Characterization of the Metabolic Activation of Hepatitis C Virus Nucleoside Inhibitor β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and Identification of a Novel Active 5′-Triphosphate Species
2007; Elsevier BV; Volume: 282; Issue: 41 Linguagem: Inglês
10.1074/jbc.m705274200
ISSN1083-351X
AutoresHan Ma, Wen-Rong Jiang, Nicole L. Robledo, Vincent Lévêque, Samir Ali, Teresa Lara‐Jaime, Mohammad R. Masjedizadeh, David B. Smith, Nick Cammack, Klaus Klumpp, Julian Symons,
Tópico(s)HIV/AIDS drug development and treatment
Resumoβ-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) replication in the subgenomic HCV replicon system, and its corresponding 5′-triphosphate is a potent inhibitor of the HCV RNA polymerase in vitro. In this study the formation of PSI-6130-triphosphate was characterized in primary human hepatocytes. PSI-6130 and its 5′-phosphorylated derivatives were identified, and the intracellular concentrations were determined. In addition, the deaminated derivative of PSI-6130, β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine (RO2433, PSI-6026) and its corresponding phosphorylated metabolites were identified in human hepatocytes after incubation with PSI-6130. The formation of the 5′-triphosphate (TP) of PSI-6130 (PSI-6130-TP) and RO2433 (RO2433-TP) increased with time and reached steady state levels at 48 h. The formation of both PSI-6130-TP and RO2433-TP demonstrated a linear relationship with the extracellular concentrations of PSI-6130 up to 100 μm, suggesting a high capacity of human hepatocytes to generate the two triphosphates. The mean half-lives of PSI-6130-TP and RO2433-TP were 4.7 and 38 h, respectively. RO2433-TP also inhibited RNA synthesis by the native HCV replicase isolated from HCV replicon cells and the recombinant HCV polymerase NS5B with potencies comparable with those of PSI-6130-TP. Incorporation of RO2433-5′-monophosphate (MP) into nascent RNA by NS5B led to chain termination similar to that of PSI-6130-MP. These results demonstrate that PSI-6130 is metabolized to two pharmacologically active species in primary human hepatocytes. β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) replication in the subgenomic HCV replicon system, and its corresponding 5′-triphosphate is a potent inhibitor of the HCV RNA polymerase in vitro. In this study the formation of PSI-6130-triphosphate was characterized in primary human hepatocytes. PSI-6130 and its 5′-phosphorylated derivatives were identified, and the intracellular concentrations were determined. In addition, the deaminated derivative of PSI-6130, β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine (RO2433, PSI-6026) and its corresponding phosphorylated metabolites were identified in human hepatocytes after incubation with PSI-6130. The formation of the 5′-triphosphate (TP) of PSI-6130 (PSI-6130-TP) and RO2433 (RO2433-TP) increased with time and reached steady state levels at 48 h. The formation of both PSI-6130-TP and RO2433-TP demonstrated a linear relationship with the extracellular concentrations of PSI-6130 up to 100 μm, suggesting a high capacity of human hepatocytes to generate the two triphosphates. The mean half-lives of PSI-6130-TP and RO2433-TP were 4.7 and 38 h, respectively. RO2433-TP also inhibited RNA synthesis by the native HCV replicase isolated from HCV replicon cells and the recombinant HCV polymerase NS5B with potencies comparable with those of PSI-6130-TP. Incorporation of RO2433-5′-monophosphate (MP) into nascent RNA by NS5B led to chain termination similar to that of PSI-6130-MP. These results demonstrate that PSI-6130 is metabolized to two pharmacologically active species in primary human hepatocytes. Hepatitis C is a major health problem affecting ∼170 million people worldwide of which around 3 million chronically infected patients reside within the United States (1Alter M.J. Kruszon-Moran D. Nainan O.V. McQuillan G.M. Gao F. Moyer L.A. 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Virol. 2003; 77: 2029-2037Crossref PubMed Scopus (72) Google Scholar, 6Ma H. Leveque V. De Witte A. Li W. Hendricks T. Clausen S.M. Cammack N. Klumpp K. Virology. 2005; 332: 8-15Crossref PubMed Scopus (59) Google Scholar). The HCV NS5B protein, the RNA-dependent RNA polymerase, is the catalytic subunit of the HCV replication complex and is responsible for the synthesis of the RNA progeny and, hence, is a prime target of anti-viral inhibition. Nucleoside analogs have been established as successful antiviral agents targeting the active site of DNA polymerases for the treatment of other viral diseases, including human immunodeficiency virus, hepatitis B virus, and herpes simplex virus (7Vivet-Boudou V. Didierjean J. Isel C. Marquet R. Cell. Mol. Life Sci. 2006; 63: 163-186Crossref PubMed Scopus (60) Google Scholar). The majority of marketed antiviral nucleoside analogs need to be converted to the active 5′-triphosphate forms in the target cells. These nucleotide triphosphate analogs then serve as alternative substrates for the viral DNA polymerases and compete with the incorporation of the corresponding natural nucleotide triphosphates. Upon incorporation by the viral DNA polymerases, the lack of the 3′-hydroxyl group in the deoxyribose moiety leads to the termination of the nascent viral DNA (chain termination). In the past few years a number of ribonucleoside analogs with 2′-C-methyl, 2′-O-methyl, or 4′-azido substituents on the ribose moiety have been reported to be inhibitors of HCV replication in the subgenomic replicon system (8Carroll S.S. Tomassini J.E. Bosserman M. Getty K. Stahlhut M.W. Eldrup A.B. Bhat B. Hall D. Simcoe A.L. LaFemina R. 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, 9Klumpp K. Leveque V. Le Pogam S. Ma H. Jiang W.R. Kang H. Granycome C. Singer M. 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Chem. 2004; 47: 5284-5297Crossref PubMed Scopus (190) Google Scholar, 12Le Pogam S. Jiang W.R. Leveque V. Rajyaguru S. Ma H. Kang H. Jiang S. Singer M. Ali S. Klumpp K. Smith D. Symons J. Cammack N. Najera I. Virology. 2006; 351: 349-359Crossref PubMed Scopus (118) Google Scholar, 13Smith D.B. Martin J.A. Klumpp K. Baker S.J. Blomgren P.A. Devos R. Granycome C. Hang J. Hobbs C.J. Jiang W.R. Laxton C. Pogam S.L. Leveque V. Ma H. Maile G. Merrett J.H. Pichota A. Sarma K. Smith M. Swallow S. Symons J. Vesey D. Najera I. Cammack N. Bioorg. Med. Chem. Lett. 2007; 17: 2570-2576Crossref PubMed Scopus (88) Google Scholar). Prodrugs of two nucleoside analogs, 2′-C-methylcytidine (NM107) and 4′-azidocytidine (R1479), have successfully progressed into clinical development and shown efficacy in HCV-infected patients (14Afdhal, N., Rodriguez-Torres, M., Lawitz, E., Godofsky, E., Chao, G., Fielman, B., Knox, S., and N., B. (2005) in 40th Annual Meeting of the European Association for the Study of the Liver, April 13–17, 2005, European Association for the Study of the Liver (EASL), Paris, abstract 93Google Scholar, 15Roberts s. Cooksley G. Dore G. Robson R. Shaw D. Berns H. Brandl M. Fettner S. Hill G. Ipe D. Klumpp K. Mannino M. O'Mara E. Tu Y. Washington C. Hepatology. 2006; 44 (abstr): 692Google Scholar). The corresponding nucleotide triphosphate analogs are substrates for HCV polymerase NS5B and inhibit RNA synthesis activity of HCV NS5B in vitro. The incorporation of the nucleotide analogs into nascent HCV RNA strongly reduces the efficiency of further RNA elongation by NS5B, resulting in termination of the nascent RNA product. Therefore, these nucleoside analogs are non-obligatory chain terminators despite the presence of a 3′-hydroxyl group. Recently, β-d-2′-deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) has been identified as a potent and selective inhibitor of HCV replication in the subgenomic replicon system with little or no cytotoxicity in various human cell lines or bone marrow precursor cells (16Stuyver L.J. McBrayer T.R. Tharnish P.M. Clark J. Hollecker L. Lostia S. Nachman T. Grier J. Bennett M.A. Xie M.Y. Schinazi R.F. Morrey J.D. Julander J.L. Furman P.A. Otto M.J. Antiviral Chem. Chemother. 2006; 17: 79-87Crossref PubMed Scopus (97) Google Scholar). The corresponding triphosphate of PSI-6130 is an inhibitor of HCV NS5B competitive with natural CTP (17Murakami E. Bao H. Ramesh M. McBrayer T.R. Whitaker T. Micolochick Steuer H.M. Schinazi R.F. Stuyver L.J. Obikhod A. Otto M.J. Furman P.A. Antimicrob. Agents Chemother. 2007; 51: 503-509Crossref PubMed Scopus (86) Google Scholar). Conversion to the active 5′-triphosphate form by cellular kinases is an important part of the mechanism of action for nucleoside analogs. In this study we determined the metabolism of β-d-2′-deoxy-2′-fluoro-2′-C-methylcytidine in primary human hepatocytes isolated from several donors. We show that β-d-2′-deoxy-2′-fluoro-2′-C-methylcytidine was converted to β-d-2′-deoxy-2′-fluoro-2′-C-methylcytidine 5′-triphosphate and β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine 5′-triphosphate via deamination of the phosphorylated cytidylates. Furthermore, we determined the kinetics of the formation of the two active triphosphates and the potency of the two triphosphates against the native HCV replicase and NS5B as well as the molecular mechanism of action of the two triphosphates. Compounds—β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) was provided by Pharmasset, Inc. (18Clark J.L. Hollecker L. Mason J.C. Stuyver L.J. Tharnish P.M. Lostia S. McBrayer T.R. Schinazi R.F. Watanabe K.A. Otto M.J. Furman P.A. Stec W.J. Patterson S.E. Pankiewicz K.W. J. Med. Chem. 2005; 48: 5504-5508Crossref PubMed Scopus (183) Google Scholar). A stock solution of 10 mm PSI-6130 was prepared in Dulbecco's phosphate-buffered saline and stored at –20 °C. Tritium-labeled PSI-6130 was synthesized at Roche Palo Alto LLC. The tritiated compound was dissolved in 50% (v/v) ethanol at the concentration of 0.97 mCi/ml with a specific activity of 25.78 Ci/mmol. The stock solution was stored at –20 °C. The phosphorylated derivatives of PSI-6130, namely PSI-6130-MP, -DP, and -TP, were provided by Pharmasset, Inc. β-d-2′-Deoxy-2′-fluoro-2′-C-methyluridine (RO2433) was synthesized at Roche Palo Alto LLC. RO2433-MP, -DP, and -TP were synthesized by TriLink BioTechnologies (San Diego, CA). Compound stock solutions were prepared in nuclease-free H2O and stored at –20 °C. 3′-dCTP was purchased from TriLink BioTechnologies. Cell Culture of Primary Human Hepatocytes—Plated fresh human hepatocytes or hepatocyte suspensions were obtained either from CellzDirect, Inc. or from In Vitro Technologies, Inc. Fresh human hepatocytes obtained from each company were plated or cultured on 6-well collagen coated plates (BD Biosciences #356400) at 1.5 million cells per well using complete serum containing medium obtained from the respective companies. Cells were allowed to recover for at least 18 h before the addition of the compound. All incubations were carried out at 37 °C in a humidified 5% CO2 atmosphere. To determine the time course of uptake and phosphorylation of PSI-6130, human primary hepatocytes were incubated with 3H-labeled PSI-6130 at a final concentration of 2 μm and 10 μCi/ml. The compound was added 72, 48, 24, 16, 6 and 1 h before cell harvesting. All time points and untreated cell controls were set up in duplicates. To determine the dose response of the phosphorylation of PSI-6130, human primary hepatocytes were incubated with 3H-labeled PSI-6130 at 0, 2, 10, 25, 50, 100, and 250 μm for 24 h. Final concentrations of PSI-6130 were achieved by supplementing 3H-labeled PSI-6130 with non-radiolabeled PSI-6130. Duplicate cell samples were harvested after 24 h of incubation. To determine the half-life of the triphosphates of PSI-6130 and RO2433, human primary hepatocytes were incubated for 24 h with 3H-labeled PSI-6130 at 2 μm and 10 μCi/ml. The cell monolayer was washed once with the cell culture medium without PSI-6130 and then incubated with fresh medium without PSI-6130 at 0-, 0.5-, 1-, 2-, 4-, 6-, 8-, 24-, 48-, and 72-h time points after the removal of PSI-6130. Duplicate cell samples were set up for each time point. The viable cell numbers of the untreated cell controls for each experiment were determined at the end of the experiment using the trypan blue exclusion method. Preparation of Cell Extract for High Performance Liquid Chromatography (HPLC) Analysis—At the time of cell harvest the cell culture medium was aspirated, and the cells were washed once with cold phosphate-buffered saline. The cells were scraped into 1 ml of pre-chilled 60% (v/v) methanol and extracted in methanol for 24 h at –20 °C. The extracted samples were then centrifuged at 10,000 × g for 15 min to remove cell debris. The supernatant was transferred to new tubes and evaporated in a speed vacuum at room temperature. The pellets were stored at –80 °C until analysis. The dried pellets of cell extracts were dissolved in H2O and filtered though a nanosep MF centrifugal device (Pall Life Sciences #ODM02C34). Before HPLC analysis, cell extract samples were spiked with unlabeled reference standards PSI-6130, RO2433, and their phosphorylated derivatives. HPLC—The phosphorylated derivatives of PSI-6130 were separated by ion exchange HPLC with a Whatman Partisil 10 SAX (4.6 × 250 mm) column coupled to a radiometric detector (β-RAM, IN/US Systems, Inc.). The mobile phase gradient changed linearly from 0% buffer A (H2O) to 100% buffer B (0.5 m KH2PO4 + 0.8 m KCl) between 4 and 8 min. 100% buffer B ran from 8 to 18 min and changed back to 100% A in 1 min. Buffer A ran until 25 min. The flow rate was 1 ml/min. A ratio of 5:1 Flo Scint IV or Ultima-Flo™ AP (PerkinElmer Life Sciences) to column eluent was used for the detection of radiolabeled species in the β-RAM detector (IN/US Systems, Inc.). The separation of PSI-6130 and RO2433 was performed by reverse phase chromatography with a Zorbax SB-C8 column (4.6 × 250 mm, 5 μm) coupled to a radiometric detector (β-RAM). The gradient changed linearly from 100% buffer A (0.01 m heptane sulfonic acid, sodium salt, 0.1% (v/v) acetic acid in water) to 10% buffer B (0.01 m heptane sulfonic acid sodium salt, 0.1% (v/v) acetic acid in 1:1 methanol water) between 0 and 3 min and then changed linearly from 10% buffer B to 95% buffer B between 3 and 18 min. 95% buffer B ran from 18 to 22 min and changed back to 100% A in 0.1 min. Buffer A ran until 25 min. The flow rate was 1 ml/min. PSI-6130 and its intercellular metabolites were identified by comparison of the retention times of the intracellular species in the radiochromatogram with the retention times of nonradioactive reference standards spiked in the cell extract samples and detected by UV absorption at 270 nm. Acid Phosphatase Treatment of Cell Extracts—Hepatocyte cell extracts were incubated with acid phosphatase (Sigma #P-0157) at a final concentration of 0.05 mg/ml (23.9 units/ml) at 37 °C for 2.5 h to dephosphorylate any phosphorylated metabolites of PSI-6130. After digestion the samples were analyzed by reversed phase HPLC. HCV Replicon Assay—The 2209-23 cell line containing a bicistronic HCV subgenomic replicon (genotype 1b, Con1 strain), which expresses a Renilla luciferase reporter gene as an index of HCV RNA replication, has been described before (9Klumpp K. Leveque V. Le Pogam S. Ma H. Jiang W.R. Kang H. Granycome C. Singer M. Laxton C. Hang J.Q. Sarma K. Smith D.B. Heindl D. Hobbs C.J. Merrett J.H. Symons J. Cammack N. Martin J.A. Devos R. Najera I. J. Biol. Chem. 2006; 281: 3793-3799Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The analysis of inhibition of HCV replication by nucleoside analogs and IC50 determinations were performed as described (12Le Pogam S. Jiang W.R. Leveque V. Rajyaguru S. Ma H. Kang H. Jiang S. Singer M. Ali S. Klumpp K. Smith D. Symons J. Cammack N. Najera I. Virology. 2006; 351: 349-359Crossref PubMed Scopus (118) Google Scholar). HCV Replicase Assay—The membrane-associated native HCV replication complexes were isolated from 2209-23 replicon cell lines as described (6Ma H. Leveque V. De Witte A. Li W. Hendricks T. Clausen S.M. Cammack N. Klumpp K. Virology. 2005; 332: 8-15Crossref PubMed Scopus (59) Google Scholar). The inhibition of the RNA synthesis activity of the HCV replicases by PSI-6130-TP was determined as described (6Ma H. Leveque V. De Witte A. Li W. Hendricks T. Clausen S.M. Cammack N. Klumpp K. Virology. 2005; 332: 8-15Crossref PubMed Scopus (59) Google Scholar) except that 5 μl of cytoplasmic replicase complex (2.5 × 106 replicon cell equivalent) was added to a 20-μl reaction for 60 min. The inhibition of the RNA synthesis activity of the HCV replicases by RO2433-TP was determined in reactions containing 6.25 μl of cytoplasmic replicase complex (3.1 × 106 replicon cell equivalent), 50 mm HEPES, pH 7.5, 10 mm KCl, 10 mm dithiothreitol, 5 mm MgCl2, 20 μg/ml actinomycin D, 1 mm ATP, GTP, and CTP, 24 μCi of (0.4 μm) [α-33P]UTP (PerkinElmer #NEG607H), 1 units/μl SUPERase. In (Ambion), 10 mm creatine phosphate, 200 μg/ml creatine phosphokinase with or without the nucleotide triphosphate inhibitor in a final volume of 20 μl for 90 min. HCV Polymerase Assay—The inhibition potency of PSI-6130-TP on the RNA-dependent RNA polymerase activity of recombinant NS5B570-Con1 (genotype 1b, GenBank™ accession number AJ242654) was measured as the incorporation of radiolabeled nucleotide monophosphate into acid-insoluble RNA products as described (6Ma H. Leveque V. De Witte A. Li W. Hendricks T. Clausen S.M. Cammack N. Klumpp K. Virology. 2005; 332: 8-15Crossref PubMed Scopus (59) Google Scholar) with the following modifications; IC50 determinations were carried out using 200 nmin vitro transcribed complementary internal ribosome entry site RNA template, 1 μCi of tritiated UTP (42 Ci/mmol), 500 μm ATP, 500 μm GTP, 1 μm CTP, 1× TMDN buffer (40 mm Tris-HCl, pH 8.0, 4 mm MgCl2, 4 mm dithiothreitol, 40 mm NaCl) and 200 nm NS5B570-Con1. The inhibition potency of RO2433-TP was determined as described above with the following modification of NTP concentrations: 1 μCi of tritiated CTP (39 Ci/mmol), 500 μm ATP, 500 μm GTP, 1 μm UTP. The compound concentration at which the enzyme-catalyzed rate is reduced by 50% (IC50) was calculated using equation, Y=%Min+(%Max-%Min)1+XIC50(Eq. 1) where Y corresponds to the relative enzyme activity, % Min is the residual relative activity at saturating compound concentration, % Max is the relative maximum enzymatic activity, and X corresponds to the compound concentration. The apparent Michaelis constants (Km(app)) for UTP or CTP were measured using assay conditions above with the following modifications; Km(app) for CTP was measured using 2 μCi of tritiated UTP (0.93 μm), 4.07 μm unlabeled UTP, 50 μm ATP, 50 μm GTP, and 5 nm to 50 μm CTP; Km(app) for UTP was measured using 2 μCi of tritiated CTP (1.67 μm), 3.33 μm unlabeled CTP, 50 μm ATP, 50 μm GTP, and 5 nm to 50 μm UTP. Apparent Km(app) values were calculated by nonlinear fitting using Equation 2, Y=(Vmax(app))XKm(app)+X(Eq. 2) where Y corresponds to the rate of RNA synthesis by NS5B, Vmax(app) is the maximum rate or RNA synthesis at saturating substrate concentration, and X corresponds to CTP or UTP concentration. Ki values were derived from the Cheng-Prusoff Equation (Equation 3) for competitive inhibition, Ki=IC501+[NTP]Km(app)(Eq. 3) where [NTP] is the concentration of CTP or UTP, and Km(app) is the apparent Michaelis constant for CTP or UTP. Mean Ki values were averaged from independent measurements at 0.2, 1, 5, and 25 μm CTP or UTP concentrations in triplicate experiments. Gel-based Nucleotide Incorporation Assay—The RNA template-directed nucleotide incorporation and extension of nucleotide triphosphates and nucleotide triphosphate analogs by HCV polymerase was performed with a 19-nucleotide RNA oligo (5′-AUGUAUAAUUAUUGUAGCC-3′) under assay conditions as described (9Klumpp K. Leveque V. Le Pogam S. Ma H. Jiang W.R. Kang H. Granycome C. Singer M. Laxton C. Hang J.Q. Sarma K. Smith D.B. Heindl D. Hobbs C.J. Merrett J.H. Symons J. Cammack N. Martin J.A. Devos R. Najera I. J. Biol. Chem. 2006; 281: 3793-3799Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The incorporation and extension of CTP and CTP analogs were determined with 5′-end-radiolabeled GG primer and nucleotide triphosphates at the indicated concentrations. The incorporation and extension of UTP and UTP analogs were performed similarly with the same RNA oligo template, 5′-end-radiolabeled GGC primer and nucleotide triphosphates at the indicated concentrations. The radio-labeled RNA products were separated on a TBE-urea acrylamide gel and analyzed using phosphorimaging (GE Healthcare). Metabolic Profile of PSI-6130—Cellular extracts from primary human hepatocytes incubated with tritium-labeled β-d-2′-deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) were resolved by ion exchange HPLC. PSI-6130 and metabolites derived from PSI-6130 were identified by comparing the retention times of radiolabeled species with the retention times of unlabeled reference compounds (Fig. 1A). As shown in Fig. 1B, PSI-6130 (3.0 min) and the 5′-phosphorylated derivatives PSI-6130-DP (13.2 min) and PSI-6130-TP (16.8 min) were identified in human hepatocytes incubated with PSI-6130. In addition, metabolites with retention times corresponding to those of the deaminated product of PSI-6130, β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine (RO2433, 3.8 min) and its corresponding 5′-phosphates RO2433-DP (12.5 min) and RO2433-TP (14.8 min), were detected (Fig. 1B). The monophosphates of the cytidine and uridine analogs PSI-6130-MP and RO2433-MP were not separated sufficiently under the chromatography conditions and, therefore, co-eluted as a single radioactive peak at 10.6 min. It has been reported that 2′-O-methylcytidine was extensively metabolized to CTP and UTP due to deamination coupled with demethylation of the 2′-substituent or base swapping after glycosidic bond cleavage (19Tomassini J.E. Getty K. Stahlhut M.W. Shim S. Bhat B. Eldrup A.B. Prakash T.P. Carroll S.S. Flores O. MacCoss M. McMasters D.R. Migliaccio G. Olsen D.B. Antimicrob. Agents Chemother. 2005; 49: 2050-2058Crossref PubMed Scopus (37) Google Scholar). None of the intracellular metabolites of PSI-6130 was eluted with retention time corresponding to those of 2′-C-methyl-CTP, CTP, and UTP (data not shown). Therefore, there was no evidence for metabolism of PSI-6130 at the 2′-position or evidence for hydrolysis at the glycosidic bond. These data suggest that the primary routes of PSI-6130 metabolism in human hepatocytes were phosphorylation at the 5′-position and deamination at the base. The hepatocyte extracts were also analyzed by reversed phase HPLC to identify unphosphorylated metabolites of PSI-6130. Two unphosphorylated species with retention times corresponding to PSI-6130 (20.3 min) and its uridine metabolite RO2433 (11.9 min) were observed, with PSI-6130 being the predominant intracellular species (Fig. 2B). There was no evidence of formation of uracil, uridine, cytosine, cytidine, or 2′-deoxycytidine (data not shown). These data agree well with the ion exchange HPLC analysis result and suggest the absence of metabolism at the 2′ position and at the glycosidic bond. The phosphorylated metabolites, with the exception of PSI-6130-MP, were not resolved by reversed phase HPLC and co-eluted early as a single broad peak (Fig. 2, A, and B). Acid phosphatase treatment completely converted all the intracellular metabolites to PSI-6130 and RO2433. Therefore, all detected intracellular metabolites represent phosphorylated derivatives of PSI-6130 and RO2433. These results established that PSI-6130 could be phosphorylated to its pharmacologically active 5′-triphosphate analog and that PSI-6130 and/or its phosphates could be deaminated to the corresponding uridine analogs in primary human hepatocytes. In Vitro Potency of RO2433 and RO2433-TP—As described above, incubation of human hepatocytes with PSI-6130 resulted in the formation of substantial concentrations of the triphosphate of its uridine analog, RO2433-TP. Next we determined whether the PSI-6130-derived uridine analog RO2433 could inhibit HCV replication targeting NS5B polymerase. Huh7 cells containing a subgenomic genotype 1b Con1 strain HCV replicon were incubated with RO2433 or PSI-6130 for 72 h, and dose-dependent inhibition of luciferase reporter activity was determined. RO2433 did not inhibit the HCV replication in the HCV subgenomic replicon system at concentrations up to 100 μm, whereas PSI-6130 inhibited HCV replication with a mean IC50 of 0.6 μm under the same assay conditions (Table 1). The lack of potency in the replicon could be related to inefficient compound phosphorylation. To address whether the triphosphate of RO2433 directly inhibits the HCV RNA polymerase, the RNA synthesis activity of the native membrane-associated HCV replication complexes isolated from the same replicon cells was tested in the presence of RO2433-TP. RO2433-TP inhibited the RNA synthesis activity of HCV replicase with a mean IC50 of 1.19 μm, whereas PSI-6130-TP inhibited HCV replicase with a mean IC50 of 0.34 μm (Table 1). RO2433-TP also inhibited the RNA synthesis activity of the recombinant HCV Con1 NS5B on a heteropolymeric RNA template derived from the 3′-end of the negative strand of the HCV genome with an IC50 of 0.52 μm and Ki of 0.141 μm, as compared with an IC50 of 0.13 μm and Ki of 0.023 μm for PSI-6130-TP under the same assay conditions (Table 1). These results established that both RO2433-TP and PSI-6130-TP are intrinsically potent inhibitors of RNA synthesis by HCV polymerase.TABLE 1Potency of PSI-6130 and RO2433 and their 5′-triphosphatesCompoundIC50Ki NS5B GT1bKm NS5B GT1bReplicon GT1bNS5B GT1bReplicase GT1bμmμmμmPSI-61300.6 ± 0.04NDNDNDNDRO2433>100NDNDNDNDPSI-6130-TPND0.13 ± 0.010.34 ± 0.10.023 ± 0.0020.22 (CTP)RO2433-TPND0.52 ± 0.111.19 ± 0.090.141 ± 0.030.37 (UTP) Open table in a new tab To investigate the molecular mechanism of HCV polymerase inhibition by PSI-6130-TP and RO2433-TP, we measured their incorporation and chain -termination properties. The incorporation of nucleotide and nucleotide analogs by HCV polymerase NS5B was determined in a gel-based assay using a short RNA template (Fig. 3A). Incorporation of CMP, PSI-6130-MP, and 3′-dCMP was initiated from a 33P-labeled GG primer (Fig. 3A). Incorporated CMP (Fig. 3B, lane 6) could be further extended in the presence of the next nucleotide UTP (Fig. 3B, lanes 7–10). PSI-6130-TP and 3′-dCTP could also serve as substrates for HCV NS5B and were incorporated into the nascent RNA product (Fig. 3B, lane 11 and 16). After incorporation of PSI-6130-MP or 3′-dCMP, further extension in the presence of the next nucleotide UTP was completed blocked even when UTP was present at concentrations up to 1 mm (Fig. 3B, lane 12–15 and lanes 16–20, respectively). Incorporation of UMP, RO2433-MP, and 3′-dUMP was initiated from a 33P-labeled GGC primer using the same RNA template (Fig. 3A). Incorporated UMP (Fig. 3C, lane 6) could be further extended in the presence of the next nucleotide ATP (Fig. 3C, lanes 7–10). Full-length RNA product was observed in the presence of UTP and ATP but absence of CTP (Fig. 3C, lanes 7–10), possibly due to misincorporation by NS5B through G-U wobble base-pairing. HCV NS5B was also able to incorporate RO2433-MP (Fig. 3C, lane 11) and 3′-dUMP (Fig. 3C
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