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Colony-forming assays reveal enhanced suppression of hepatitis C virus replication using combinations of direct-acting antivirals
Journal of Virological Methods 174 (2011) 153–157
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Colony-forming assays reveal enhanced suppression of hepatitis C virus replication using combinations of direct-acting antivirals Emily J.S. Graham, Rachael Hunt, Stephen M. Shaw, Chris Pickford, Jennifer Hammond, Mike Westby, Paul Targett-Adams ∗ Pfizer Global Research & Development, Sandwich Laboratories, Sandwich, Kent CT13 9NJ, UK
a b s t r a c t Article history: Received 17 February 2011 Received in revised form 25 March 2011 Accepted 28 March 2011 Available online 5 April 2011 Keywords: HCV NS5A Filibuvir Colony-forming assay Direct-acting antiviral Combination
The current standard of care for patients infected with hepatitis C virus (HCV) is not effective universally and is associated with severe side effects. Direct-acting antiviral molecules have potential to transform treatment of HCV-infected individuals but emergence of drug-resistant virus will be problematic. It is anticipated that, to limit the emergence of drug-resistant virus, future HCV therapies must consist of multiple direct-acting antivirals. In the present study, cell culture-based colony-forming assays were used to demonstrate enhanced suppression of HCV RNA replication following simultaneous treatment of HCV replicon-containing cells with two direct-acting antivirals. Specifically, combinations of NS5Ai and Filibuvir (small molecule inhibitors of HCV-encoded NS5A and NS5B proteins respectively) were able to suppress colony formation fully at concentrations that individually they could not. HCV replicon RNA isolated from colonies that emerged following treatment with suboptimal concentrations of NS5Ai were found to encode resistance substitutions in the NS5A gene, which rendered them insensitive to subsequent high doses of NS5Ai. Furthermore, both NS5Ai and Filibuvir were effective at suppressing colony formation in combination with BILN 2061, an inhibitor of HCV-encoded NS3. Collectively, these data underscore the increased inhibitory capacity of direct-acting antivirals to suppress HCV RNA replication when present in combination. © 2011 Elsevier B.V. All rights reserved.
Hepatitis C virus (HCV) is a global health concern; 2.2–3% of the world’s population, (130–170 million individuals) are infected chronically with the virus (Lavanchy, 2009; The Global Burden of Hepatitis C Working Group, 2004). Many of these individuals will develop severe HCV-associated liver disease, such as cirrhosis and hepatocellular carcinoma, the burden of which is set to rise over the next 20 years (Alberti et al., 1999; Deuffic-Burban et al., 2007). No HCV vaccine is available, or likely to be in the near future. The current standard of care involves lengthy treatment with ribavirin and injected pegylated interferon, but variable efficacy and severe side effects limit effectiveness. Many direct-acting antiviral molecules are in clinical evaluation and the most advanced (Telaprevir and Boceprevir) are set to treat patients infected with HCV from 2011 (Hezode et al., 2009; Kwo et al., 2010). However, drug-resistant HCV variants are particularly problematic for directacting antiviral therapies. To limit emergence of drug-resistant virus, effective future HCV treatments must consist of multiple direct-acting antivirals to increase the relative genetic barrier of resistance, especially if interferon-sparring regimens are to be realized (Rong et al., 2010).
∗ Corresponding author. Tel.: +44 01304 648416; fax: +44 01304 651819. E-mail address: paul.targett-adams@pfizer.com (P. Targett-Adams). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.03.031
HCV subgenomic replicon systems allow the activity of directacting antivirals to be assessed conveniently in tissue culture. Although replicon systems do not establish a productive virus infection in tissue culture, they are widely accepted as the most predictive translational tool for the development of direct-acting antiviral drugs that target HCV RNA replication. HCV replicons are commonly dicistronic subgenomic RNA molecules that consist of the HCV internal ribosome entry site, which directs expression of the G418-selectable marker neor in the first cistron, and the encephalomyocarditis virus internal ribosome entry site to control production of HCV-encoded proteins required for genome replication (NS3, NS4A, NS4B, NS5A, and NS5B) in the second cistron. Following G418 selection of Huh-7 human hepatoma cells containing in vitro-transcribed subgenomic replicon RNA, clonal lines capable of supporting constitutive virus replication are isolated (Lohmann et al., 1999). In the presence of inhibitors of HCV RNA synthesis, production of replicon RNA is diminished and cells die from loss of resistance to G418. Following prolonged exposure of replicon cells to HCV direct-acting antivirals, cells refractory to inhibition, due to the selection of resistant replicon variants, form colonies upon the surface of a tissue culture plate. Hence, the number of colonies formed following long-term treatment of replicon-containing cells with specific anti-HCV direct-acting antivirals provides a quantitate measure for the capacity of the
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molecules to both inhibit HCV genome synthesis, and suppress the emergence of resistant variants (Einav et al., 2010). The present study aimed to determine whether colony-forming assays could be used as a pre-clinical tool to reveal the effectiveness of regimens containing combinations of direct-acting antivirals. Accordingly, 100 mm culture plates were seeded with 8 × 104 Huh-7 or genotype 1b (con1 strain) replicon-containing cells (hereafter referred to as ‘Replicon 1b cells’) in Dulbecco’s-modified Eagle medium supplemented with 10% heat-inactivated foetal bovine serum, 1× non-essential amino acids, 2 mM l-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, 100 g/ml streptomycin, and 500 g/ml G418 (G418 was omitted for Huh-7 cells). The following day, the cells were washed with phosphate-buffered saline and medium containing HCV inhibitors (prepared in 100% DMSO and added to medium to achieve the desired molarity with a final DMSO concentration of 0.5% (v/v)) was added. Inhibitors were added at multiples of their IC50 , which was determined as the concentration of compound at which 50% of HCV RNA replication was inhibited in short-term (48 h) replication assays. Control cells received DMSO only. Medium was changed twice weekly (new medium contained freshly prepared inhibitors) and when cell monolayers approached 80–90% confluence, cells were split 1:6 (a maximum of twice per assay). After 3 weeks of compound exposure, remaining cell colonies were fixed and stained (0.6 g/L Coomassie brilliant blue in 50% (v/v) methanol, 10% (v/v) acetic acid). Images of culture plates were captured digitally and the level of cell coverage was determined using densitometry. The small molecules used in the present study were Filibuvir (FBV), NS5Ai, and BILN 2061. FBV is a non-nucleoside inhibitor of the HCV-encoded RNA dependent RNA polymerase, NS5B (Beaulieu, 2010), NS5Ai is a small molecule targeting the HCV NS5A protein (example molecule selected from compound series described in patent WO 2011/004276), and BILN 2061 is an inhibitor of the HCV-encoded NS3 protease (Lamarre et al., 2003). Firstly, to demonstrate NS5Ai and FBV did not cause cell death through cellular toxicity, parental Huh-7 cells were treated with the compounds at 20× their IC50 values, which are 6 pM and 20 nM for NS5Ai and FBV respectively (data not shown). Results revealed no Huh-7 cell death was apparent at these concentrations of NS5Ai and FBV (Fig. 1A and B respectively). Therefore, cell death observed in Replicon 1b cells in the presence of G418 and the HCV-targeting compounds (up to 20× IC50 concentrations) would result from antiviral activity of the inhibitors and not through any potential cellular toxicity. Next, Replicon 1b cells were treated with NS5Ai (Fig. 1A) or FBV (Fig. 1B) at 0 (DMSO only), 1×, or 4× IC50 concentrations to determine the capacity of the compounds to suppress colony formation as single agents at low concentrations. At 1× IC50 , no reduction in Replicon 1b cell numbers was observed for NS5Ai and FBV (Fig. 1A and B). It should be noted that for some experiments small reductions were observed at 1× IC50 (e.g. Fig. 3A and B), but generally, 1× IC50 had little effect upon Replicon 1b cells in the colony-forming assay format. Following treatment of Replicon 1b cells with 4× IC50 NS5Ai and FBV, cell density decreased to 17% and 83% of DMSO-treated controls respectively (Fig. 1A and B). To ensure no large variations in cell densities were evident over a number of repeated experiments (n = 6), average reductions in cell densities following administration of 4× IC50 NS5Ai or FBV to Replicon 1b cells were calculated to be 30 ± 13% and 86 ± 5% of control values respectively (data not shown). To determine whether a combination of NS5Ai and FBV could further reduce colony formation, Replicon 1b cells were treated with NS5Ai and FBV simultaneously (Fig. 1C). At 4× IC50 NS5Ai and FBV, cell density was reduced further and was 7% of untreated controls (Fig. 1C). Taken together, these data highlighted the application of colony-forming assays to assess the ability of direct-acting antivirals to suppress HCV RNA replication. Additionally, the data revealed that combination of 4× IC50
each of NS5Ai and FBV can suppress colony formation to a greater extent than treatment with the inhibitors in isolation. To investigate the enhanced suppression of colony formation evident from treatment of Replicon 1b cells with NS5Ai and FBV in combination further, and to ascertain whether total abrogation of colony formation was possible at low inhibitor concentrations, Replicon 1b cells were treated with different concentrations of NS5Ai (1× to 20× IC50 ) alone, or in combination with 8× IC50 FBV (Fig. 2A). At all concentrations of NS5Ai, enhanced suppression of colony formation was evident when FBV was present in combination (Fig. 2A). Notably, cell density was reduced to just 2% of DMSO-treated controls following treatment with a combined regimen of 16× and 8× IC50 NS5Ai and FBV respectively (Fig. 2A). Next, a similar experiment was conducted by titrating the concentration of FBV alone (1× to 20× IC50 ), or in combination with 4× IC50 NS5Ai (Fig. 2B). A lower concentration of NS5Ai was selected for these experiments because NS5Ai was more effective at colony suppression than FBV at low IC50 multiples (Fig. 1A and B). However, similar cell densities were evident following treatment with NS5Ai or FBV at higher (20× IC50 ) concentrations; 17% and 15% respectively (Fig. 2A and B). Furthermore, results revealed that 16× and 4× IC50 of FBV and NS5Ai respectively was sufficient to abrogate colony formation in the assay; the addition of 4× IC50 NS5Ai, in combination with 16× IC50 FBV resulted in a decrease in cell density from 41% to 0% (Fig. 2B). This finding illustrated the ability of direct-acting antiviral combinations to suppress HCV RNA replication completely at concentrations that individually allowed colony growth. To determine what impact resistance substitutions in the HCV genome had upon colony formation, Replicon 1b cells were either treated with DMSO or 8× IC50 NS5Ai for 3 weeks (Fig. 2C; images i and iii respectively). Consistent with previous results, DMSO had no inhibitory effect upon colony formation whereas 8× IC50 NS5Ai reduced cell density to 18% (Fig. 2C; images i and iii). RNA from cells on both these plates was extracted and cDNA was prepared with a replicon-specific primer (5 -GGATGGCCTATTGGCCTGGA3 ). The NS5A gene was amplified by PCR using gene-specific primers (forward, 5 -CCCCACGCACTATGTGCCTG-3 ; reverse, 5 TGTGGTGACGGAGCAAAGAG-3 ) and the nucleotide sequence of the resulting amplicons was determined (data not shown). A mutation was identified in the NS5A gene from Replicon 1b cells treated with 8× IC50 NS5Ai, which was not present in RNA derived from DMSO-treated cells. The nucleotide change conferred substitution from tyrosine to histidine at position 93 (Y93H) in the NS5A primary amino acid sequence. Y93H has been identified previously as a resistance substitution that reduces the capacity of NS5A-targeting molecules to inhibit NS5A-encoded functions (Fridell et al., 2010; Lemm et al., 2010). Hence, exposure of Replicon 1b cells to suboptimal inhibitor concentrations resulted in the emergence of resistant variants. To demonstrate the significance of resistance, the DMSOand 8× IC50 NS5Ai-treated Replicon 1b cells were subsequently treated with 2000× IC50 NS5Ai for a further 3 weeks (Fig. 2C; images ii and iv respectively). This level of compound reduced cell density of the DMSO-treated population to 1% (Fig. 2C, image ii). However, in Replicon 1b cells harbouring HCV RNA encoding a Y93H substitution in NS5A, no reduction in cell density was observed in response to treatment with 2000× IC50 NS5Ai after 3 weeks; cell monolayers grew to confluence (Fig. 2C; image iv). Finally, the ability of NS5Ai and FBV to suppress colony formation in the presence of an inhibitor of the HCV-encoded NS3 protease (BILN 2061; 1× IC50 of 1 nM) was assessed (Fig. 3). Following treatment of Replicon 1b cells with 4× IC50 BILN 2061, cell density was reduced to 19% of DMSO-treated controls, similar to the levels of inhibition evident with 4× IC50 NS5Ai (Fig. 3A). Consistent with previous findings (Figs. 1B and 2B), treatment with 4× IC50 FBV revealed only modest inhibition; cell densities were 79% of DMSO-treated controls (Fig. 3B). The differences in the levels of
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Fig. 1. NS5Ai and FBV inhibit colony formation. Huh-7 or Replicon 1b cells were treated with NS5Ai (A) or FBV (B) alone (A and B) or in combination (C) at the indicated concentrations. After 3 weeks, remaining cells were stained and digitally captured images were analyzed using densitometry. Representative images are shown from n = 3 analysis.
colony suppression observed following treatment of Replicon 1b cells with 4× IC50 NS5Ai or FBV may be related to their distinct mode of actions and/or differences in their respective genetic barrier to resistance. However, when 4× IC50 BILN 2061 was added in combination with equivalent IC50 multiples of NS5Ai and FBV, cell densities were reduced further to 4% and 8% of controls respectively (Fig. 3A and B). These data confirmed the enhanced suppression of colony formation observed following treatment of Replicon 1b cells with NS5Ai and FBV in combination was not limited to these specific molecules only. Furthermore, colony-forming assays revealed that NS5A- and NS5B-targeting molecules operated in concert with NS3 inhibitors to reduce colony formation. The current study employed direct-acting antivirals NS5Ai and FBV to demonstrate the rationale for combination therapy
using colony-forming assays; a convenient, straight-forward, and visually-appealing assay format that, when analyzed using simple densitometry software, provided a quantitative measure of the capacity for direct-acting antivirals to inhibit HCV RNA replication and suppress emergence of drug-resistant variants. The present study also established an assay platform that can be used to evaluate the capacity of other direct-acting antiviral combinations, including triple therapies, to suppress HCV RNA replication. Colony-forming assays differ from short-term transient HCV replication assays, which use luciferase reporter gene activity to monitor levels of HCV genome synthesis (Lohmann et al., 2003; Targett-Adams and McLauchlan, 2005), because short-term transient replication assays do not assess the capacity of inhibitory molecules to suppress the emergence of resistant replicon variants.
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Fig. 2. Enhanced reduction of colony formation using combinations of NS5Ai and FBV. (A and B) Replicon 1b cells were treated with NS5Ai (A) or FBV (B) at the indicated concentrations alone (upper panels) or in combination with 8× IC50 FBV (A) or 4× IC50 NS5Ai (B) (lower panels). Representative images are shown from n = 3 analysis. (C) Replicon 1b cells were treated with either DMSO (i) or 8× IC50 NS5Ai (iii) for 3 weeks. Cells from i and iii were subsequently treated for an additional 3 weeks with 2000× IC50 NS5Ai (ii and iv). Y93H in iii indicates amino acid substitution in the NS5A protein evident upon nucleotide sequencing of replicon RNA obtained from cells treated with 8× IC50 NS5Ai (iii). Cells were stained and analyzed as described for Fig. 1.
Although the use of colony-forming assays to assess inhibition of HCV RNA replication is not novel; in vitro data on HCV combination therapies is lacking and the emerging era of direct-acting antivirals makes application of this assay pertinent for investigating inhibitor combinations. Accordingly, NS5Ai and FBV reduced colony formation to a greater extent in combination than when present alone at equivalent concentrations. Similar findings were evident when BILN 2061 was used in combination with either NS5Ai or FBV. Molecules targeting NS3 are expected to be the first direct-acting antivirals to gain regulatory approval as add-on therapy to current standard of care and data described herein indicated that both NS5A- and NS5B-targeting molecules could complement the use of protease inhibitors to enhance suppression of HCV RNA repli-
cation and the emergence of drug-resistant variants. The present study also highlighted the problem of drug-resistance; when suboptimal levels of NS5Ai were administered to Replicon 1b cells, resistance substitutions in the NS5A gene emerged that rendered the remaining population of replicon-containing cells impervious to subsequent high doses of the compound. Clinical findings have revealed that generation of resistant HCV variants occur rapidly following exposure of HCV-infected individuals to direct-acting antivirals (Rong et al., 2010). This is likely driven by the high replicative ability of the virus in vivo; up to a trillion HCV particles can be produced per day in chronic infection (Neumann et al., 1998), combined with the error-prone nature of the virus-encoded RNAdependent RNA polymerase, which is ∼10−4 for a single mutation
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direct-acting antiviral. Since therapeutic regimens composed of a combination of direct-acting antivirals from different mechanistic classes will be required to combat drug resistance, colony-forming assays represent a useful tool to assess the effectiveness of specific inhibitor combinations prior to clinical evaluation. References
Fig. 3. Suppression of colony formation using NS5Ai or FBV in combination with a HCV protease inhibitor. Replicon 1b cells were treated 0, 1×, and 4× IC50 NS5Ai (A) or FBV (B) in combination with 0, 1×, and 4× IC50 BILN 2061 (A and B). Cells were stained and analyzed as described for Fig. 1. Representative images are shown from n = 2 analysis.
(Ogata et al., 1991). As a result, HCV exhibits a high level of genetic diversity in chronic infections and it is likely that every possible point mutation in the virus genome is generated at least once a day. This is compounded by the fact that variation at only one or two nucleotide residues in the HCV genome is often sufficient to prevent recognition of the corresponding viral protein by a specific
Alberti, A., Chemello, L., Benvegnu, L., 1999. Natural history of hepatitis C. Journal of Hepatology 31 (Suppl. 1), 17–24. Beaulieu, P.L., 2010. Filibuvir, a non-nucleoside NS5B polymerase inhibitor for the potential oral treatment of chronic HCV infection. IDrugs 13, 938–948. Deuffic-Burban, S., Poynard, T., Sulkowski, M.S., Wong, J.B., 2007. Estimating the future health burden of chronic hepatitis C and human immunodeficiency virus infections in the United States. Journal of Viral Hepatitis 14, 107–115. Einav, S., Sobol, H.D., Gehrig, E., Glenn, J.S., 2010. The hepatitis C virus (HCV) NS4B RNA binding inhibitor clemizole is highly synergistic with HCV protease inhibitors. The Journal of Infectious Diseases 202, 65–74. Fridell, R.A., Qiu, D., Wang, C., Valera, L., Gao, M., 2010. Resistance analysis of the hepatitis C virus NS5A inhibitor BMS-790052 in an in vitro replicon system. Antimicrobial Agents and Chemotherapy 54, 3641–3650. Hezode, C., Forestier, N., Dusheiko, G., Ferenci, P., Pol, S., Goeser, T., Bronowicki, J.P., Bourliere, M., Gharakhanian, S., Bengtsson, L., McNair, L., George, S., Kieffer, T., Kwong, A., Kauffman, R.S., Alam, J., Pawlotsky, J.M., Zeuzem, S., 2009. Telaprevir and peginterferon with or without ribavirin for chronic HCV infection. The New England Journal of Medicine 360, 1839–1850. Kwo, P.Y., Lawitz, E.J., McCone, J., Schiff, E.R., Vierling, J.M., Pound, D., Davis, M.N., Galati, J.S., Gordon, S.C., Ravendhran, N., Rossaro, L., Anderson, F.H., Jacobson, I.M., Rubin, R., Koury, K., Pedicone, L.D., Brass, C.A., Chaudhri, E., Albrecht, J.K., 2010. Efficacy of boceprevir, an NS3 protease inhibitor, in combination with peginterferon alfa-2b and ribavirin in treatment-naive patients with genotype 1 hepatitis C infection (SPRINT-1): an open-label, randomised, multicentre phase 2 trial. Lancet 376, 705–716. Lamarre, D., Anderson, P.C., Bailey, M., Beaulieu, P., Bolger, G., Bonneau, P., Bos, M., Cameron, D.R., Cartier, M., Cordingley, M.G., Faucher, A.M., Goudreau, N., Kawai, S.H., Kukolj, G., Lagace, L., LaPlante, S.R., Narjes, H., Poupart, M.A., Rancourt, J., Sentjens, R.E., St George, R., Simoneau, B., Steinmann, G., Thibeault, D., Tsantrizos, Y.S., Weldon, S.M., Yong, C.L., Llinas-Brunet, M., 2003. An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature 426, 186–189. Lavanchy, D., 2009. The global burden of hepatitis C. Liver International 29 (Suppl. 1), 74–81. Lemm, J.A., O’Boyle 2nd, D., Liu, M., Nower, P.T., Colonno, R., Deshpande, M.S., Snyder, L.B., Martin, S.W., St Laurent, D.R., Serrano-Wu, M.H., Romine, J.L., Meanwell, N.A., Gao, M., 2010. Identification of hepatitis C virus NS5A inhibitors. Journal of Virology 84, 482–491. Lohmann, V., Hoffmann, S., Herian, U., Penin, F., Bartenschlager, R., 2003. Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. Journal of Virology 77, 3007–3019. Lohmann, V., Korner, F., Koch, J., Herian, U., Theilmann, L., Bartenschlager, R., 1999. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113, New York, N.Y. Neumann, A.U., Lam, N.P., Dahari, H., Gretch, D.R., Wiley, T.E., Layden, T.J., Perelson, A.S., 1998. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy. Science 282, 103–107, New York, N.Y. Ogata, N., Alter, H.J., Miller, R.H., Purcell, R.H., 1991. Nucleotide sequence and mutation rate of the H strain of hepatitis C virus. Proceedings of the National Academy of Sciences of the United States of America 88, 3392–3396. Rong, L., Dahari, H., Ribeiro, R.M., Perelson, A.S., 2010. Rapid emergence of protease inhibitor resistance in hepatitis C virus. Science Translational Medicine 2, 30ra32. Targett-Adams, P., McLauchlan, J., 2005. Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon. The Journal of General Virology 86, 3075–3080. The Global Burden of Hepatitis C Working Group, 2004. Global burden of disease (GBD) for hepatitis C. Journal of Clinical Pharmacology 44, 20–29.
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Colony-forming assays reveal enhanced suppression of hepatitis C virus replication using combinations of direct-acting antivirals Emily J.S. Graham, Rachael Hunt, Stephen M. Shaw, Chris Pickford, Jennifer Hammond, Mike Westby, Paul Targett-Adams ∗ Pfizer Global Research & Development, Sandwich Laboratories, Sandwich, Kent CT13 9NJ, UK
a b s t r a c t Article history: Received 17 February 2011 Received in revised form 25 March 2011 Accepted 28 March 2011 Available online 5 April 2011 Keywords: HCV NS5A Filibuvir Colony-forming assay Direct-acting antiviral Combination
The current standard of care for patients infected with hepatitis C virus (HCV) is not effective universally and is associated with severe side effects. Direct-acting antiviral molecules have potential to transform treatment of HCV-infected individuals but emergence of drug-resistant virus will be problematic. It is anticipated that, to limit the emergence of drug-resistant virus, future HCV therapies must consist of multiple direct-acting antivirals. In the present study, cell culture-based colony-forming assays were used to demonstrate enhanced suppression of HCV RNA replication following simultaneous treatment of HCV replicon-containing cells with two direct-acting antivirals. Specifically, combinations of NS5Ai and Filibuvir (small molecule inhibitors of HCV-encoded NS5A and NS5B proteins respectively) were able to suppress colony formation fully at concentrations that individually they could not. HCV replicon RNA isolated from colonies that emerged following treatment with suboptimal concentrations of NS5Ai were found to encode resistance substitutions in the NS5A gene, which rendered them insensitive to subsequent high doses of NS5Ai. Furthermore, both NS5Ai and Filibuvir were effective at suppressing colony formation in combination with BILN 2061, an inhibitor of HCV-encoded NS3. Collectively, these data underscore the increased inhibitory capacity of direct-acting antivirals to suppress HCV RNA replication when present in combination. © 2011 Elsevier B.V. All rights reserved.
Hepatitis C virus (HCV) is a global health concern; 2.2–3% of the world’s population, (130–170 million individuals) are infected chronically with the virus (Lavanchy, 2009; The Global Burden of Hepatitis C Working Group, 2004). Many of these individuals will develop severe HCV-associated liver disease, such as cirrhosis and hepatocellular carcinoma, the burden of which is set to rise over the next 20 years (Alberti et al., 1999; Deuffic-Burban et al., 2007). No HCV vaccine is available, or likely to be in the near future. The current standard of care involves lengthy treatment with ribavirin and injected pegylated interferon, but variable efficacy and severe side effects limit effectiveness. Many direct-acting antiviral molecules are in clinical evaluation and the most advanced (Telaprevir and Boceprevir) are set to treat patients infected with HCV from 2011 (Hezode et al., 2009; Kwo et al., 2010). However, drug-resistant HCV variants are particularly problematic for directacting antiviral therapies. To limit emergence of drug-resistant virus, effective future HCV treatments must consist of multiple direct-acting antivirals to increase the relative genetic barrier of resistance, especially if interferon-sparring regimens are to be realized (Rong et al., 2010).
∗ Corresponding author. Tel.: +44 01304 648416; fax: +44 01304 651819. E-mail address: paul.targett-adams@pfizer.com (P. Targett-Adams). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.03.031
HCV subgenomic replicon systems allow the activity of directacting antivirals to be assessed conveniently in tissue culture. Although replicon systems do not establish a productive virus infection in tissue culture, they are widely accepted as the most predictive translational tool for the development of direct-acting antiviral drugs that target HCV RNA replication. HCV replicons are commonly dicistronic subgenomic RNA molecules that consist of the HCV internal ribosome entry site, which directs expression of the G418-selectable marker neor in the first cistron, and the encephalomyocarditis virus internal ribosome entry site to control production of HCV-encoded proteins required for genome replication (NS3, NS4A, NS4B, NS5A, and NS5B) in the second cistron. Following G418 selection of Huh-7 human hepatoma cells containing in vitro-transcribed subgenomic replicon RNA, clonal lines capable of supporting constitutive virus replication are isolated (Lohmann et al., 1999). In the presence of inhibitors of HCV RNA synthesis, production of replicon RNA is diminished and cells die from loss of resistance to G418. Following prolonged exposure of replicon cells to HCV direct-acting antivirals, cells refractory to inhibition, due to the selection of resistant replicon variants, form colonies upon the surface of a tissue culture plate. Hence, the number of colonies formed following long-term treatment of replicon-containing cells with specific anti-HCV direct-acting antivirals provides a quantitate measure for the capacity of the
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molecules to both inhibit HCV genome synthesis, and suppress the emergence of resistant variants (Einav et al., 2010). The present study aimed to determine whether colony-forming assays could be used as a pre-clinical tool to reveal the effectiveness of regimens containing combinations of direct-acting antivirals. Accordingly, 100 mm culture plates were seeded with 8 × 104 Huh-7 or genotype 1b (con1 strain) replicon-containing cells (hereafter referred to as ‘Replicon 1b cells’) in Dulbecco’s-modified Eagle medium supplemented with 10% heat-inactivated foetal bovine serum, 1× non-essential amino acids, 2 mM l-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, 100 g/ml streptomycin, and 500 g/ml G418 (G418 was omitted for Huh-7 cells). The following day, the cells were washed with phosphate-buffered saline and medium containing HCV inhibitors (prepared in 100% DMSO and added to medium to achieve the desired molarity with a final DMSO concentration of 0.5% (v/v)) was added. Inhibitors were added at multiples of their IC50 , which was determined as the concentration of compound at which 50% of HCV RNA replication was inhibited in short-term (48 h) replication assays. Control cells received DMSO only. Medium was changed twice weekly (new medium contained freshly prepared inhibitors) and when cell monolayers approached 80–90% confluence, cells were split 1:6 (a maximum of twice per assay). After 3 weeks of compound exposure, remaining cell colonies were fixed and stained (0.6 g/L Coomassie brilliant blue in 50% (v/v) methanol, 10% (v/v) acetic acid). Images of culture plates were captured digitally and the level of cell coverage was determined using densitometry. The small molecules used in the present study were Filibuvir (FBV), NS5Ai, and BILN 2061. FBV is a non-nucleoside inhibitor of the HCV-encoded RNA dependent RNA polymerase, NS5B (Beaulieu, 2010), NS5Ai is a small molecule targeting the HCV NS5A protein (example molecule selected from compound series described in patent WO 2011/004276), and BILN 2061 is an inhibitor of the HCV-encoded NS3 protease (Lamarre et al., 2003). Firstly, to demonstrate NS5Ai and FBV did not cause cell death through cellular toxicity, parental Huh-7 cells were treated with the compounds at 20× their IC50 values, which are 6 pM and 20 nM for NS5Ai and FBV respectively (data not shown). Results revealed no Huh-7 cell death was apparent at these concentrations of NS5Ai and FBV (Fig. 1A and B respectively). Therefore, cell death observed in Replicon 1b cells in the presence of G418 and the HCV-targeting compounds (up to 20× IC50 concentrations) would result from antiviral activity of the inhibitors and not through any potential cellular toxicity. Next, Replicon 1b cells were treated with NS5Ai (Fig. 1A) or FBV (Fig. 1B) at 0 (DMSO only), 1×, or 4× IC50 concentrations to determine the capacity of the compounds to suppress colony formation as single agents at low concentrations. At 1× IC50 , no reduction in Replicon 1b cell numbers was observed for NS5Ai and FBV (Fig. 1A and B). It should be noted that for some experiments small reductions were observed at 1× IC50 (e.g. Fig. 3A and B), but generally, 1× IC50 had little effect upon Replicon 1b cells in the colony-forming assay format. Following treatment of Replicon 1b cells with 4× IC50 NS5Ai and FBV, cell density decreased to 17% and 83% of DMSO-treated controls respectively (Fig. 1A and B). To ensure no large variations in cell densities were evident over a number of repeated experiments (n = 6), average reductions in cell densities following administration of 4× IC50 NS5Ai or FBV to Replicon 1b cells were calculated to be 30 ± 13% and 86 ± 5% of control values respectively (data not shown). To determine whether a combination of NS5Ai and FBV could further reduce colony formation, Replicon 1b cells were treated with NS5Ai and FBV simultaneously (Fig. 1C). At 4× IC50 NS5Ai and FBV, cell density was reduced further and was 7% of untreated controls (Fig. 1C). Taken together, these data highlighted the application of colony-forming assays to assess the ability of direct-acting antivirals to suppress HCV RNA replication. Additionally, the data revealed that combination of 4× IC50
each of NS5Ai and FBV can suppress colony formation to a greater extent than treatment with the inhibitors in isolation. To investigate the enhanced suppression of colony formation evident from treatment of Replicon 1b cells with NS5Ai and FBV in combination further, and to ascertain whether total abrogation of colony formation was possible at low inhibitor concentrations, Replicon 1b cells were treated with different concentrations of NS5Ai (1× to 20× IC50 ) alone, or in combination with 8× IC50 FBV (Fig. 2A). At all concentrations of NS5Ai, enhanced suppression of colony formation was evident when FBV was present in combination (Fig. 2A). Notably, cell density was reduced to just 2% of DMSO-treated controls following treatment with a combined regimen of 16× and 8× IC50 NS5Ai and FBV respectively (Fig. 2A). Next, a similar experiment was conducted by titrating the concentration of FBV alone (1× to 20× IC50 ), or in combination with 4× IC50 NS5Ai (Fig. 2B). A lower concentration of NS5Ai was selected for these experiments because NS5Ai was more effective at colony suppression than FBV at low IC50 multiples (Fig. 1A and B). However, similar cell densities were evident following treatment with NS5Ai or FBV at higher (20× IC50 ) concentrations; 17% and 15% respectively (Fig. 2A and B). Furthermore, results revealed that 16× and 4× IC50 of FBV and NS5Ai respectively was sufficient to abrogate colony formation in the assay; the addition of 4× IC50 NS5Ai, in combination with 16× IC50 FBV resulted in a decrease in cell density from 41% to 0% (Fig. 2B). This finding illustrated the ability of direct-acting antiviral combinations to suppress HCV RNA replication completely at concentrations that individually allowed colony growth. To determine what impact resistance substitutions in the HCV genome had upon colony formation, Replicon 1b cells were either treated with DMSO or 8× IC50 NS5Ai for 3 weeks (Fig. 2C; images i and iii respectively). Consistent with previous results, DMSO had no inhibitory effect upon colony formation whereas 8× IC50 NS5Ai reduced cell density to 18% (Fig. 2C; images i and iii). RNA from cells on both these plates was extracted and cDNA was prepared with a replicon-specific primer (5 -GGATGGCCTATTGGCCTGGA3 ). The NS5A gene was amplified by PCR using gene-specific primers (forward, 5 -CCCCACGCACTATGTGCCTG-3 ; reverse, 5 TGTGGTGACGGAGCAAAGAG-3 ) and the nucleotide sequence of the resulting amplicons was determined (data not shown). A mutation was identified in the NS5A gene from Replicon 1b cells treated with 8× IC50 NS5Ai, which was not present in RNA derived from DMSO-treated cells. The nucleotide change conferred substitution from tyrosine to histidine at position 93 (Y93H) in the NS5A primary amino acid sequence. Y93H has been identified previously as a resistance substitution that reduces the capacity of NS5A-targeting molecules to inhibit NS5A-encoded functions (Fridell et al., 2010; Lemm et al., 2010). Hence, exposure of Replicon 1b cells to suboptimal inhibitor concentrations resulted in the emergence of resistant variants. To demonstrate the significance of resistance, the DMSOand 8× IC50 NS5Ai-treated Replicon 1b cells were subsequently treated with 2000× IC50 NS5Ai for a further 3 weeks (Fig. 2C; images ii and iv respectively). This level of compound reduced cell density of the DMSO-treated population to 1% (Fig. 2C, image ii). However, in Replicon 1b cells harbouring HCV RNA encoding a Y93H substitution in NS5A, no reduction in cell density was observed in response to treatment with 2000× IC50 NS5Ai after 3 weeks; cell monolayers grew to confluence (Fig. 2C; image iv). Finally, the ability of NS5Ai and FBV to suppress colony formation in the presence of an inhibitor of the HCV-encoded NS3 protease (BILN 2061; 1× IC50 of 1 nM) was assessed (Fig. 3). Following treatment of Replicon 1b cells with 4× IC50 BILN 2061, cell density was reduced to 19% of DMSO-treated controls, similar to the levels of inhibition evident with 4× IC50 NS5Ai (Fig. 3A). Consistent with previous findings (Figs. 1B and 2B), treatment with 4× IC50 FBV revealed only modest inhibition; cell densities were 79% of DMSO-treated controls (Fig. 3B). The differences in the levels of
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Fig. 1. NS5Ai and FBV inhibit colony formation. Huh-7 or Replicon 1b cells were treated with NS5Ai (A) or FBV (B) alone (A and B) or in combination (C) at the indicated concentrations. After 3 weeks, remaining cells were stained and digitally captured images were analyzed using densitometry. Representative images are shown from n = 3 analysis.
colony suppression observed following treatment of Replicon 1b cells with 4× IC50 NS5Ai or FBV may be related to their distinct mode of actions and/or differences in their respective genetic barrier to resistance. However, when 4× IC50 BILN 2061 was added in combination with equivalent IC50 multiples of NS5Ai and FBV, cell densities were reduced further to 4% and 8% of controls respectively (Fig. 3A and B). These data confirmed the enhanced suppression of colony formation observed following treatment of Replicon 1b cells with NS5Ai and FBV in combination was not limited to these specific molecules only. Furthermore, colony-forming assays revealed that NS5A- and NS5B-targeting molecules operated in concert with NS3 inhibitors to reduce colony formation. The current study employed direct-acting antivirals NS5Ai and FBV to demonstrate the rationale for combination therapy
using colony-forming assays; a convenient, straight-forward, and visually-appealing assay format that, when analyzed using simple densitometry software, provided a quantitative measure of the capacity for direct-acting antivirals to inhibit HCV RNA replication and suppress emergence of drug-resistant variants. The present study also established an assay platform that can be used to evaluate the capacity of other direct-acting antiviral combinations, including triple therapies, to suppress HCV RNA replication. Colony-forming assays differ from short-term transient HCV replication assays, which use luciferase reporter gene activity to monitor levels of HCV genome synthesis (Lohmann et al., 2003; Targett-Adams and McLauchlan, 2005), because short-term transient replication assays do not assess the capacity of inhibitory molecules to suppress the emergence of resistant replicon variants.
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Fig. 2. Enhanced reduction of colony formation using combinations of NS5Ai and FBV. (A and B) Replicon 1b cells were treated with NS5Ai (A) or FBV (B) at the indicated concentrations alone (upper panels) or in combination with 8× IC50 FBV (A) or 4× IC50 NS5Ai (B) (lower panels). Representative images are shown from n = 3 analysis. (C) Replicon 1b cells were treated with either DMSO (i) or 8× IC50 NS5Ai (iii) for 3 weeks. Cells from i and iii were subsequently treated for an additional 3 weeks with 2000× IC50 NS5Ai (ii and iv). Y93H in iii indicates amino acid substitution in the NS5A protein evident upon nucleotide sequencing of replicon RNA obtained from cells treated with 8× IC50 NS5Ai (iii). Cells were stained and analyzed as described for Fig. 1.
Although the use of colony-forming assays to assess inhibition of HCV RNA replication is not novel; in vitro data on HCV combination therapies is lacking and the emerging era of direct-acting antivirals makes application of this assay pertinent for investigating inhibitor combinations. Accordingly, NS5Ai and FBV reduced colony formation to a greater extent in combination than when present alone at equivalent concentrations. Similar findings were evident when BILN 2061 was used in combination with either NS5Ai or FBV. Molecules targeting NS3 are expected to be the first direct-acting antivirals to gain regulatory approval as add-on therapy to current standard of care and data described herein indicated that both NS5A- and NS5B-targeting molecules could complement the use of protease inhibitors to enhance suppression of HCV RNA repli-
cation and the emergence of drug-resistant variants. The present study also highlighted the problem of drug-resistance; when suboptimal levels of NS5Ai were administered to Replicon 1b cells, resistance substitutions in the NS5A gene emerged that rendered the remaining population of replicon-containing cells impervious to subsequent high doses of the compound. Clinical findings have revealed that generation of resistant HCV variants occur rapidly following exposure of HCV-infected individuals to direct-acting antivirals (Rong et al., 2010). This is likely driven by the high replicative ability of the virus in vivo; up to a trillion HCV particles can be produced per day in chronic infection (Neumann et al., 1998), combined with the error-prone nature of the virus-encoded RNAdependent RNA polymerase, which is ∼10−4 for a single mutation
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direct-acting antiviral. Since therapeutic regimens composed of a combination of direct-acting antivirals from different mechanistic classes will be required to combat drug resistance, colony-forming assays represent a useful tool to assess the effectiveness of specific inhibitor combinations prior to clinical evaluation. References
Fig. 3. Suppression of colony formation using NS5Ai or FBV in combination with a HCV protease inhibitor. Replicon 1b cells were treated 0, 1×, and 4× IC50 NS5Ai (A) or FBV (B) in combination with 0, 1×, and 4× IC50 BILN 2061 (A and B). Cells were stained and analyzed as described for Fig. 1. Representative images are shown from n = 2 analysis.
(Ogata et al., 1991). As a result, HCV exhibits a high level of genetic diversity in chronic infections and it is likely that every possible point mutation in the virus genome is generated at least once a day. This is compounded by the fact that variation at only one or two nucleotide residues in the HCV genome is often sufficient to prevent recognition of the corresponding viral protein by a specific
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