Inhibition of Hepatitis C Virus RNA Replication by 2′-Modified Nucleoside Analogs

Abstract

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2′-substituted nucleosides as inhibitors of HCV replication. The 5′-triphosphates of 2′-C-methyladenosine and 2′-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2′-O-methylcytidine inhibit HCV RNA replication. The 5′-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2′-C-methyladenosine triphosphate than 2′-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2′-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitroenzyme assays. Thus, the 2′-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication. The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2′-substituted nucleosides as inhibitors of HCV replication. The 5′-triphosphates of 2′-C-methyladenosine and 2′-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2′-O-methylcytidine inhibit HCV RNA replication. The 5′-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2′-C-methyladenosine triphosphate than 2′-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2′-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitroenzyme assays. Thus, the 2′-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication. hepatitis C virus nonstructural protein 5B, the RNA-dependent RNA polymerase NS5BΔ55, NS5B proteins with C-terminal truncations of 21 and 55 amino acids, respectively single-strand heteromeric RNA of 500-nucleotide length reverse phase high-performance liquid chromatography liquid chromatography/mass spectrometry dithiothreitol room temperature Hepatitis C virus (HCV)1infection is the leading cause of sporadic, post-transfusion, non-A non-B hepatitis (1Choo Q.L. Kuo G. Weiner A.J. Overby L.R. Bradley D.W. Houghton M. Science. 1989; 244: 359-362Google Scholar, 2Kuo G. Choo Q.L. Alter H.J. Gitnick G.L. Redeker A.G. Purcell R.H. Miyamura T. Dienstag J.L. Alter M.J. Stevens C.E. Bonino F. Colombo M. Lee W.-S. Kuo Berger C., K. Shuster J.R. Bradley D.W. Houghton M. Science. 1989; 244: 362-364Google Scholar). One hundred seventy million people worldwide are thought to be infected with hepatitis C virus of which an estimated 4 million reside in the United States (3Alter M.J. Kruszon-Moran D. Nainan O.V. McQuillan G.M. Gao F. Moyer L.A. Kaslow R.A. Margolis H.S. N. Engl. J. Med. 1999; 341: 556-562Google Scholar). Approximately 807 of infected individuals progress to chronic infection. Long term chronic HCV infection can lead to liver cirrhosis and to hepatocellular carcinoma (4Tong M.J. el-Farra N.S. Reikes A.R. Co R.L. N. Engl. J. Med. 1995; 332: 1463-1466Google Scholar, 5Poynard T. Bedossa P. Opolon P for the OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Lancet. 1997; 349: 825-832Google Scholar, 6Darby S.C. Ewart D.W. Giangrande P.L.F. Spooner R.J. Rizza C.R. Dusheiko G.M. Lee C.A. Ludlam C.A. Preston F.E. Lancet. 1997; 350: 1425-1431Google Scholar). Currently, the recommended therapy is treatment with a combination of interferon α2b and ribavirin, which results in a sustained viral response in 407 of patients (7McHutchison J.G. Gordon S.C. Schiff E.R. Shiffman M.L. Lee W.M. Rustgi V.K. Goodman Z. Ling M.-H. Cort S. Albrecht J. N. Engl. J. Med. 1998; 339: 1485-1492Google Scholar, 8Poynard T. Marcellin P. Lee S.S. Niederau C. Minuk G.S. Ideo G. Bain V. Heathcote J. Zeuzem S. Trepo C. Albrecht J. Lancet. 1998; 352: 1426-1432Google Scholar). Investigational therapies using a combination of pegylated interferon and ribavirin have lead to an sustained viral response in 547 of patients, but the response rate (427) of patients harboring HCV genotype 1 is lower (9Zeuzem S. Feinman S.V. Rasenack J. Heathcote E.J. Lai M.Y. Gane E. O'Grady J. Reichen J. Diago M. Lin A. Hoffman J. Brunda M. N. Engl. J. Med. 2000; 343: 1666-1672Google Scholar, 10Manns M.P. McHutchison J.G. Gordon S.C. Rustgi V.K. Shiffman M. Reindollar R. Goodman Z.D. Koury K. Ling M. Albrecht J.K. the International Hepatitis Interventional Therapy Group Lancet. 2001; 358: 958-966Google Scholar). Consequently, additional therapies for HCV infection are needed. Antiviral chemotherapies based on administration of analogs of deoxynucleosides have been widely successful as treatment for HIV, herpes virus, and hepatitis B infection (11De Clercq E. J. Clin. Virol. 2001; 22: 73-89Google Scholar, 12Wright M. Main J. Thomas H.C. Antivir. Chem. Chemother. 2001; 12: 201-212Google Scholar). Intracellular phosphorylation of the nucleoside analog to the triphosphate creates the active form of the inhibitor that then serves as a substrate for the viral polymerase. Generally, incorporation of the nucleotide analog at the 3′-end of the replicating viral DNA causes termination of DNA synthesis, owing to the lack of the 3′-hydroxyl required for extension. These successes suggest that an investigation of ribonucleoside analogs as inhibitors of HCV replication would be worthwhile. The HCV NS5B protein, the RNA-dependent polymerase responsible for the synthesis of the viral RNA genome, is an attractive target for the development of antiviral agents (13De Francesco R. Behrens S. Tomei L. Altamura S. Jiricny J. Methods Enzymol. 1996; 275: 58-68Google Scholar). The enzymatic activity of NS5B has been extensively characterized in vitro(13De Francesco R. Behrens S. Tomei L. Altamura S. Jiricny J. Methods Enzymol. 1996; 275: 58-68Google Scholar, 14Hong Z. Cameron C.E. Walker M.P. Castor C. Yao N. Lau J.Y.N. Zhong W. Virology. 2001; 285: 6-11Google Scholar, 15Luo G. Hamatake R.K. Mathis D. Racela J. Rigat K., L. Lemm J. Colonno R.J. J. Virol. 2000; 74: 851-863Google Scholar, 29Ohno Y. Spriggs D. Matsukage A. Ohno T. Kufe D. Cancer Res. 1989; 49: 2077-2081Google Scholar). Additionally, cell lines that harbor subgenomic replicons capable of supporting HCV replication are available to assess inhibition of replication by compounds within the cellular environment (16Lohmann V. Korner F. Koch J. Herian U. Theilmann L. Bartenschlager R. Science. 1999; 285: 110-113Google Scholar, 17Blight K.J. Kolykhalov A.A. Rice C.M. Science. 2000; 290: 1972-1974Google Scholar). The antiviral effect of interferon α has been documented in these lines (18Guo J.-T. Bichko V. Seeger C. J. Virol. 2001; 75: 8516-8523Google Scholar). Screens of available nucleosides for inhibitors in the cell-based bicistronic replicon assay have identified two nucleoside analogs, 2′-C-methyladenosine and 2′-O-methylcytidine, that specifically inhibit HCV RNA replication in the absence of cytotoxicity. The biochemical basis for the inhibition by these nucleoside analogs has been investigated. When added to replicon cells growing in culture, the nucleoside analogs resulted in the intracellular formation of the corresponding triphosphates that were shown to be potent, competitive inhibitors of NS5B-catalyzed reactionsin vitro. This study demonstrates the utility of 2′-substituted nucleosides in the inhibition of HCV RNA replication. Nucleotides, α- or γ-32P- and -33P-labeled, were purchased from PerkinElmer Life Sciences. Ultrapure nucleoside triphosphates were purchased fromAmersham Biosciences (Piscataway, NJ). 2′-O-Methylcytidine triphosphate was purchased from Trilink (San Diego, CA). 2′-O-Methylcytidine and 2′-O-methyl-5-iodocytidine were purchased from Berry Associates (Dexter, MI). 2′-C-methyladenosine from the Merck chemical collection was synthesized as previously described (19Walton E. Jenkins S. Nutt R.F. Holly F.W. J. Org. Chem. 1969; 12: 306-309Google Scholar, 20Franchetti P. Cappellacci L. Marchetti S. Trincavelli L. Martini C. Mazzoni M.R. Lucacchini A. Grifantini M. J. Med. Chem. 1998; 41: 1708-1715Google Scholar). The structures of the nucleoside analogs were verified by mass spectrometry and gradient-enhanced homonuclear correlation NMR. 2′-C-Methyladenosine triphosphate was synthesized according to the general procedures previously described (21Burgess K. Cook P.D. Chem. Rev. 2000; 10: 2047-2059Google Scholar). The triphosphate was purified by anion exchange chromatography using a 30- × 100-mm Mono Q column (Amersham Biosciences) with a buffer system of 50 mm Tris, pH 8. The elution gradient was 40 mmto 0.8 m NaCl in two column volumes. Appropriate fractions from Mono Q chromatography were collected and desalted by reverse-phase (RP) chromatography using a Luna C18 250- × 21-mm column (Phenomenex) with an elution gradient from 17 to 957 methanol in 5 mmtriethylammonium acetate. Mass spectra of the purified triphosphate were determined using in-line RP HPLC mass spectrometry on a Hewlett-Packard (Palo Alto, CA) MSD 1100. The molecular mass was determined using the Hewlett-Packard Chemstation analysis package. LC/MS: 520.0 (calc. for C11H17N5O13P3: 520.0036). The purity of the nucleoside triphosphate was determined with analytical RP and anion exchange HPLC to be 1007. 8-Bromo-2′-C-methyladenosine was synthesized from 2′-C-methyladenosine by addition ofN-bromosuccinimide in dimethylformamide. The crude product was purified on silica gel using methanol/dichloromethane (1:9) as eluent. 1H NMR (Me2SO-d6): δ 0.86 (s,3H), 3.78 (m, 2H), 3.89 (m, 1H), 4.45 (dd, 1H), 5.11 (t, 1H), 5.18 (s, 1H), 5.32 (d, 1H), 5.93 (s, 1H), 7.55 (s br, 1H), 8.09 (s, 1H). MS/ES: 360.0299 (calc. for C11H14BrN5O4 + H+: 360.0307). [8-3H]-2′-C-Methyladenosine and [5-3H]-2′-O-methylcytidine were prepared by the Tritium Custom Preparation group at Amersham Biosciences (Cardiff, Wales), starting with 8-bromo-2′-C-methyladenosine and 5-iodo-2′-O-methylcytidine, respectively. The specific activity of the [5-3H]-2′-O-methylcytidine was 24 Ci/mmol and the specific activity of the [8-3H]-2′-C-methyladenosine was 42 Ci/mmol. DNA polymerase α was supplied by T. Wang (Stanford University). DNA polymerase ॆ was purchased from AB Peptides (St. Louis, MO). DNA polymerase γ was supplied by W. Copeland (NIEHS, National Institutes of Health). HCV (BK strain) NS5BΔ21 (GenBankTM accession number M58335) was expressed inEscherichia coli BL21(DE3) harboring plasmid pT7(NS5BΔ21) and purified as previously described (22Carroll S.S. Sardana V. Yang Z. Jacobs A.R. Mizenko C. Hall D. Hill L. Zugay-Murphy J. Kuo L.C. Biochemistry. 2000; 39: 8243-8249Google Scholar). HCV (BK strain) NS5BΔ55 (23Bressanelli S. Tomei L. Roussel A. Incitti I. Vitale R.L. Mathieu M. De Francesco R. Rey F.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13034-13039Google Scholar) was expressed in E. coli BL21(DE3) harboring plasmid pT7(NS5BΔ55). Plasmid pT7(NS5BΔ55) was constructed from plasmid pT7(NS5BΔ21) by introduction of a stop codon after Leu-537. Protein expression and purification of HCV (BK strain) NS5BΔ55 followed the same protocol as that used for expression and purification of HCV (BK strain) NS5BΔ21 (22Carroll S.S. Sardana V. Yang Z. Jacobs A.R. Mizenko C. Hall D. Hill L. Zugay-Murphy J. Kuo L.C. Biochemistry. 2000; 39: 8243-8249Google Scholar). Protein concentration was determined with use of quantitative amino acid analysis. RNA polymerase activity was determined in reactions catalyzed by NS5BΔ21 and NS5BΔ55 by measuring the incorporation of radiolabeled NTPs into a heteromeric RNA template via a copy-back mechanism. Template t500 was generated by T7 runoff transcription as previously described (22Carroll S.S. Sardana V. Yang Z. Jacobs A.R. Mizenko C. Hall D. Hill L. Zugay-Murphy J. Kuo L.C. Biochemistry. 2000; 39: 8243-8249Google Scholar), using commercially available kits (Ambion) following the manufacturer's instructions. The t500 was purified with RNeasy kits (Qiagen) and was quantified using absorbance at 260 nm. NS5B-catalyzed reaction conditions included 500 nm NS5BΔ21 or nm NS5BΔ55 in a reaction of mm Tris, pH mm mm 5 mm 50 1 NTPs and of either or or were by the addition of a of after of the reaction for at for at and were by addition of formation was determined by as previously described (22Carroll S.S. Sardana V. Yang Z. Jacobs A.R. Mizenko C. Hall D. Hill L. Zugay-Murphy J. Kuo L.C. Biochemistry. 2000; 39: 8243-8249Google Scholar, Biochemistry. 22: Scholar). The inhibitor concentration at which the rate is by was determined by the rate to the Hill 1 is the reaction in the of is the reaction in the absence of and is the Hill was with use of The incorporation into RNA and of nucleoside analogs catalyzed by HCV NS5B was determined in reactions The of analogs of triphosphate was in reactions on template which is to into a with the available template as a in the The concentration of was determined by absorbance at 260 nm and the was with in reactions catalyzed by or as previously described S.S. F. Methods Enzymol. 1996; 275: Scholar). NS5BΔ55 was with template in reaction buffer mm Tris, pH mm 5 mm 50 for at room were by the addition of included or 2′-C-methyladenosine triphosphate with or were to for or and then were and with of gel the RNA at for substrate and or were on m and using a (Amersham In a similar manner analogs of triphosphate were for in reactions with and were synthesized using purified using and according to the manufacturer's instructions. DNA α and ॆ were in reactions DNA DNA polymerase α or mm 50 pH mm mm and Copeland L. Methods Enzymol. 1995; Scholar). catalyzed by DNA polymerase ॆ mm for 1 at in a reaction DNA polymerase γ was in reactions mm Tris, pH mm mm 5 × polymerase and either 1 or were by the addition of of m and for product formation with use of (22Carroll S.S. Sardana V. Yang Z. Jacobs A.R. Mizenko C. Hall D. Hill L. Zugay-Murphy J. Kuo L.C. Biochemistry. 2000; 39: 8243-8249Google Scholar, Biochemistry. 22: Scholar). cells G. G. A. C. L. M.R. E. G. Virology. Scholar) were and as previously E. E. K. S. and G. for cells were at and at a cell of in (Amersham Biosciences) in and 0.8 in was added to the cells at a concentration of 17 and for 24 at were in in and then with an RNA × in buffer (Amersham Biosciences) at 50 The RNA was generated with T7 runoff transcription and a to of the NS5B were with at room temperature for with buffer at room temperature and then at for and then in a was as previously The cells were in in at the same and the was added as the One was added for 1 at and then absorbance was at nm in a cell and were used for intracellular of and is a cell line, and a from cells that the HCV bicistronic cells were in and cells in the same at × that cells were 807 at the of was at in the cell for or were with and The cells were then in mm mm and The was and radiolabeled were using an reverse phase HPLC on a system to an in-line The HPLC of with mm and methanol mm with mm identification was by of to NS5B-catalyzed incorporation of in reactions with template t500 a copy-back or product (22Carroll S.S. Sardana V. Yang Z. Jacobs A.R. Mizenko C. Hall D. Hill L. Zugay-Murphy J. Kuo L.C. Biochemistry. 2000; 39: 8243-8249Google Scholar), as previously described for RNA Tomei L. De Francesco R. J. 1996; Scholar). The rate of product formation catalyzed by NS5BΔ21 or NS5BΔ55 was in the of either 2′-C-methyladenosine triphosphate or 2′-O-methylcytidine triphosphate shown in with as shown in as determined by the incorporation of radiolabeled nucleotide as described The Hill significantly from The potency of inhibition by either nucleotide analog was the radiolabeled nucleoside triphosphate was or that of the by the nucleoside analog was responsible for the of NS5B-catalyzed reactions in by nucleoside analog are from at in a are from at the of inhibition by 2′-C-methyladenosine triphosphate and 2′-O-methylcytidine triphosphate of RNA synthesis catalyzed by reactions were in which the concentrations of and were the concentrations of the NTPs at concentrations of the as shown in competitive inhibition of activity by 2′-C-methyladenosine triphosphate and 2′-O-methylcytidine triphosphate with and respectively. The as determined from a of the of the was for 2′-C-methyladenosine triphosphate and for 2′-O-methylcytidine triphosphate. NS5BΔ55 is capable of the nucleoside analogs into a growing RNA gel-based of reactions using RNA were The of the RNA template was that an the incorporation of followed by NS5BΔ55 a greater ability to incorporate the RNA than The activity of NS5BΔ21 with the is a of the of NS5BΔ21 S.S. Sardana V. Yang Z. Jacobs A.R. Mizenko C. Hall D. Hill L. Zugay-Murphy J. Kuo L.C. Biochemistry. 2000; 39: 8243-8249Google Scholar) with NS5BΔ55 The incorporation of to the of a product In reactions that included and the product from the incorporation of was by addition of NS5BΔ55 was capable of 2′-C-methyladenosine the 3′-end of the RNA at the nucleotide concentration 1 However, as shown in NS5BΔ55 the template a of product was In a similar NS5BΔ55 was capable of 2′-O-methylcytidine the 3′-end of RNA as shown in However, product was the next nucleoside was added to the the of inhibition the activity of DNA and γ were in the of 2′-C-methyladenosine triphosphate and 2′-O-methylcytidine triphosphate. than inhibition of the activity of DNA or γ was detected at 50 of either nucleoside triphosphate 2′-C-methyladenosine and 2′-O-methylcytidine were for activity in a cell-based replicon assay using a cell line, which the replication of HCV RNA and The effect of the nucleosides RNA replication in a G. G. A. C. L. M.R. E. G. Virology. Scholar) was detected assay as previously of the compounds in the replicon assay are shown in compounds were active in the assay at 24 with of for the 2′-C-methyladenosine and 21 for the 2′-O-methylcytidine The antiviral activity of both compounds was in the absence of in cells as in the assay to potency and of nucleoside analogs in the HCV replicon assay in were in cell for 24 to of the of HCV replicon RNA with the in assay were to 1 to and the are the from at was determined by assay on at the same were in cell for 24 to of the of HCV replicon RNA with the in assay G. G. A. C. L. M.R. E. G. Virology. Scholar, Tomei L. De Francesco R. J. 1996; Scholar). were to 1 to and the are the from at was determined by assay on at the same in a The intracellular of the of 2′-C-methyladenosine and 2′-O-methylcytidine was in and The compounds were for or at to and HPLC as shown in The results are in 2′-C-Methyladenosine was into the cells and to its corresponding triphosphate. In of cells with 2′-O-methylcytidine triphosphate and from to that are consistent with to for and that this was to and in the of and in cell to and in the of is to and in the of is to and is to and is were in cell for the to and analysis with are expressed as cells of corresponding triphosphate and are of at to and in the of is to and is in a were in cell for the to and analysis with are expressed as cells of corresponding triphosphate and are of at The of the bicistronic replicon system (16Lohmann V. Korner F. Koch J. Herian U. Theilmann L. Bartenschlager R. Science. 1999; 285: 110-113Google Scholar) as a of the replication of viral RNA within the cellular environment has the of analogs of to at inhibitors of purified NS5B in vitro. of available nucleosides for inhibition of viral replication in the replicon assay identified two 2′-C-methyladenosine and The triphosphates of 2′-C-methyladenosine and 2′-O-methylcytidine inhibit the activity of purified HCV RNA polymerase with of and respectively of HCV RNA polymerase two C-terminal truncations that were have significantly with NS5BΔ55 greater specific activity than However, the two for the nucleoside analog triphosphates investigated. the two nucleoside analog triphosphates were competitive inhibitors with nucleoside that were of the incorporation of the nucleoside analogs a growing RNA was using RNA that are to into NS5BΔ55 is capable of both 2′-C-methyladenosine and 2′-O-methylcytidine the RNA that both triphosphates can to the in the substrate and is additional room in the of the and the in the active that HCV NS5B to either the or The of of inhibition of the viral RNA polymerase inhibition of the DNA incorporation of the nucleotide NS5BΔ55 is capable of the incorporated analog by addition of the next that with this template the nucleotide analogs as despite the in both of a The results suggest that after incorporation the is able to on the of the investigation is to the molecular of the termination by incorporation of nucleotide analogs that a 3′-hydroxyl has previously been in the inhibition of DNA polymerase ॆ by triphosphate Y. Spriggs D. Matsukage A. Ohno T. Kufe D. Cancer Res. 1989; 49: 2077-2081Google Scholar) and triphosphate Y. Spriggs D. Matsukage A. Ohno T. Kufe D. Cancer Res. Scholar). The nucleosides were intracellularly to the corresponding in as specific inhibitors of HCV RNA The potency of inhibition of HCV replication in cells with the of triphosphates of the intracellular 2′-O-methylcytidine triphosphate was detected than the 2′-C-methyladenosine and the is in the potency of 2′-O-methylcytidine in despite the inhibition of the purified by the two The potency of inhibition by 2′-C-methyladenosine in the cell-based replicon assay is greater than the potency of the corresponding triphosphate in the assay The greater potency in the cell-based assay a combination of the intracellular concentration of the corresponding triphosphate that is cells in the of and the that the analog as a the nucleotide analog is incorporated into the replicon the RNA in the absence of a is as a template for of viral RNA However, in the assay, the nucleotide analog has been the RNA is as the the in assay is conditions that from is that the assay be of the use of in assays is by the of the inhibition of purified HCV NS5B by the triphosphates of nucleoside analogs that are capable of HCV replication in cell in the absence of cytotoxicity. correlation of inhibition with antiviral effect in the replicon assay be are identified that both in and in the cell the replicon assay be a of antiviral activity in has to be at this is the of 2′-C-methyladenosine or 2′-O-methylcytidine have and that are attractive to development as HCV However, the the inhibition of HCV RNA polymerase activity by leading to inhibition of HCV replication in V. Sardana and J. Zugay-Murphy for the purification of HCV NS5BΔ55 and M. Sardana for amino acid analysis.