Cytotoxic and cytostatic effects of TMC647078
The cytotoxic effect of TMC647078 on HEK-293T, HepG2, Huh7.5 and MRC5 cells was analyzed after 3 to 4 days of compound exposure by incubation of cells with resazurin (Sigma-Aldrich, St. Louis, MO) followed by resorfurin fluorescence detection in a fluorescence reader. Inhibition curves were generated and CC50 values calculated (50% cytotoxic concentration).
To test the cytostatic effect of TMC647078, Huh7 cells (1.5x103cells/well) were incubated for 3days in a 96-well plate containing five serial dilutions of the compound in triplicate. The effects of the compound on cell proliferation were quantified by the level of 5-bromo-2’-deoxyuridine (BrdU, thymidine analogue) incorporation during DNA synthesis using the Biotrak cell proliferation enzyme-linked immunosorbent assay (ELISA) system according to the manufacturer’s protocol (Biotrak cell proliferation ELISA system version 2, GE Healthcare). The levels of incorporated BrdU correlated directly with the amount of DNA synthesis and thereby the number of proliferating cells in culture. The compound concentration that reduced cell proliferation by 50% in comparison with non-treated cell controls was determined.
The effect of TMC647078 on cell cycle progression was examined in MT4 cells incubated with test compound (24-well plate, 8x104cells/well). After 3 days, nuclei were stained using propidium iodide, a fluorogenic substance which binds stoichiometrically to nucleic acids, followed by flow cytometry.
Determination of mitochondrial (mt) toxicity
HepG2 cells were seeded at 2.5x103 cells/well in a collagen-coated 96-well plate containing four serial dilutions of the compound with the highest test concentration of 200 µM. Cells were incubated for 14 days with an intermediate renewal of medium and compound on day 7. On day14, total DNA and total RNA were extracted using the ABI6100 nucleic acid extraction robot according to the manufacturer’s protocol (Applied Biosystems).
For the mitochondrial toxcity assay, the mitochondrial cytochrome c oxidase subunit II (COXII) gene and the nuclear β-actin gene were selected as indicators of mitochondrial toxicity and cellular toxicity respectively. The COXII and βactin primer and probe sequences described by Stuyver and colleagues were used (1), with the minor adaptation that 6carboxy-fluorescein (6-FAM) was used as a 5’-fluorescent dye for the COXII probe and JOE was used as a 5’-fluorescent dye for the β-actin probe. In addition, for both probes, 3’-black hole quenchers replaced TAMRA (BHQ1, Eurogentec, Seraing, Belgium). A duplex realtime PCR was performed using 5 µl of extracted DNA in a 25µl mix containing 1X Roche Lightcycler 480 mastermix (Roche Applied Science, Germany), 200 nM primers and 100 nM of each probe. Primers and probe for relative mitochondrial RNA quantification were designed in the ND6 mRNA, using the Primer Express Software (Applied Biosystems): ND6-181F, 5’-CACAACCACCACCCCATCA-3’; ND6-269R, 5’-TTGAGGTCTTGGTGAGTGTTTTAGTG-3’; ND6-207P, 5'-FAM-TCACCCACAGCACCAATCCTACCTCC-BHQ1-3'. As a reference for mRNA transcribed from a nuclear gene with a stable expression in HepG2 cells, the large ribosomal protein PO (RPLPO) mRNA was selected, and the primers and probe were purchased from Applied Biosystems (Human RPLPO Endogenous Control (FAM / MGB Probe, Non-Primer Limited), Applied Biosystems). Prior to the duplex real-time PCR assay, the extracted RNA was transcribed into cDNA using the High Capacity cDNA RT kit using RT random primers (Applied Biosystems). The PCR program was run on a Roche Lightcycler 480 (Roche Applied Science, Germany): 2min at 50°C, 10 min at 95°C, and 50cycles of 95°C for 50 seconds and 60°C for 1 min. Delta Ct values (Ct mt COXII DNA – Ct nuclear β-actin DNA or Ct mt ND6 mRNA – Ct nuclear RPLPO mRNA) were calculated for compoundtreated wells and non-compoundtreated control wells, and the comparative CT method was used for the generation of dose-response curves and the calculation of CC50 values.
Colony formation assay with Huh7-luc replicon cells
Briefly, Huh7-Luc replicon cells were seeded at a concentration of 2x104 per 10cm dish and treated with different concentrations of a single compound or a combination of two compounds in the presence of 1000 µg/ml G418. Medium was refreshed twice weekly and fresh compound was added. After 2 to 3 weeks, when significant cell death occurred, the remaining colonies were stained with neutral red and counted.
Clearance of HCV RNA from replicon-containing cells
For this experiment Huh7-Luc replicon-containing cells were seeded at a concentration of 3x105/dish in 10cm dishes and treated with different concentrations of single compound or a combination of two compounds for 2weeks in the absence of G418 (clearance phase). Cells were subcultured twice per week upon reaching confluency of 70-80% and cells were collected for RNA extraction followed by cDNA synthesis and real-time PCR to determine HCV RNA levels. After 2 weeks, cells were incubated in the absence of a compound for an additional period of 3 weeks in the presence of 250 µg/ml G418 to enable viral rebound to occur. Where HCV RNA was completely cleared upon compound treatment during the clearance phase, G418 resulted in cell death, and in conditions where cells harboured the replicon, colonies grew in the presence of G418. Culture medium was refreshed every 3-4 days and cells were sub-cultured when 70-80% confluency was reached. Cell pellets of each passage during the rebound phase were collected, followed by RNA extraction, cDNA generation and real-time PCR to monitor HCV RNA levels.
In vitro selection of resistant replicons
Huh7-Luc replicon cells or Huh7-SG1a H77 cells were seeded at a concentration of 3x105cells per 10cm dish and treated with fixed doses of 2.5x or 5x the EC50 or a serial increase from 2.5x to 10x the EC50 for up to 12 weeks in the presence of 1000 µg/ml G418. Medium was refreshed twice weekly and fresh compound was added. Cells were sub-cultured when necessary and significant cell death typically occurred after 2to 3 weeks. Surviving cell colonies were individually picked or pooled and expanded. As soon as the quantity of cells obtained was sufficient, RNA was extracted, amplified and the NS5B region sequenced by standard sanger sequencing at a population level.
Recombinant enzyme expression and purification
The HCV NS5B (con1) sequence was inserted into the expression vector pET-21 (Novagen). A 6xHis-tag was added to the C-terminus to facilitate purification. The plasmid encoding the C-terminal truncated enzyme (Δ21) was then transformed into Escherichia coli BL21(DE3) cells. Induction of protein expression was undertaken with the addition of 0.25 mM isopropyl-β-D-thiogalactopyranoside at 25°C and purified using a Ni-chelating Sepharose column.
Kinase enzyme preparation and purification
Recombinant human deoxycytidine kinase (dCK), deoxythymidine kinase 1 (TK1), mitochondrial thymidine kinase 2 (TK2), deoxyguanosine kinase (dGK) and uridine-cytidine kinases 1 and 2 (UCK-1 and UCK-2) were expressed and purified using a bacterial vector system Escherichia coli BL21 (DE3) pLysS. This vector facilitates expression of target proteins with anpolyhistidine tag enabling efficient purification in one step using a Ni2+-nitrilotriacetic acid-agarose resin. The vector also encodes a thrombin cleavage site enabling removal of the 6xHis tag. Enzyme purity was determined using SDS-PAGE.
1. Stuyver, L. J., S. Lostia, M. Adams, J. S. Mathew, B. S. Pai, J. Grier, P. M. Tharnish, Y. Choi, Y. Chong, H. Choo, C. K. Chu, M. J. Otto, and R. F. Schinazi. 2002. Antiviral activities and cellular toxicities of modified 2',3'-dideoxy-2',3'-didehydrocytidine analogues. Antimicrob Agents Chemother 46:3854-60.