Downloaded from
`
`http://www.jbc.org/
`
` by guest on January 13, 2015
`
`THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 41, pp. 29812–29820, October 12, 2007
`© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
`
`Characterization of the Metabolic Activation
`of Hepatitis C Virus Nucleoside Inhibitor
`␤-D-2ⴕ-Deoxy-2ⴕ-fluoro-2ⴕ-C-methylcytidine
`(PSI-6130) and Identification of a Novel Active
`5ⴕ-Triphosphate Species*
`
`Received for publication, June 27, 2007, and in revised form, August 6, 2007 Published, JBC Papers in Press, August 13, 2007, DOI 10.1074/jbc.M705274200
`Han Ma1, Wen-Rong Jiang, Nicole Robledo, Vincent Leveque, Samir Ali, Teresa Lara-Jaime,
`Mohammad Masjedizadeh, David B. Smith, Nick Cammack, Klaus Klumpp, and Julian Symons
`From the Roche Palo Alto LLC, Palo Alto, California 94304
`
`␤-D-2ⴕ-Deoxy-2ⴕ-fluoro-2ⴕ-C-methylcytidine (PSI-6130) is a
`potent inhibitor of hepatitis C virus (HCV) replication in the
`subgenomic HCV replicon system, and its corresponding
`5ⴕ-triphosphate is a potent inhibitor of the HCV RNA polymer-
`ase in vitro. In this study the formation of PSI-6130-triphos-
`phate was characterized in primary human hepatocytes. PSI-
`6130 and its 5ⴕ-phosphorylated derivatives were identified, and
`the intracellular concentrations were determined. In addition,
`the deaminated derivative of PSI-6130, ␤-D-2ⴕ-deoxy-2ⴕ-fluoro-
`2ⴕ-C-methyluridine (RO2433, PSI-6026) and its corresponding
`phosphorylated metabolites were identified in human hepato-
`cytes after incubation with PSI-6130. The formation of the
`5ⴕ-triphosphate (TP) of PSI-6130 (PSI-6130-TP) and RO2433
`(RO2433-TP) increased with time and reached steady state lev-
`els at 48 h. The formation of both PSI-6130-TP and RO2433-TP
`demonstrated a linear relationship with the extracellular con-
`centrations of PSI-6130 up to 100 ␮M, 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 compa-
`rable with those of PSI-6130-TP. Incorporation of RO2433-5ⴕ-
`monophosphate (MP) into nascent RNA by NS5B led to chain
`termination similar to that of PSI-6130-MP. These results dem-
`onstrate 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 (1). The cur-
`rent standard treatment for hepatitis C consisting of pegylated
`interferon-␣and ribavirin only results in about a 50% sustained
`virological response in patients infected with genotype 1 hepa-
`
`* The costs of publication of this article were defrayed in part by the payment
`of page charges. This article must therefore be hereby marked “advertise-
`ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
`1 To whom correspondence should be addressed: Roche Palo Alto LLC, 3431
`Hillview Ave., Palo Alto, CA 94304. Tel.: 650-852-3190; Fax: 650-354-7554;
`E-mail: han.ma@roche.com.
`
`titis C virus (HCV),2 the most predominant genotype in the
`United States and Europe (2–4). New treatment options with
`improved clinical efficacy and greater tolerability are urgently
`needed. Novel antiviral agents targeting essential processes of
`HCV replication as part of optimized combination regimens
`could achieve increased clinical efficacy and potentially
`improved adverse event profiles as well as shortened treatment
`duration as compared with the current standard of care.
`HCV RNA replication is mediated by a membrane-associ-
`ated multiprotein replication complex (5, 6). 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 estab-
`lished 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 (7). 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 ter-
`mination 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 rep-
`lication in the subgenomic replicon system (8–13). 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 (14, 15). The corresponding nucleotide triphosphate
`analogs are substrates for HCV polymerase NS5B and inhibit
`RNA synthesis activity of HCV NS5B in vitro. The incorpora-
`tion of the nucleotide analogs into nascent HCV RNA strongly
`reduces the efficiency of further RNA elongation by NS5B,
`
`2 The abbreviations used are: HCV, hepatitis C virus; MP, 5⬘-monophosphate;
`DP, 5⬘-diphosphate; TP, 5⬘-triphosphate; HPLC, high performance liquid
`chromatography.
`
`29812 JOURNAL OF BIOLOGICAL CHEMISTRY
`
`VOLUME 282 • NUMBER 41 • OCTOBER 12, 2007
`
`IPR2018-00119
`
`Page 1 of 10
`
`I-MAK 1005
`
`

`

`Downloaded from
`
`http://www.jbc.org/
`
` by guest on January 13, 2015
`
`Metabolism and Mechanism of Action of ␤-D-2ⴕ-Deoxy-2ⴕ-fluoro-2ⴕ-C-methylcytidine
`
`resulting in termination of the nascent RNA product. There-
`fore, these nucleoside analogs are non-obligatory chain termi-
`nators despite the presence of a 3⬘-hydroxyl group.
`Recently, ␤-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 (16). The corresponding triphosphate of PSI-
`6130 is an inhibitor of HCV NS5B competitive with natural
`CTP (17). 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 metab-
`olism of ␤-D-2⬘-deoxy-2⬘-fluoro-2⬘-C-methylcytidine in pri-
`mary human hepatocytes isolated from several donors. We
`show that ␤-D-2⬘-deoxy-2⬘-fluoro-2⬘-C-methylcytidine was
`converted to ␤-D-2⬘-deoxy-2⬘-fluoro-2⬘-C-methylcytidine
`5⬘-triphosphate and ␤-D-2⬘-deoxy-2⬘-fluoro-2⬘-C-methyluri-
`dine 5⬘-triphosphate via deamination of the phosphorylated
`cytidylates. Furthermore, we determined the kinetics of the for-
`mation 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.
`
`EXPERIMENTAL PROCEDURES
`Compounds—␤-D-2⬘-Deoxy-2⬘-fluoro-2⬘-C-methylcytidine
`(PSI-6130) was provided by Pharmasset, Inc. (18). A stock solu-
`tion 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 com-
`pound 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 deriv-
`atives of PSI-6130, namely PSI-6130-MP, -DP, and -TP, were
`provided by Pharmasset, Inc. ␤-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 com-
`plete 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 ␮M and 10
`␮Ci/ml. The compound was added 72, 48, 24, 16, 6 and 1 h
`before cell harvesting. All time points and untreated cell con-
`trols 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 ␮M for 24 h.
`Final concentrations of PSI-6130 were achieved by supple-
`menting 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 ␮M and 10 ␮Ci/ml. The cell
`monolayer was washed once with the cell culture medium with-
`out 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 sam-
`ples 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 Sci-
`ences #ODM02C34). Before HPLC analysis, cell extract sam-
`ples 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
`(␤-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-FloTM AP (PerkinElmer Life Sciences) to
`column eluent was used for the detection of radiolabeled spe-
`cies in the ␤-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 ␮m) coupled to a radiometric detector
`(␤-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 intercel-
`lular metabolites were identified by comparison of the reten-
`tion times of the intracellular species in the radiochromato-
`gram with the retention times of nonradioactive reference
`standards spiked in the cell extract samples and detected by UV
`absorption at 270 nm.
`
`OCTOBER 12, 2007 • VOLUME 282 • NUMBER 41
`
`JOURNAL OF BIOLOGICAL CHEMISTRY 29813
`
`IPR2018-00119
`
`Page 2 of 10
`
`I-MAK 1005
`
`

`

`Downloaded from
`
`http://www.jbc.org/
`
` by guest on January 13, 2015
`
`Metabolism and Mechanism of Action of ␤-D-2ⴕ-Deoxy-2ⴕ-fluoro-2ⴕ-C-methylcytidine
`
`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 ana-
`lyzed 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 (9).
`The analysis of inhibition of HCV replication by nucleoside
`analogs and IC50 determinations were performed as described
`(12).
`HCV Replicase Assay—The membrane-associated native
`HCV replication complexes were isolated from 2209-23 repli-
`con cell lines as described (6). The inhibition of the RNA syn-
`thesis activity of the HCV replicases by PSI-6130-TP was deter-
`mined as described (6) except that 5 ␮l of cytoplasmic replicase
`complex (2.5 ⫻ 106 replicon cell equivalent) was added to a
`20-␮l 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 ␮l of cytoplasmic replicase com-
`plex (3.1 ⫻ 106 replicon cell equivalent), 50 mM HEPES, pH 7.5,
`10 mM KCl, 10 mM dithiothreitol, 5 mM MgCl2, 20 ␮g/ml acti-
`nomycin D, 1 mM ATP, GTP, and CTP, 24 ␮Ci of (0.4 ␮M)
`[␣-33P]UTP (PerkinElmer #NEG607H), 1 units/␮l SUPERase.
`In (Ambion), 10 mM creatine phosphate, 200 ␮g/ml creatine
`phosphokinase with or without the nucleotide triphosphate
`inhibitor in a final volume of 20 ␮l 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, GenBankTM acces-
`sion number AJ242654) was measured as the incorporation of
`radiolabeled nucleotide monophosphate into acid-insoluble
`RNA products as described (6) with the following modifica-
`tions; IC50 determinations were carried out using 200 nM in
`vitro transcribed complementary internal ribosome entry site
`RNA template, 1 ␮Ci of tritiated UTP (42 Ci/mmol), 500 ␮M
`ATP, 500 ␮M GTP, 1 ␮M 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 fol-
`lowing modification of NTP concentrations: 1 ␮Ci of tritiated
`CTP (39 Ci/mmol), 500 ␮M ATP, 500 ␮M GTP, 1 ␮M UTP. The
`compound concentration at which the enzyme-catalyzed rate is
`reduced by 50% (IC50) was calculated using equation,
`共% Max ⫺ % Min兲
`X
`
`Y ⫽ % Min ⫹
`
`冉1 ⫹
`
`共IC50兲冊
`
`(Eq. 1)
`
`where Y corresponds to the relative enzyme activity, % Min is
`the residual relative activity at saturating compound concentra-
`tion, % 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 ␮Ci of
`
`tritiated UTP (0.93 ␮M), 4.07 ␮M unlabeled UTP, 50 ␮M ATP, 50
`␮M GTP, and 5 nM to 50 ␮M CTP; Km(app) for UTP was meas-
`ured using 2 ␮Ci of tritiated CTP (1.67 ␮M), 3.33 ␮M unlabeled
`CTP, 50 ␮M ATP, 50 ␮M GTP, and 5 nM to 50 ␮M UTP. Appar-
`ent Km(app) values were calculated by nonlinear fitting using
`Equation 2,
`
`Y ⫽
`
`共Vmax共app兲兲 X
`Km共app兲 ⫹ X
`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,
`
`(Eq. 2)
`
`(Eq. 3)
`
`Ki ⫽
`
`冉1 ⫹
`
`冊
`
`IC50
`关NTP兴
`Km共app兲
`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 ␮M CTP or UTP concentrations in triplicate
`experiments.
`Gel-based Nucleotide Incorporation Assay—The RNA tem-
`plate-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 (9). The incorporation and extension of
`CTP and CTP analogs were determined with 5⬘-end-radiola-
`beled 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 nucleo-
`tide triphosphates at the indicated concentrations. The radio-
`labeled RNA products were separated on a TBE-urea acrylam-
`ide gel and analyzed using phosphorimaging (GE Healthcare).
`
`RESULTS
`Metabolic Profile of PSI-6130—Cellular extracts from
`primary human hepatocytes incubated with tritium-labeled
`␤-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 reten-
`tion 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 identi-
`fied in human hepatocytes incubated with PSI-6130. In addi-
`tion, metabolites with retention times corresponding to
`those of the deaminated product of PSI-6130, ␤-D-2⬘-deoxy-
`2⬘-fluoro-2⬘-C-methyluridine (RO2433, 3.8 min) and its cor-
`responding 5⬘-phosphates RO2433-DP (12.5 min)
`and
`RO2433-TP (14.8 min), were detected (Fig. 1B). The mono-
`phosphates of the cytidine and uridine analogs PSI-6130-MP
`and RO2433-MP were not separated sufficiently under the
`
`29814 JOURNAL OF BIOLOGICAL CHEMISTRY
`
`VOLUME 282 • NUMBER 41 • OCTOBER 12, 2007
`
`IPR2018-00119
`
`Page 3 of 10
`
`I-MAK 1005
`
`

`

`Metabolism and Mechanism of Action of ␤-D-2ⴕ-Deoxy-2ⴕ-fluoro-2ⴕ-C-methylcytidine
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on January 13, 2015
`
`FIGURE 2. Identification of PSI-6130 and its metabolites by reversed
`phase HPLC and acid phosphatase treatment. A, reversed phase HPLC sep-
`aration of standard compounds: PSI-6130 (a, 20 min), RO2433 (b, 11.7 min),
`PSI-6130-MP (c, 7.5 min), (d, PSI-6130-DP, PSI-6130-TP, RO2433-MP, RO2433-
`DP, and RO2433-TP). B, reversed phase HPLC profile of an extract from hepa-
`tocytes incubated with 25 ␮M extracellular PSI-6130 for 24 h. C, reversed
`phase HPLC profile of the same extract after acid phosphatase treatment.
`
`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 deter-
`mined 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 rep-
`lication in the HCV subgenomic replicon system at concentra-
`tions up to 100 ␮M, whereas PSI-6130 inhibited HCV replica-
`tion with a mean IC50 of 0.6 ␮M 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 iso-
`lated 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 ␮M, whereas PSI-
`6130-TP inhibited HCV replicase with a mean IC50 of 0.34 ␮M
`(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 ␮M and Ki of 0.141 ␮M, as
`compared with an IC50 of 0.13 ␮M and Ki of 0.023 ␮M 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.
`
`FIGURE 1. Ion exchange HPLC profile of an extract of primary human
`hepatocytes incubated with PSI-6130. A, HPLC separation and retention
`times of reference compounds PSI-6130 (a, 2.5 min), RO2433 (b, 3.4 min),
`RO2433-MP (c, 10.2 min), PSI-6130-MP (d, 10.4 min), RO2433-DP (e, 12.2 min),
`PSI-6130-DP (f, 12.8 min), RO2433-TP (g, 14.4 min), and PSI-6130-TP (h, 16.3
`min). B, HPLC profile of an extract from hepatocytes incubated with 3H-la-
`beled PSI-6130 at 2 ␮M for 24 h. The retention times in min of the intracellular
`species are indicated above the radioactive peaks. Intracellular PSI-6130 and
`its metabolites were identified by comparing the retention times of the radio-
`active peaks with those of the UV absorption peaks of reference compounds.
`There is a calibrated 0.3– 0.4-min delay in the retentions times of radioactive
`trace compared with those of UV trace due to sample traveling from UV
`detector to radiometric detector.
`
`chromatography conditions and, therefore, co-eluted as a sin-
`gle 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⬘-sub-
`stituent or base swapping after glycosidic bond cleavage (19).
`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 evi-
`dence 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 cor-
`responding 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 evi-
`dence 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 metabo-
`lites to PSI-6130 and RO2433. Therefore, all detected intracel-
`lular 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 phos-
`phates could be deaminated to the corresponding uridine ana-
`logs in primary human hepatocytes.
`
`OCTOBER 12, 2007 • VOLUME 282 • NUMBER 41
`
`JOURNAL OF BIOLOGICAL CHEMISTRY 29815
`
`IPR2018-00119
`
`Page 4 of 10
`
`I-MAK 1005
`
`

`

`Metabolism and Mechanism of Action of ␤-D-2ⴕ-Deoxy-2ⴕ-fluoro-2ⴕ-C-methylcytidine
`
`TABLE 1
`Potency of PSI-6130 and RO2433 and their 5ⴕ-triphosphates
`Values presented are the mean ⫾ S.D. from greater than three independent experiments. ND, not determined.
`IC50
`Compound
`NS5B GT1b
`␮M
`ND
`ND
`0.13 ⫾ 0.01
`0.52 ⫾ 0.11
`
`PSI-6130
`RO2433
`PSI-6130-TP
`RO2433-TP
`
`Replicon GT1b
`
`0.6 ⫾ 0.04
`⬎100
`ND
`ND
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on January 13, 2015
`
`Ki NS5B GT1b
`
`Km NS5B GT1b
`
`␮M
`ND
`ND
`0.023 ⫾ 0.002
`0.141 ⫾ 0.03
`
`␮M
`ND
`ND
`0.22 (CTP)
`0.37 (UTP)
`
`Replicase GT1b
`
`ND
`ND
`0.34 ⫾ 0.1
`1.19 ⫾ 0.09
`
`FIGURE 3. PSI-6130-TP and RO2433-TP are substrates of HCV polymerase
`and chain-terminators. A, sequence of the RNA template and primers. B, the
`incorporation of CTP and CTP analogs was initiated with GG primer according
`as described under “Experimental Procedures.” The nucleotide triphosphates
`included in the reactions were as follows: 10 ␮M CTP, ATP, and UTP (lane 1); 2,
`10, 100, and 1000 ␮M UTP (lanes 2–5); 10 ␮M CTP without UTP (lane 6) or with
`2, 10, 100, and 1000 ␮M UTP (lanes 7–10); 10 ␮M PSI-6130-TP without UTP (lane
`11) or with 2, 10, 100, and 1000 ␮M UTP (lanes 12–15); 10 ␮M 3⬘-dCTP without
`UTP (lane 16) or with 2, 10, 100, and 1000 ␮M UTP (lane 17–20). FL, full-length.
`C, incorporation of UTP and UTP analogs initiated with GGC primer. The
`nucleotide triphosphates included in the reactions were as follows: 100
`␮M CTP, ATP, and UTP (lane 1); 20, 50, and 200 ␮M ATP (lanes 2–5); 100 ␮M
`UTP without ATP (lane 6) or with 2, 20, 50, and 200 ␮M ATP (lanes 7–10); 100
`␮M RO2433-TP without ATP (lane 11) or with 20, 50, and 200 ␮M ATP (lanes
`12–15); 100 ␮M 3⬘-dUTP without ATP (lane 16) or with 20, 50, and 200 ␮M
`ATP (lanes 17–20).
`
`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 incorpo-
`ration of nucleotide and nucleotide analogs by HCV polymer-
`ase 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 sub-
`strates 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). Incorpo-
`rated 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, lane 16) but was unable to
`further extend the incorporated RO2433-MP and 3⬘-dUMP in
`the presence of the next nucleotide ATP (Fig. 3C, lanes 12–15
`and lanes 17–20, respectively). The control samples with GG
`primer and UTP-only as well as GGC primer and ATP-only did
`not lead to further extension of the primers, suggesting the
`incorporation of PSI-6130-MP and RO2433-MP was base-spe-
`cific. Taken together, these results demonstrate that PSI-
`6130-TP and RO2433-TP serve as alternative substrates for
`HCV NS5B and act as functional chain terminators once incor-
`porated into nascent RNA. While this manuscript was under
`preparation it was reported that the incorporation of PSI-
`6130-MP into the nascent RNA by HCV polymerase led to
`chain termination, in agreement with our observations (17).
`Kinetics of Phosphorylation of PSI-6130 in Primary Human
`Hepatocytes—To determine the steady state level of the two
`triphosphates in hepatocytes after exposure to PSI-6130, pri-
`mary human hepatocytes from 4 different donors were incu-
`bated with 2 ␮M PSI-6130 for up to 72 h. The uptake of PSI-6130
`by human hepatocytes was fast, and total intracellular activity
`reached steady state levels at 1 h ofPSI-6130 incubation, the
`earliest time point in this study (Table 2). PSI-6130-TP was
`detectable in hepatocytes from all 4 donors at 6 h after PSI-6130
`incubation and increased with time to reach steady state levels
`
`29816 JOURNAL OF BIOLOGICAL CHEMISTRY
`
`VOLUME 282 • NUMBER 41 • OCTOBER 12, 2007
`
`IPR2018-00119
`
`Page 5 of 10
`
`I-MAK 1005
`
`

`

`Metabolism and Mechanism of Action of ␤-D-2ⴕ-Deoxy-2ⴕ-fluoro-2ⴕ-C-methylcytidine
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on January 13, 2015
`
`FIGURE 4. Time course of uptake and metabolism of PSI-6130. A, time
`course of uptake of PSI-6130 and formation of PSI-6130-TP and RO2433-TP.
`B, time course of formation of PSI-6130-MP ⫹ RO2433-TP, PSI-6130-DP, and
`RO2433-TP. The data show mean values from four experiments performed
`with primary hepatocytes from four donors.
`
`PSI-6130-TP and RO2433-TP were quantified at different time
`points up to 72 h (see “Experimental Procedures”).
`The PSI-6130-TP level remained constant up to 1 h after the
`removal of extracellular PSI-6130. Thereafter, PSI-6130-TP
`decreased after a single-phase exponential decay kinetics pat-
`tern with a mean half-life of 4.7 ⫾ 0.6 h (Fig. 6, Table 4). The
`PSI-6130-TP level was below quantification limit at 48 h after
`the removal of extracellular PSI-6130. The RO2433-TP level
`increased slightly to reach steady state level at 1–2 h after extra-
`cellular PSI-6130 removal and remained at steady state level for
`up to 6 h before decreasing toward base-line levels. RO2433-TP
`decreased with a mean half-life of 38.1 ⫾ 16.1 h (Fig. 6, Table 4).
`The levels of unphosphorylated species PSI-6130 and RO2433
`decreased rapidly after removal of extracellular RO1656 (data
`
`TABLE 2
`Time course of uptake and phosphorylation of PSI-6130
`Levels of total intracellular species, PSI-6130-TP, and RO2433-TP were determined
`in hepatocytes incubated with 2 ␮M PSI-6130 for the indicated lengths of time. Data
`shown are mean ⫾ S.D. derived from four independent experiments using hepato-
`cytes from four donors. BQL, below the quantification limit.
`RO2433-TP
`Time
`Total intracellular species
`PSI-6130-TP
`pmol/million cells
`h
`pmol/million cells
`pmol/million cells
`BQL
`1
`30.4 ⫾ 18.8
`BQL
`0.3 ⫾ 0.01a
`6
`32.9 ⫾ 16.8
`0.6 ⫾ 0.5
`0.6 ⫾ 0.3
`16
`34.4 ⫾ 20.8
`0.7 ⫾ 0.6
`1.3 ⫾ 1.1
`24
`36.4 ⫾ 21.3
`1.1 ⫾ 0.7
`2.0 ⫾ 1.1
`48
`31.9 ⫾ 17.1
`1.3 ⫾ 0.6
`2.0 ⫾ 1.3
`72
`34.6 ⫾ 15.4
`1.0 ⫾ 0.4
`a Mean ⫾ S.D. derived from 2 donors as RO2433-TP was only detectable in 2 out of
`4 donors at the 6-h time point.
`
`at 24–48 h. The formation of the triphosphate of the uridine
`metabolite, RO2433-TP, demonstrated a delayed time course
`relative to that of PSI-6130-TP. RO2433-TP was detectable in
`hepatocytes from only 2 of 4 donors at 6 h and in hepatocytes of
`all 4 donors at 16 h. RO2433-TP formation reached steady
`state levels at 48–72 h. RO2433-TP concentrations were
`lower than those of PSI-6130-TP at time points earlier than
`16 h but surpassed those of PSI-6130-TP at 24 h and beyond.
`The mean steady state level concentrations of PSI-6130-TP
`and RO2433-TP after incubation with 2 ␮M PSI-6130 were
`1.3 ⫾ 0.6 and 2.0 ⫾ 1.1 pmol/106 cells at 48 h, respectively.
`Unchanged PSI-6130 was the major intracellular species at
`all time points tested after incubation of human hepatocytes
`with radiolabeled PSI-6130 (Fig. 4).
`To determine whether increasing the exposure of hepato-
`cytes to PSI-6130 will lead to increased uptake of PSI-6130 and
`formation of PSI-6130-TP and RO2433-TP, primary human
`hepatocytes from 3 different donors were incubated with PSI-
`6130 at different concentrations up to a maximal concentration
`of 250 ␮M for 24 h. Total intracellular species, determined by
`total intracellular radioactivity in the cell extracts, increased
`linearly with the extracellular PSI-6130 up to 250 ␮M (Table 3).
`The mean total intracellular species at 250 ␮M extracellular
`PSI-6130 reached 3591 pmol/106 cells, with the unphosphoryl-
`ated PSI-6130 being the predominant species (data not shown).
`The formation of both PSI-6130-TP and RO2433-TP increased
`linearly with the e