The Novel Nucleoside Analog R1479 (4′-Azidocytidine) Is a Potent Inhibitor of NS5B-dependent RNA Synthesis and Hepatitis C Virus Replication in Cell Culture
2005; Elsevier BV; Volume: 281; Issue: 7 Linguagem: Inglês
10.1074/jbc.m510195200
ISSN1083-351X
AutoresKlaus Klumpp, Vincent Lévêque, Sophie Le Pogam, Han Ma, Wen-Rong Jiang, Hyunsoon Kang, Caroline Granycome, Margaret Singer, Carl Laxton, Julie Q. Hang, Keshab Sarma, David B. Smith, Dieter Heindl, Chris Hobbs, John H. Merrett, Julian Symons, Nick Cammack, Joseph A. Martin, René Devos, Isabel Nájera,
Tópico(s)HIV/AIDS drug development and treatment
ResumoHepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4′-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC50 = 1.28 μm) with similar potency compared with 2′-C-methylcytidine (IC50 = 1.13 μm). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mm. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5′-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a Ki of 40 nm. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3′-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3′-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2′-C-MeATP and other 2′-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479. Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4′-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC50 = 1.28 μm) with similar potency compared with 2′-C-methylcytidine (IC50 = 1.13 μm). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mm. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5′-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a Ki of 40 nm. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3′-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3′-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2′-C-MeATP and other 2′-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479. Hepatitis C virus (HCV) 2The abbreviations used are: HCV, hepatitis C virus; IRES, internal ribosome entry site; RT, reverse transcription; cIRES, complementary internal ribosome entry site; UTR, untranslated region; TP, 5′-triphosphate.2The abbreviations used are: HCV, hepatitis C virus; IRES, internal ribosome entry site; RT, reverse transcription; cIRES, complementary internal ribosome entry site; UTR, untranslated region; TP, 5′-triphosphate. infection is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma and is currently the leading cause of liver transplantation (1.Thomson B.J. Finch R.G. Clin. Microbiol. Infect. 2005; 11: 86-94Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 2.Centers of Disease Control and PreventionMorb. Mortal. Wkly. Rep. 1998; 47: 1-39PubMed Google Scholar). Viral genome sequence analysis established six HCV genotype classes (HCV genotypes 1–6), with genotypes 1–3 being the most prevalent in the United States, Europe, and Japan. Current treatment options available to HCV-infected persons are limited, and sustained virological response rates are particularly low for HCV genotype 1-infected patients. Only ∼50% of individuals infected with HCV genotype 1 with serum viral titers of >2 × 106 copies/ml achieved sustained virological response rates when treated with a combination of pegylated interferon-α and ribavirin (3.Fried M.W. Shiffman M.L. Reddy K.R. Smith C. Marinos G. Goncales Jr., F.L. Haussinger D. Diago M. Carosi G. Dhumeaux D. Craxi A. Lin A. Hoffman J. Yu J. N. Engl. J. Med. 2002; 347: 975-982Crossref PubMed Scopus (5889) Google Scholar, 4.Manns M.P. McHutchison J.G. Gordon S.C. Rustgi V.K. Shiffman M. Rein-dollar R. Goodman Z.D. Koury K. Ling M. Albrecht J.K. Lancet. 2001; 358: 958-965Abstract Full Text Full Text PDF PubMed Scopus (5905) Google Scholar). Response rates are even lower in persons with HIV co-infection or cirrhosis and also decrease with age (1.Thomson B.J. Finch R.G. Clin. Microbiol. Infect. 2005; 11: 86-94Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 5.Pawlotsky J.-M. N. Engl. J. Med. 2004; 351: 422-423Crossref PubMed Scopus (42) Google Scholar, 6.Torriani F.J. Rodriguez-Torres M. Rockstroh J.K. Lissen E. Gonzalez-Garcia J. Lazzarin A. Carosi G. Sasadeusz J. Katlama C. Montaner J. Sette H.J. Passe S. De Pamphilis J. Duff F. Schrenk U. Dieterich D.T. N. Engl. J. Med. 2004; 351: 438-450Crossref PubMed Scopus (1197) Google Scholar, 7.Chung R.T. Andersen J. Volberding P. Robbins G.K. Liu T. Sherman K.E. Peters M.G. Koziel M.J. Bhan A.K. Alston B. Colquhoun D. Nevin T. Harb G. van der Horst C. N. Engl. J. Med. 2004; 351: 451-459Crossref PubMed Scopus (843) Google Scholar). Urgently required improvements in anti-HCV therapy will depend on the development of novel therapeutic approaches, especially in difficult to treat populations. HCV is an enveloped (+)-strand RNA virus that enters host cells via receptor-mediated endocytosis and replicates in the host cell cytoplasm. A membrane-associated replicase complex containing HCV genome-encoded nonstructural proteins and HCV genomic RNA in a tight complex is responsible for the formation of viral RNA for packaging into new virus particles during the HCV replication process. The viral NS5B protein contains the HCV polymerase active site within the replicase complex, an RNA-dependent RNA polymerase. The concept of polymerase inhibition to attain antiviral efficacy has been successfully established in other viral infections (human immunodeficiency virus, hepatitis B virus, and herpes viruses). Polymerase inhibitors are the largest class of approved antiviral drugs, and nucleosides are the largest chemical class therein. The majority of antiviral nucleoside analogs are further metabolized to the corresponding nucleoside 5′-triphosphate analogs by cellular enzymes. Nucleoside 5′-triphosphate analogs then function as alternative substrates for the viral polymerase, competitively inhibit viral nucleic acid synthesis, and can terminate nucleic acid synthesis after incorporation. A novel ribonucleoside analog, 2′-C-methylcytidine (NM107), has entered clinical development as a 3′-valinate prodrug (NM283). 3Zhou, X. J., Afdahl, N., Godofsky, E., Dienstag, J., Rustgi, V., Schick, L., McInery, D., Fielman, B. A., and Brown, N. A., Digestive Disease Week 2005, Chicago, IL, May 14–19, 2005, Abstr. S926.3Zhou, X. J., Afdahl, N., Godofsky, E., Dienstag, J., Rustgi, V., Schick, L., McInery, D., Fielman, B. A., and Brown, N. A., Digestive Disease Week 2005, Chicago, IL, May 14–19, 2005, Abstr. S926. Additional nucleoside analogs carrying the 2′-β-methyl moiety were also found to specifically inhibit HCV RNA replication in cell culture (9.Carroll S.S. Tomassini J.E. Bosserman M. Getty K. Stahlhut M.W. Eldrup A.B. Bhat B. Hall D. Simcoe A.L. LaFemina R.L. Rutkowski C.A. Wolanski B. Yang Z. Migliaccio G. De Francesco R. Kuo L.C. MacCoss M. Olsen D.B. J. Biol. Chem. 2003; 278: 11979-11984Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 10.Olsen D.B. Eldrup A.B. Bartholomew L. Bhat B. Bosserman M.R. Ceccacci A. Colwell L.F. Fay J.F. Flores O.A. Getty K.L. Grobler J.A. LaFemina R.L. Markel E.J. Migliaccio G. Prhavc M. Stahlhut M.W. Tomassini J.E. MacCoss M. Hazuda D.J. Carroll S.S. Antimicrob. Agents Chemother. 2004; 48: 3944-3953Crossref PubMed Scopus (209) Google Scholar, 11.Eldrup A.B. Prhavc M. Brooks J. Bhat B. Prakash T.P. Song Q. Bera S. Bhat N. Dande P. Cook P.D. Bennett C.F. Carroll S.S. Ball R.G. Bosserman M. Burlein C. Colwell L.F. Fay J.F. Flores O.A. Getty K. LaFemina R.L. Leone J. MacCoss M. McMasters D.R. Tomassini J.E. Von Langen D. Wolanski B. Olsen D.B. J. Med. Chem. 2004; 47: 5284-5297Crossref PubMed Scopus (190) Google Scholar). Targeted screening of a nucleoside analog library combined with rational lead optimization at Roche identified R1479 (4′-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system. This study describes the profile of potency and selectivity of 4′-azidocytidine as an antiviral agent in cell culture and the characterization of 4′-azidocytidine triphosphate as an inhibitor of native HCV replicase and recombinant HCV polymerase (NS5B). Based on its promising preclinical profile, R1479 is currently under evaluation in clinical trials as a drug candidate for the treatment of HCV infection. HCV Replicon Assays—The 2209-23 cell line was established from Huh-7 cells by stable transfection with a bicistronic replicon (genotype 1b), of which the first open reading frame (driven by the HCV internal ribosome entry site (IRES)) contains the Renilla luciferase gene fused with the neomycin phosphotransferase gene (nptII) and the second open reading frame (driven by the encephalomyocarditis virus IRES) contains the HCV nonstructural genes NS3, NS4a, NS4b, NS5A, and NS5B derived from the NK5.1 replicon backbone, originally described by Krieger et al. (12.Krieger N. Lohmann V. Bartenschlager R. J. Virol. 2001; 75: 4614-4624Crossref PubMed Scopus (461) Google Scholar). Thus, the replicon cell line 2209-23 is resistant to Geneticin (G418) and expresses the Renilla luciferase reporter gene as a marker of HCV RNA replication. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with Glutamax and 100 mg/ml sodium pyruvate (Invitrogen). The medium was further supplemented with 10% (v/v) fetal calf serum, 1% (v/v) penicillin/streptomycin, and 500 μg/ml G418. Cells were maintained at 37 °C in a humidified 5% CO2 and 95% air atmosphere. The selection and characterization of replicon clones carrying the point mutation S282T in the NS5B coding sequence will be described elsewhere. 4I. Nájera, S. Rajyaguru, S. Le Pogam, V. Lévêque, H. Kang, S. Yiang, H. Ma, K. Klumpp, J. Symons, and N. Cammack, manuscript in preparation. Nucleoside analogs were synthesized at Roche, dissolved in 100% Me2SO, and then diluted in Dulbecco's modified Eagle's medium with 5% (v/v) fetal calf serum before addition to cells. The final concentration of Me2SO was 1% (v/v) in all experiments. Quantification of Renilla luciferase activity was performed using the Renilla luciferase assay kit (Promega) according to the manufacturer's instructions. The WST-1 cell proliferation assay (Roche Diagnostics) was used to measure cell viability. Direct quantification of HCV RNA levels was performed by quantitative kinetic reverse transcription (RT)-PCR (TaqMan) using endogenous β-actin mRNA as a control. The primers used were 5′-GCTGCTATTGGGCGAAGTG-3′ (forward), 5′-GCCGCCGCATTGCA-3′ (reverse), and 5′-TCCTGCCGAGAAAGTATCCATCATGGCT-3′ (probe). Reactions were analyzed on a Model 7700 sequence detector (PE Biosystems). The genotype 1b Con1-adapted transient replicon (pKF-repPI-luc/ET) was obtained from Dr. R. Bartenschlager (13.Lohmann V. Hoffmann S. Herian U. Penin F. Bartenschlager R. J. Virol. 2003; 77: 3007-3019Crossref PubMed Scopus (345) Google Scholar). The Con1-S282T transient replicon was constructed, and assays were performed as described. 4I. Nájera, S. Rajyaguru, S. Le Pogam, V. Lévêque, H. Kang, S. Yiang, H. Ma, K. Klumpp, J. Symons, and N. Cammack, manuscript in preparation. Replicon Cell Proliferation Assay—The effect of compounds on the incorporation of tritiated thymidine into cellular DNA was measured using the [3H]thymidine incorporation scintillation proximity assay system from Amersham Biosciences. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and WST-1 assay systems (Roche Diagnostics) were used to measure cell viability. The ATP bioluminescence assay kit HSII (Roche Diagnostics) was used to measure intracellular ATP levels. HCV RNA Clearance Assay—HCV replicon cells were cultured with 6 and 12 μm R1479 in the absence of neomycin selection for 15 days (replicon clearance phase). Cells were passaged at a 1:2 ratio when they reached confluency, and samples were taken for HCV replicon RNA level determination. After 15 days, the inhibitor was removed, and cells were cultured for an additional 15 days in culture medium containing 0.25 mg/ml neomycin to allow replication of residual replicon RNA molecules remaining after treatment. The levels of HCV RNA were determined by quantitative kinetic RT-PCR using TaqMan technology as described above and are expressed as log change compared with the HCV replicon RNA level in untreated cells at day 0. HCV Replicase Assay—The membrane-associated, native HCV replicase complex was isolated from 2209-23 HCV replicon cells and a derived cell line carrying HCV replicon RNA with a S282T mutation in the NS5B coding sequence according to previously published procedures (14.Ma H. Leveque V. De Witte A. Li W. Hendricks T. Clausen S.M. Cammack N. Klumpp K. Virology. 2005; 332: 8-15Crossref PubMed Scopus (60) Google Scholar). The in vitro replicase assay contained 10 μl of cytoplasmic membrane fraction, 50 mm HEPES (pH 7.5), 10 mm KCl, 10 mm dithiothreitol, 5 mm MgCl2, 20 μg/ml actinomycin D, 1 mm ATP, 1 mm GTP, 1 mm UTP, 30 μCi of [α-33P]CTP (3000 Ci/mmol, 10 mCi/ml), 1 unit/μl SUPERase·In (Ambion, Inc.), 10 mm creatine phosphate, and 200 μg/ml creatine phosphokinase in a final volume of 25 μl. Inhibition by nucleotide analogs was determined as described (14.Ma H. Leveque V. De Witte A. Li W. Hendricks T. Clausen S.M. Cammack N. Klumpp K. Virology. 2005; 332: 8-15Crossref PubMed Scopus (60) Google Scholar). HCV Polymerase Assay—The enzyme activities of NS5B570-BK, NS5B570-Con1, and NS5B570-S282T-Con1 proteins was measured as incorporation of radiolabeled NMP into acid-insoluble RNA products as described previously (14.Ma H. Leveque V. De Witte A. Li W. Hendricks T. Clausen S.M. Cammack N. Klumpp K. Virology. 2005; 332: 8-15Crossref PubMed Scopus (60) Google Scholar). Briefly, HCV polymerase reactions contained 10 μg/ml complementary IRES (cIRES) or 3′-untranslated region (UTR) RNA template, 8.4 μg/ml poly(A)·oligo(U) template-primer or 1.6 μg/ml poly(I)·oligo(C) template-primer, 1 μm tritiated UTP or CTP (1–5 μCi), 1 μm ATP, 1 μm CTP, 1 μm GTP (with cIRES and 3′-UTR RNA templates), 40 mm Tris-HCl (pH 8.0), 4 mm dithiothreitol, and 4 mm MgCl2. NS5B570 proteins contain a C-terminal deletion of 21 amino acids, which removes a transmembrane domain and increases solubility of the protein (14.Ma H. Leveque V. De Witte A. Li W. Hendricks T. Clausen S.M. Cammack N. Klumpp K. Virology. 2005; 332: 8-15Crossref PubMed Scopus (60) Google Scholar, 15.Ferrari E. Wright-Minogue J. Fang J.W. Baroudy B.M. Lau J.Y. Hong Z. J. Virol. 1999; 73: 1649-1654Crossref PubMed Google Scholar). The HCV RNA templates used were as follows: cIRES RNA, corresponding to 377 nucleotides from the 3′-end of HCV (–)-strand RNA (14.Ma H. Leveque V. De Witte A. Li W. Hendricks T. Clausen S.M. Cammack N. Klumpp K. Virology. 2005; 332: 8-15Crossref PubMed Scopus (60) Google Scholar), with a base content of 21% Ade, 23% Ura, 28% Cyt, and 28% Gua; and 3′-UTR RNA, corresponding to 389 nucleotides from the 3′-end of HCV (+)-strand RNA, with a base content of 15% Ade, 38% Ura, 26% Cyt, and 21% Gua. RNA was transcribed in vitro using a T7 transcription kit (Ambion, Inc.) and purified either by phenol-chloroform extraction or using the Qiagen RNeasy maxi kit, with similar results. Poly(A) RNA was from Amersham Biosciences, and poly(I) RNA was from Yamasa Corp. Data were analyzed with GraphPad® Prism® and/or Microsoft® Excel®. The apparent Michaelis constant (Km(app)) of CTP for NS5B570-BK was calculated by nonlinear fitting using Equation 1, Y=(Vmax(app))XKm(app)+X (Eq. 1) where Y corresponds to the rate of RNA synthesis by NS5B570-BK (in cpm/min), Vmax(app) is the maximum rate at saturating substrate concentration, and X corresponds to CTP concentration. The compound concentration at which the enzyme-catalyzed rate was reduced by 50% (IC50) was calculated by fitting the data to Equation 2, Y=% Min+(% Max-% Min)(1+X(IC50)) (Eq. 2) where Y corresponds to the percent relative enzyme activity, % Min is the residual relative activity at saturating compound concentration, % Max is the relative maximum enzyme activity, and X corresponds to the compound concentration. The mean IC50 value was derived from the mean of several independent experiments. The S.D. was calculated from the nonbiased method using Equation 3. S.D.=n∑(IC50) 2-(∑IC50) 2n(n-1) (Eq. 3) The Ki(CTP) for Ro10482975 ′-triphosphate was derived by fitting the data to the Cheng-Prusoff equation, assuming competitive inhibition relative to CMP incorporation (Equation 4), Ki(CTP)=IC50(1+[CTP]Km(app)) (Eq. 4) where [CTP] is the initial concentration of CTP and Km(app) is the apparent Km for CTP. Gel-based Nucleotide Incorporation Assay—The RNA template-directed incorporation and extension of the nucleotide and nucleotide analogs by HCV NS5B were performed with a 19-nucleotide RNA oligonucleotide (5′-AUGUAUAAUUAUUGUAGCC-3′) and a 5′-end-labeled GG primer (Dharmacon, Inc.). The RNA template is predicted to form a single stem-loop structure with an unpaired 3-nucleotide sequence at the 3′-end. The 5′-end labeling of the GG primer was performed with [γ-33P]ATP and T4 polynucleotide kinase (Roche Applied Science). The nucleotide incorporation reactions contained 40 mm Tris-HCl (pH 8.0), 20 mm KCl, 2 mm MgCl2, 5 mm dithiothreitol, 2 μm NS5B570-BK, 5 μm RNA template, 0.15 μm end-labeled GG primer, and nucleoside triphosphates at the indicated concentrations in a volume of 10 μl. The reactions were incubated at 30 °C for 60 min and stopped by the addition of 10 μl of formamide gel loading buffer II (Ambion, Inc.). After denaturing at 95 °C for 3 min, the RNA template, primer, and extended primer were separated on a Tris borate/EDTA-urea-20% acrylamide gel. The dried gel was exposed to a storage phosphor screen and analyzed using a PhosphorImager (Amersham Biosciences). Nucleoside analogs have proven to be highly successful agents for the treatment of viral infections, including HIV, hepatitis B virus, and herpes viruses. In most cases, the nucleoside triphosphates are the biologically active metabolites, and nucleoside analogs are converted to their active triphosphate forms by cellular enzymes. The nucleoside triphosphate analogs then function as competitive substrate analogs of the viral polymerase and inhibit the synthesis of viral DNA and/or RNA molecules. The HCV subgenomic replicon provides a convenient cellular system for the assessment of nucleoside analogs as inhibitors of HCV replication (9.Carroll S.S. Tomassini J.E. Bosserman M. Getty K. Stahlhut M.W. Eldrup A.B. Bhat B. Hall D. Simcoe A.L. LaFemina R.L. Rutkowski C.A. Wolanski B. Yang Z. Migliaccio G. De Francesco R. Kuo L.C. MacCoss M. Olsen D.B. J. Biol. Chem. 2003; 278: 11979-11984Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 16.Frese M. Schwarzle V. Barth K. Krieger N. Lohmann V. Mihm S. Haller O. Bartenschlager R. Hepatology. 2002; 35: 694-703Crossref PubMed Scopus (274) Google Scholar). A bicistronic HCV replicon was developed that contains the HCV 5′-UTR directing translation of Renilla luciferase and the selectable marker neomycin phosphotransferase. The encephalomyocarditis virus IRES directs the translation of HCV proteins NS3, NS4A, NS4B, NS5A, and NS5B (12.Krieger N. Lohmann V. Bartenschlager R. J. Virol. 2001; 75: 4614-4624Crossref PubMed Scopus (461) Google Scholar, 17.Vrolijk J.M. Kaul A. Hansen B.E. Lohmann V. Haagmans B.L. Schalm S.W. Bartenschlager R. J. Virol. Methods. 2003; 110: 201-209Crossref PubMed Scopus (159) Google Scholar). The Renilla replicon system was validated using a range of different types of HCV replication inhibitors, including interferon-α, and established that the level of Renilla luciferase activity correlated with the level of HCV replicon RNA as determined by quantitative kinetic RT-PCR and Northern blot analysis. Screening of a nucleoside library identified a series of compounds that inhibited HCV subgenomic RNA replication without interfering with cell viability and cell proliferation. After further optimization, R1479 (4′-azidocytidine) (Fig. 1) was selected for further characterization based on an exceptionally high therapeutic window in HCV replicon cells. Inhibition of HCV RNA Replication by R1479—R1479 inhibited HCV RNA replication with a mean IC50 value of 1.28 μm when measured as dose-dependent reduction of Renilla luciferase activity after a 72-h incubation of proliferating replicon cells (Table 1). The potency of R1479 in this cell line was similar to that obtained for 2′-C-methylcytidine with a mean IC50 value of 1.13 μm. Similar potencies for both compounds were also obtained from the measurement of the dose-dependent reduction of HCV replicon RNA by quantitative kinetic RT-PCR (Table 1). In contrast, the structurally related cytidine analog 3′-deoxycytidine did not inhibit HCV RNA replication in replicon cells at concentrations up to 100 μm. R1479 showed only low cytotoxicity in human hepatoma-derived replicon cells. Cell viability was monitored by measuring intracellular ATP concentration or cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or succinate-tetrazolium reductase activities. No reduction in cell viability was observed with R1479 at concentrations up to 2 mm using any of the three methods (Table 1).TABLE 1Inhibition of HCV RNA replication in HCV replicon cellsCompoundIC50LauaInhibition of HCV replicon-encoded Renilla luciferase reporter activity; mean IC50 value ± S.E. determined from 101 (R1479) and 354 (2′-C-methylcytidine) experiments.TaqbInhibition of HCV RNA determined by quantitative kinetic RT-PCR; mean IC50 value ± S.E. calculated from at least four experiments.WST-1cCell viability determined from at least four experiments.[3H]ThddInhibition of tritiated thymidine incorporation into cellular DNA determined from at least four experiments.ATPeReduction of cellular ATP levels determined from four experiments.μm4′-Azidocytidine (R1479)1.28 ± 0.080.67 ± 0.13>2000>100>20002′-C-Methylcytidine1.13 ± 0.030.76 ± 0.01>100>100NDfND, not determined.3′-Deoxycytidine>100ND>100NDNDMycophenolic acid0.32 ± 0.07ND>1000.60 ± 0.02NDa Inhibition of HCV replicon-encoded Renilla luciferase reporter activity; mean IC50 value ± S.E. determined from 101 (R1479) and 354 (2′-C-methylcytidine) experiments.b Inhibition of HCV RNA determined by quantitative kinetic RT-PCR; mean IC50 value ± S.E. calculated from at least four experiments.c Cell viability determined from at least four experiments.d Inhibition of tritiated thymidine incorporation into cellular DNA determined from at least four experiments.e Reduction of cellular ATP levels determined from four experiments.f ND, not determined. Open table in a new tab It has been reported previously that ongoing cell proliferation appears to be important for HCV RNA replication in Huh-7-derived cell lines, whereas replicon RNA levels are reduced when cells reach confluency (18.Pietschmann T. Lohmann V. Rutter G. Kurpanek K. Bartenschlager R. J. Virol. 2001; 75: 1252-1264Crossref PubMed Scopus (322) Google Scholar). Cytostatic compounds therefore reduce HCV RNA levels in replicon cells in a nonspecific manner, as exemplified by the IMP dehydrogenase inhibitor mycophenolic acid. Mycophenolic acid is a potent inhibitor of cell proliferation, as measured by the inhibition of tritiated thymidine incorporation into cellular DNA (IC50 = 0.6 μm) without affecting cell viability over a 72-h incubation period at concentrations up to 100 μm (Table 1). The apparent IC50 value for the inhibition of HCV replicon RNA replication by mycophenolic acid was similar to the IC50 value for thymidine incorporation into cellular DNA, suggesting an indirect cell culture-specific effect of HCV replicon RNA reduction by mycophenolic acid. In contrast, R1479 was not cytostatic and did not inhibit tritiated thymidine incorporation at concentrations up to 1 mm, suggesting that R1479 inhibits HCV replication by a direct antiviral mechanism. The point mutation S282T in the NS5B coding sequence has been previously associated with resistance to nucleoside analogs carrying a 2′-β-methyl moiety, as shown using wild-type and mutant S282T transient replicons (10.Olsen D.B. Eldrup A.B. Bartholomew L. Bhat B. Bosserman M.R. Ceccacci A. Colwell L.F. Fay J.F. Flores O.A. Getty K.L. Grobler J.A. LaFemina R.L. Markel E.J. Migliaccio G. Prhavc M. Stahlhut M.W. Tomassini J.E. MacCoss M. Hazuda D.J. Carroll S.S. Antimicrob. Agents Chemother. 2004; 48: 3944-3953Crossref PubMed Scopus (209) Google Scholar, 19.Migliaccio G. Tomassini J.E. Carroll S.S. Tomei L. Altamura S. Bhat B. Bartholomew L. Bosserman M.R. Ceccacci A. Colwell L.F. Cortese R. De Francesco R. Eldrup A.B. Getty K.L. Hou X.S. LaFemina R.L. Ludmerer S.W. MacCoss M. McMasters D.R. Stahlhut M.W. Olsen D.B. Hazuda D.J. Flores O.A. J. Biol. Chem. 2003; 278: 49164-49170Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). In contrast, the S282T mutation did not confer resistance to HCV replication inhibition by R1479. Both stable and transient mutant replicons carrying mutation S282T in the NS5B coding region were sensitive to inhibition by R1479, suggesting absence of cross-resistance between 2′-methyl-nucleosides and R1479 (Table 2).TABLE 2Inhibition of genotype 1b wild-type Con1 and Con1-S282T stable and transient replicon replicationCompoundIC50Stable repliconsTransient repliconsWild typeaInhibition of HCV RNA replication of the genotype 1b wild-type Con1 stable replicon.S282TbInhibition of HCV RNA determined by quantitative kinetic RT-PCR; IC50 value determined from two different stable replicon cell lines containing the S282T mutation (cell lines 16 and C).Wild typecInhibition of HCV RNA replication; mean IC50 value ± S.E. determined from at least five independent experiments by quantification of replicon-encoded luciferase activity.S282TcInhibition of HCV RNA replication; mean IC50 value ± S.E. determined from at least five independent experiments by quantification of replicon-encoded luciferase activity.Cell line 16Cell line CμmμmR14790.67 ± 0.130.30.271.9 ± 0.260.53 ± 0.1a Inhibition of HCV RNA replication of the genotype 1b wild-type Con1 stable replicon.b Inhibition of HCV RNA determined by quantitative kinetic RT-PCR; IC50 value determined from two different stable replicon cell lines containing the S282T mutation (cell lines 16 and C).c Inhibition of HCV RNA replication; mean IC50 value ± S.E. determined from at least five independent experiments by quantification of replicon-encoded luciferase activity. Open table in a new tab Clearance of HCV RNA from Replicon Cells—Prolonged incubation of replicon cells with HCV polymerase inhibitors can result in the selection of inhibitor-resistant replicon variants with point mutations in the NS5B protein coding sequence.4 In contrast, prolonged incubation of HCV replicon cells with R1479 resulted in a continued decrease in HCV RNA to undetectable levels (Fig. 2). To determine the kinetics of the HCV subgenomic replicon inhibition by R1479, HCV replicon cells were cultured in the presence of inhibitor at 6 and 12 μm and in the absence of neomycin selection for 15 days (replicon clearance phase). During this phase in the absence of neomycin, cells were able to proliferate even in the absence of active replicon replication. After this time and during the rebound phase, the inhibitor was removed, and cells were cultured for an additional 30 days in culture medium containing 0.25 mg/ml neomycin (15 days for the untreated control). In the presence of neomycin, only cells harboring active replicating replicons are able to proliferate. HCV RNA levels were monitored every 3 days by quantitative kinetic RT-PCR as described under "Experimental Procedures." HCV RNA levels remained constant throughout the viral clearance and the rebound phases in the untreated cells. In the presence of R1479 at 6 and 12 μm (approximately 5 × IC50 and 10 × IC50, respectively), the HCV RNA was reduced by >5 log units and became undetectable by RT-PCR after the 15-day incubation period. In addition, cells had lost the ability to grow in the presence of neomycin during the rebound phase (Fig. 2). Therefore, R1479 at concentrations of approximately its IC90 was able to completely clear the HCV subgenomic replicon RNA from the replicon cells within a 15-day incubation period. R1479 5′-Triphosphate (TP) Inhibits Native HCV Replicase in Vitro—The highly specific inhibition of HCV RNA replication by R1479 suggested HCV polymerase NS5B as a possible target of the corresponding R1479-TP. NS5B likely forms large multiprotein-RNA complexes with the other HCV-encoded nonstructural proteins and the HCV genomic RNA during the assembly of the functional HCV replicase (20.Moradpour D. Gosert R. Egger D. Penin F. Blum H.E. Bienz K. Antiviral Res. 2003; 60: 103-109Crossref PubMed Scopus (130) Google Scholar). Membrane-associated, native HCV replicase complexes were isolated from HCV replicon cells according to published procedures (14.Ma H. Leveque V. De Witte
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