50=log concentration of HPP-[(HPP-50)/(HPP-LPP)×log d HPP: highest protective percentage closest to 50% LPP: Lowest protective percentage closest to 50% d: dilution factor

HCV replicon genotype 1b cells were passaged in the presence of G418 alone (medium 694 control) or G418 with miravirsen, SPC4729 (oligonucleotide negative control) or telaprevir for 28 days in fixed concentrations at a multiple (X) of the EC₅₀ of miravirsen or telaprevir. Colony formation was assessed by staining surviving cells with crystal violet.

Genetic barriers of JTK-853, NNI-A, PF-868554, VX-222, PSI-6130, BMS-790052, and TMC435 for Con1. VX-222 and the other DAAs were treated at 1/3xEC90 for 17 days.

Western blot analysis of HCV NS3 protease cleavage of MAVS. The catalytic efficiency of four proteases (H, H-A156T, 41 and 41-A156T) from individuals H and 41 were tested in the absence or presence of an NS3 protease inhibitor (danoprevir). Expression of the HCV NS3 proteases resulted in cleavage of the lambda cI repressor with MAVS cleavage site. Expression of the protease was induced with IPTG for 3 h. The lambda cI repressor with MAVS cleavage site was not cleaved by an NS3 protease that included a substitution in catalytic residue S139A. Similarly, different catalytic efficiencies were observed with different proteases.

Representative confocal microscope image showing phosphorylated Drp1 translocation to mitochondria and mitochondrial fission in the presence of Sofosbuvir. At 24 hours after treatment with Sofosbuvir (100 nM), Huh7 cells were immunostained with antibodies against TOM20 (red) and p-Drp1 (S616) (green). Nuclei are demarcated with white dotted circles. Treated (+) and untreated (–) cells are marked. In the zoomed images, the arrows indicate the colocalization of TOM20 and p-Drp1 (S616) in Sofosbuvir-treated cells (yellow spots).

(C) Impact of HCV clearance by DAA treatment on the Nrf2 nuclear translocation. HCV infected Huh-7.5 cells day 12 were given two rounds of antiviral treatment with IFN-α, sofosbuvir, ledipasvir or combination of sofosbuvir plus ledipasvir. After 72 hours, cells were split, and then treated again with the same antiviral agent. After two rounds of antiviral treatment, the expressions of HCV core and pNrf2 were measured by immunostaining.