Abstract
It is estimated that more than 170 million people are infected with hepatitis C virus (HCV) worldwide1,2. Clinical trials have demonstrated that, for the first time in human history, the potential exists to eradicate a chronic viral disease using combination therapies that contain only direct-acting antiviral agents3. HCV non-structural protein 5A (NS5A) is a multifunctional protein required for several stages of the virus replication cycle4. NS5A replication complex inhibitors, exemplified by daclatasvir (DCV; also known as BMS-790052 and Daklinza), belong to the most potent class of direct-acting anti-HCV agents described so far, with in vitro activity in the picomolar (pM) to low nanomolar (nM) range. The potency observed in vitro has translated into clinical efficacy, with HCV RNA declining by ~3–4 log10 in infected patients after administration of single oral doses of DCV. Understanding the exceptional potency of DCV was a key objective of this study. Here we show that although DCV and an NS5A inhibitor analogue (Syn-395) are inactive against certain NS5A resistance variants, combinations of the pair enhance DCV potency by >1,000-fold, restoring activity to the pM range. This synergistic effect was validated in vivo using an HCV-infected chimaeric mouse model. The cooperative interaction of a pair of compounds suggests that NS5A protein molecules communicate with each other: one inhibitor binds to resistant NS5A, causing a conformational change that is transmitted to adjacent NS5As, resensitizing resistant NS5A so that the second inhibitor can act to restore inhibition. This unprecedented synergistic anti-HCV activity also enhances the resistance barrier of DCV, providing additional options for HCV combination therapy and new insight into the role of NS5A in the HCV replication cycle.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Mohd Hanafiah, K., Groeger, J., Flaxman, A. D. & Wiersma, S. T. Global epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seroprevalence. Hepatology 57, 1333–1342 (2013)
Shepard, C. W., Finelli, L. & Alter, M. J. Global epidemiology of hepatitis C virus infection. Lancet Infect. Dis. 5, 558–567 (2005)
Pawlotsky, J. M. NS5A inhibitors in the treatment of hepatitis C. J. Hepatol . 59, 375–382 (2013)
Guedj, J. et al. Modeling shows that the NS5A inhibitor daclatasvir has two modes of action and yields a shorter estimate of the hepatitis C virus half-life. Proc. Natl Acad. Sci. USA 110, 3991–3996 (2013)
Macdonald, A. & Harris, M. Hepatitis C virus NS5A: tales of a promiscuous protein. J. Gen. Virol. 85, 2485–2502 (2004)
He, Y., Staschke, K. A. & Tan, S.-L. in Hepatitis C Viruses Genomes and Molecular Biology (ed. Tan, S.-L. ) Ch. 9, 267–291 (Horizon Bioscience, 2006)
Tellinghuisen, T. L., Marcotrigiano, J. & Rice, C. M. Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase. Nature 435, 374–379 (2005)
Love, R. A., Brodsky, O., Hickey, M. J., Wells, P. A. & Cronin, C. N. Crystal structure of a novel dimeric form of NS5A domain I protein from hepatitis C virus. J. Virol. 83, 4395–4403 (2009)
Lambert, S. M. et al. The crystal structure of NS5A domain 1 from genotype 1a reveals new clues to the mechanism of action for dimeric HCV inhibitors. Protein Sci. 23, 723–734 (2014)
Gao, M. et al. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 465, 96–100 (2010)
Quinkert, D., Bartenschlager, R. & Lohmann, V. Quantitative analysis of the hepatitis C virus replication complex. J. Virol. 79, 13594–13605 (2005)
Pietschmann, T., Lohmann, V., Rutter, G., Kurpanek, K. & Bartenschlager, R. Characterization of cell lines carrying self-replicating hepatitis C virus RNAs. J. Virol. 75, 1252–1264 (2001)
O’Boyle, D. R. II et al. Characterizations of HCV NS5A replication complex inhibitors. Virology 444, 343–354 (2013)
Fridell, R. A., Qiu, D., Wang, C., Valera, L. & Gao, M. Resistance analysis of the hepatitis C virus NS5A inhibitor BMS-790052 in an in vitro replicon system. Antimicrob. Agents Chemother. 54, 3641–3650 (2010)
Pelosi, L. A., Voss, S., Liu, M., Gao, M. & Lemm, J. A. Effect on hepatitis C virus replication of combinations of direct-acting antivirals, including NS5A inhibitor daclatasvir. Antimicrob. Agents Chemother. 56, 5230–5239 (2012)
Nettles, R. E. et al. BMS-824393 is a potent HCV NS5A inhibitor with substantial antiviral activity when given as monotherapy in subjects with chronic G1 HCV infection. 61th Ann. Meeting AASLD (2010)
Sandgren, E. P. et al. Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene. Cell 66, 245–256 (1991)
Hernandez, D., Zhou, N., Ueland, J., Monikowski, A. & McPhee, F. Natural prevalence of NS5A polymorphisms in subjects infected with hepatitis C virus genotype 3 and their effects on the antiviral activity of NS5A inhibitors. J. Clin. Virol. 57, 13–18 (2013)
McCarville, J. F., Seifer, M., Standring, D. N., Mayers, D. L. & IDX-06A–001 Investigator Team. Treatment-emergent variants following 3 days of monotherapy with IDX719, a potent, pan-genotypic NS5A inhibitor, in subjects infected with HCV genotypes 1–4. J. Hepatol . 58 (suppl. 1), S491–S492 (2013)
Everson, G. T. et al. Phase 2b study of the interferon-free and ribavirin-free combination of daclatasvir, asunaprevir, and BMS-791325 for 12 weeks in treatment-naive patients with chronic HCV genotype 1 infection. 64th Ann. Meeting AASLD (2013)
Penin, F. et al. Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A. J. Biol. Chem. 279, 40835–40843 (2004)
Pack, S. K. et al. Hepatitis C virus inhibitors. US patent 9,006,455 B2 (2015)
St. Laurent, D. R. et al. HCV NS5A replication complex inhibitors. Part 4. Optimization for genotype 1a replicon inhibitory activity. J. Med. Chem. 57, 1976–1994 (2014)
Seebach, D., Naef, R. & Calderari, G. α-Alkylation of α-heterosubstituted carboxylic acids without racemization. Tetrahedron 40, 1313–1324 (1984)
Nagase, R., Oguni, Y., Misaki, T. & Tanabe, Y. Practical and robust method for the preparation of Seebach and Frater’s chiral template, cis-2-substituted 5-methyl (or phenyl)-1,3-dioxolan-4-ones. Synthesis 22, 3915–3917 (2006)
Reddy, L. R., Gupta, A. P. & Liu, Y. Asymmetric synthesis of α-amino acids by reduction of N-tert-butanesulfinyl ketimine esters. J. Org. Chem. 76, 3409–3415 (2011)
Link, J. O. et al. Discovery of ledipasvir (GS-5885): a potent, once-daily oral NS5A inhibitor for the treatment of hepatitis C virus infection. J. Med. Chem. 57, 2033–2046 (2014)
Baldick, C. J. et al. A novel small molecule inhibitor of hepatitis C virus entry. PLoS Pathog. 6, e1001086 (2010)
Acknowledgements
We thank J. Pizzano for conducting in vivo experiments and K. Snow for bioanalytical support.
Author information
Authors and Affiliations
Contributions
J.-H.S. and M.G. initiated synergy combination experiments. J.-H.S., D.R.O., R.A.F., C.W., S.B.R., P.N., M.B., Y.-K.W., M.L., K.R., J.A.L. and M.G. designed and performed the replicon screen for identifying synergy compounds, experiments with infectious HCV, genotype coverage, colony elimination, inhibitor-binding, isolation and mapping of resistant variants, and in vitro combination studies. M.G., M.B., J.K., N.A.M. and M.C. designed the overall virology studies and provided input to the overall research direction. D.R.L. constructed models of HCV NS5A and D.R.L., N.A.M. and M.G. contributed to the development of mechanistic hypotheses. B.M.J., F.M., M.J.N., M.B. and M.K. designed and interpreted in vivo studies. Y.T., P.H., M.B. and N.A.M. designed and synthesized the discussed compounds.
Corresponding author
Ethics declarations
Competing interests
The authors are or were, at the time this work was conducted, employees of Bristol-Myers Squibb, a company that markets antiviral therapies for the treatment of HCV.
Extended data figures and tables
Extended Data Figure 1 Structures.
Structures of the Bristol-Myers Squibb (BMS) compounds used to characterize NS5A synergy: DCV (BMS-790052; also known as daclatasvir and Daklinza), BMS-313, BMS-665, BMS-393, BMS-671 (biotin-tagged), Syn-395 and Syn-776.
Extended Data Figure 2 Quantification of HCV NS5A in GT-1b (Con1) cells.
Cells were counted (4,050 to 49,500) and harvested at the indicated time points (17 to 113 h after seeding). The harvested cells were lysed and subjected to immunoblot analysis with polyclonal anti-NS5A sera13. NS5A in each sample was quantified using a standard curve derived from purified NS5A protein. On the basis of the average concentration of NS5A in cells (189 nM) and the concentration of DCV at half-maximal inhibition (EC50 0.004 nM), the ratio of NS5A to DCV was 47,000:1. The immunoblot is representative of two independent experiments.
Extended Data Figure 3 Inhibition and compound binding to HCV NS5A.
a, EC50 values for BMS-671/BMS-665 on GT-1b wild-type and Y93H mutant are 33/22 nM and 7,800/1,800 nM, respectively. Increasing amounts of biotinylated inhibitor BMS-671 (0.05 to 3 μM) were added to wild-type (top) and Y93H resistant (bottom) GT-1b replicon cells for 16 h. Approximately 1 of 17 of the lysed cells were used as input controls and the remaining lysates were mixed with streptavidin-agarose beads. Proteins bound to streptavidin beads were fractionated on polyacrylamide gels and detected by immunoblot with polyclonal anti-NS5A sera13. Bound NS5A was quantified by scanning the signal intensity of each band. Results from two independent experiments are shown. b, Western blot of NS5A wild type and Y93H bound to biotinylated BMS-671 in the presence of increasing concentrations of the competitor BMS-665. Proteins were detected and quantified as described earlier. Results from two independent experiments are shown. c, The relative intensity of NS5A in each sample shown in b expressed as a percentage of the signal from cells treated with BMS-671 alone. Values (wild type/Y93H = 3.9/1.4 μM and 7.8/2.8 μM) represent the BMS-665 concentration required to reduce the binding of biotinylated BMS-671 to wild-type and Y93H NS5A (GT-1b) by 50%.
Extended Data Figure 4 Synergistic effect of DCV and Syn-395 on a GT-1a Y93N replicon.
EC50 values of Syn-395 were determined in the absence or presence of different concentrations of DCV. Individual values and the average of four independent experiments are shown.
Extended Data Figure 5 Colony elimination studies with GT-1a replicon cells.
a, The combination of DCV and Syn-395 enhances the resistance barrier compared to the resistance barrier of monotherapies14. GT-1a colonies surviving treatment for 7 days with DMSO (control), Syn-395 (100 nM), DCV (30 nM) or DCV (10 nM) plus Syn-395 (100 or 10 nM) were visualized after crystal violet staining. Surviving cells in duplicate plates (10 nM DCV plus 10 nM Syn-395 plus G418) were expanded for genotypic (resistance mapping) and phenotypic (EC50 determination) analysis. No consensus substitutions were observed in Syn-395-treated cells. In DCV-selected cells, most variants contained a single amino acid substitution (M28T or Y93H) with lower level resistance (EC50 of 19 nM for DCV). In cells treated with the DCV plus Syn-395 combination, higher level resistance was observed (EC50 of 352 nM for DCV), and linked substitutions containing M28T (M28T–Q30R, M28T–S38F or M28T–Q30R–S38F) were detected in 100% of the replicon cells. The pattern shown is representative of three independent experiments. b, Synergistic activity was confirmed with an alternative combination, BMS-393 and Syn-776. EC50 values of BMS-393 (0.003 ± 0.002 nM) and Syn-776 (544 ± 179 nM) in GT-1a replicon cells provided a basis for selecting inhibitor concentrations for two-way titrations. The pattern shown is representative of two independent experiments.
Extended Data Figure 6 PXB mouse study.
a, Body weights of PXB mice that had BMS-393 and Syn-776 administered concomitantly (0.4 + 5, and 4 + 25 mg kg−1) were stable during the in-life phase (90% or more compared to day 0). Data are presented as the mean and standard deviation (s.d.) for five mice. b, Serum concentration–time profiles of BMS-393 and Syn-776 (0.4 and 5 mg kg−1) administered concomitantly to PXB mice (day 1) are shown in the graph and pharmacokinetic (PK) parameter values are summarized in the adjacent box. To assess the systemic exposure to BMS-393 and Syn-776, pharmacokinetic parameters were calculated using non-compartmental methods. Each area under the curve (AUC) was calculated using the linear trapezoidal rule. Owing to small sample volumes, serum samples from three mice were pooled at each time point before analysis. Data are presented as the concentration (nM) of each pooled sample. c, Change in HCV titres for PXB mice dosed with vehicle only (filled circle), Syn-776 only (square; 15 mg kg−1), BMS-393 only (diamond; 0.4 mg kg−1) or a combination of BMS-393 plus Syn-776 (triangle; 0.4 + 15 mg kg−1) were plotted. Data for each group of mice (five) at each time point are presented as the percentage of baseline and the error bars indicate per cent variation at each time point. Mice dosed with either vehicle or Syn-776 had similar variations in titre, indicating no significant virus suppression. On day 14, 13% inhibition was observed for the group of mice treated with BMS-393 alone, versus 87% inhibition for the group treated with the BMS-393 plus Syn-776 combination. Genotypic analysis by population sequencing of day 14 samples revealed only wild-type sequences in samples derived from cohorts receiving vehicle or Syn-776 alone; a single Q30H substitution was predominant with BMS-393 treatment; a mixture of substitutions (mainly Q30H and Y93H) was observed with the combination of BMS-393 plus Syn-776. The genotypic analysis confirms that the BMS-393 plus Syn-776 combination confers a higher genetic barrier to the emergence of resistance than DCV alone, an in vivo validation of the synergistic effect.
Extended Data Figure 7 Synergistic effect of LDV (GS-5885) and Syn-395.
EC50 values of LDV were determined by titrating LDV in the absence and presence of 40 nM Syn-395 using resistant replicon cells of different genotypes (GT-1a Q30R, Y93H, Y93N and GT-1b L31V–Y93H). Values are from a single experiment.
Extended Data Figure 8 Direct comparison of synergistic combinations versus DCV-3DAA.
A synergist (Syn-395; EC50 214 nM) was used to replace either an NS3 protease inhibitor (ASV; EC50 6 nM) or NS5B polymerase inhibitor (BCV; EC50 5 nM) in a three-drug combination to eliminate HCV replicon colonies in vitro. GT-1a replicon cells were treated with HCV inhibitors as indicated, and surviving colonies were visualized by crystal violet staining. Concentration (nM) of DAAs increased as indicated by arrow: ASV + DCV + BCV (row 1: 6 + 0.04 + 5; row 2: 18 + 0.12 + 15; row 3: 60 + 0.4 + 50; row 4: 180 + 1.2 + 150); BCV + DCV + Syn-395 (row 1: 5 + 0.04 + 5; row 2: 15 + 0.12 + 15; row 3: 50 + 0.4 + 50; row 4: 150 + 1.3 + 150); and ASV + DCV + Syn-395 (row 1: 6 + 0.04 + 5; row 2: 18 + 0.12 + 15; row 3: 60 + 0.4 + 50; row 4: 180 + 1.2 + 150). The profile is representative of four independent experiments.
Extended Data Figure 9 Comparing synergy for a specific pair of NS5A compounds.
Experiments were performed on a GT-1a Y93N replicon as described in the legend of Fig. 1b. Unlike the synergy observed between DCV (SSSS stereochemistry) and BMS-395, no synergy was observed at concentrations ≤40 nM when a diastereomer of DCV, BMS-313 (RSSR stereochemistry) was paired with BMS-395. Individual values and the average from four independent experiments are shown.
Supplementary information
Supplementary Information
This file contains a description of synthesis of BMS compounds. (PDF 317 kb)
Rights and permissions
About this article
Cite this article
Sun, JH., O’Boyle II, D., Fridell, R. et al. Resensitizing daclatasvir-resistant hepatitis C variants by allosteric modulation of NS5A. Nature 527, 245–248 (2015). https://doi.org/10.1038/nature15711
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature15711
This article is cited by
-
Reflections on a 40-year career in drug design and discovery
Medicinal Chemistry Research (2023)
-
Hepatitis C virus nonstructural protein 5A perturbs lipid metabolism by modulating AMPK/SREBP-1c signaling
Lipids in Health and Disease (2019)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
