From the Departments of Biological Chemistry and
Structural Biology, Merck Research Laboratories, West Point,
Pennsylvania 19486, the
Department of
Medicinal Chemistry, Merck Research Laboratories, Rahway, New
Jersey 07065, the ** Istituto di Ricerche di Biologia
Molecolare, P. Angeletti, Pomezia (Roma) 00040, Italy, and
¶ Isis Pharmaceuticals, Carlsbad, California 92008
Received for publication, October 25, 2002, and in revised form, January 21, 2003
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ABSTRACT |
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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 vitro
enzyme assays. Thus, the 2'-modifications of natural substrate
nucleosides transform these molecules into potent inhibitors of HCV replication.
Hepatitis C virus (HCV)1
infection is the leading cause of sporadic, post-transfusion, non-A
non-B hepatitis (1, 2). 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 (3). Approximately 80% of
infected individuals progress to chronic infection. Long term chronic
HCV infection can lead to liver cirrhosis and to hepatocellular
carcinoma (4-6). Currently, the recommended therapy is treatment with
a combination of interferon Antiviral chemotherapies based on administration of analogs of
deoxynucleosides have been widely successful as treatment for HIV,
herpes virus, and hepatitis B infection (11, 12). 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 (13). The enzymatic activity of NS5B has been extensively characterized in vitro
(13-15, 29). 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 (16, 17). The antiviral effect of interferon 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 reactions in vitro. This study demonstrates the utility of
2'-substituted nucleosides in the inhibition of HCV RNA replication.
Materials--
Nucleotides,
2'-C-Methyladenosine triphosphate was synthesized according
to the general procedures previously described (21). 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 mM
to 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 1% to 95% methanol in 5 mM
triethylammonium 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 100%.
8-Bromo-2'-C-methyladenosine was synthesized from
2'-C-methyladenosine by addition of
N-bromosuccinimide in dimethylformamide. The crude product
was purified on silica gel using methanol/dichloromethane (1:9) as
eluent. 1H NMR
(Me2SO-d6):
[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.
Human DNA Polymerases--
DNA polymerase HCV NS5B Expression/Purification--
HCV (BK strain) NS5B NS5B Enzyme Assay on Template t500--
RNA polymerase activity
was determined in reactions catalyzed by NS5B Gel-based Incorporation Assay--
The incorporation into
synthetic RNA and extension of nucleoside analogs catalyzed by HCV NS5B
was determined in reactions utilizing 5'-end-labeled
oligoribonucleotides. The incorporation/extension of analogs of
adenosine triphosphate was analyzed in reactions on template 68N
(sequence 5'-AGAUGGCCCGGUUUUCCGGGCC-3'), which is designed
to fold into a hairpin structure with the first available template base
as a U (underlined in the sequence). The concentration of
oligonucleotide 68N was determined by absorbance at 260 nm and
the oligonucleotide was 5'-end-labeled with
[ Counterscreen Human DNA Polymerases--
Human DNA
polymerases In Situ Ribonuclease Protection Assay--
HBI10A cells (27)
were grown and assayed as previously
described.2 Replicon cells
were passaged at 1:5 and plated at a cell density of 40,000 cells/well in Cytostar plates (Amersham Biosciences) in complete
Dulbecco's modified Eagle's medium media containing 10% FBS and 0.8 mg/ml G418. Compound dissolved in Me2SO was added to
the cells at a final Me2SO concentration of 1% and
incubated for 24 h at 37 °C/5% CO2. Cells were
fixed in 10% formalin/phosphate-buffered saline, permeabilized in
0.25% Triton X-100, and then hybridized with an antisense
33P-labeled RNA probe (1.0-2.0 × 104
cpm/µl) in formamide hybridization buffer (Amersham Biosciences) at
50 °C overnight. The RNA probe was generated with T7 runoff transcription and had a sequence complementary to nucleotides 1184-1481 of the NS5B gene. Plates were treated with 20 µg/ml RNase
A at room temperature for 30 min, washed with 0.25× SSC buffer at room
temperature and then at 65 °C for 20 min each wash, and then counted
in a TopCount plate reader.
Cytotoxicity Assay--
Cytotoxicity was assayed as previously
described.2 The cells were plated in 96-well plates in
parallel at the same density, and the compound was added as above. At
the indicated times, Promega Cell Titer 96 Aqueous One Solution Reagent
(MTS) was added for 1 h at 37 °C/5% CO2 and
then absorbance was read at 490 nm in a plate reader.
Intracellular Metabolism Studies--
Two cell lines, Huh-7 and
HBI10A, were used for intracellular metabolism studies of
5-[3H]-2'-O-methylcytidine and
7-[3H]-2'-C-methyladenosine. Huh-7 is a human
hepatoma cell line, and HBI10A denotes a clonal line derived from Huh-7
cells that harbors the HCV bicistronic replicon. Huh-7 cells were
plated in complete Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and HBI10A cells in the same containing G418 (0.8 mg/ml) at 1.5 × 106 cells/60-mm dish such that cells
were 80% confluent at the time of compound addition. Tritiated
compound was incubated at 2 µM in the cell medium for 3 or 23 h. Cells were collected, washed with phosphate-buffered
saline, and counted. The cells were then extracted in 70% methanol, 20 mM EDTA, 20 mM EGTA, and centrifuged. The
lysate was dried, and radiolabeled nucleotides were analyzed using an
ion-pair reverse phase (C-18) HPLC on a Waters Millenium system
connected to an in-line Inhibition of NS5B Enzyme Activity--
NS5B-catalyzed
incorporation of nucleotides in reactions with template t500 generates
a copy-back or hairpin product (22), as previously described for other
RNA templates (28). The rate of product formation catalyzed by
NS5B Mode of Inhibition--
To determine the mode of inhibition by
2'-C-methyladenosine triphosphate and
2'-O-methylcytidine triphosphate of RNA synthesis catalyzed
by NS5B Gel-based Incorporation Assay--
To determine whether NS5B Inhibition of DNA Polymerases Nucleosides 2'-C-Methyladenosine and 2'-O-Methylcytidine Inhibit
HCV RNA Replication in Cells--
Nucleosides
2'-C-methyladenosine and 2'-O-methylcytidine were
tested for inhibitory activity in a cell-based replicon assay using a
stable Huh-7 human hepatoma cell line, which supports the replication
of HCV RNA and proteins. The effect of the nucleosides upon RNA
replication in a clonal line designated HBI10A (27) was detected by
in situ ribonuclease protection assay as previously described.2 Representative titrations of the compounds in
the replicon assay are shown in Fig. 4.
Both compounds were active in the assay at 24 h with
IC50 values of 0.3 µM for the
2'-C-methyladenosine and 21 µM for the
2'-O-methylcytidine (Table
II). The antiviral activity of both
compounds was observed in the absence of cytotoxicity in HBI10A cells
as measured in the MTS assay when tested up to 100 µM.
Intracellular Metabolism to the Active Triphosphate--
The
intracellular metabolism of the tritiated versions of
2'-C-methyladenosine and 2'-O-methylcytidine was
studied in Huh-7 and HBI10A cells. The compounds were incubated for 3 or 23 h at 2 µM prior to extraction and HPLC
analysis, as shown in Fig. 5. The results
are summarized in Table III.
2'-C-Methyladenosine was efficiently taken up
into the cells and converted to its corresponding triphosphate. In
contrast, incubation of HBI10A cells with
2'-O-methylcytidine yielded very little triphosphate and
suffered from extensive metabolism to species that are consistent with
(by comparison to retention times for appropriate controls) UTP and
CTP, indicating that this molecule was subject to deamination and base
swapping in the cell.
The advent of the cell-based, subgenomic, bicistronic replicon
system (16) as a means of assessing the replication of viral RNA within
the cellular environment has permitted the evaluation of analogs of
ribonucleosides to complement ongoing efforts aimed at identifying
inhibitors of purified NS5B in vitro. Screening of available
nucleosides for inhibition of viral replication in the replicon assay
identified two inhibitory compounds, 2'-C-methyladenosine and 2'-O-methylcytidine.
The triphosphates of 2'-C-methyladenosine and
2'-O-methylcytidine inhibit the catalytic activity of
purified HCV RNA polymerase with IC50 values of 1.9 and 3.8 µM, respectively (Table I). Forms of HCV RNA polymerase
having two different C-terminal truncations that were investigated have
significantly different catalytic efficiencies, with NS5B Analysis of the incorporation of the nucleoside analogs onto a growing
RNA strand was carried out using synthetic RNA templates that are
designed to fold into intramolecular hairpins. NS5B The nucleosides were converted intracellularly to the corresponding
triphosphates, which, in turn, functioned as specific inhibitors of HCV
RNA synthesis. The potency of inhibition of HCV replication observed in
replicon-containing cells correlated with the levels of triphosphates
formed intracellularly. A lesser amount of the intracellular
2'-O-methylcytidine triphosphate was detected than the
2'-C-methyladenosine triphosphate, and the difference is
reflected in the reduced potency of 2'-O-methylcytidine in replicon-containing cells, despite the equivalent inhibition of the
purified enzyme by the two triphosphates.
The potency of inhibition by 2'-C-methyladenosine in the
cell-based replicon assay (0.3 µM) is greater than the
potency of the corresponding triphosphate in the enzyme assay (1.9 µM). The greater potency in the cell-based assay likely
reflects a combination of the high intracellular concentration of the
corresponding triphosphate that is achieved (105 pmol/million cells in
the presence of 2 µM extracellular nucleoside) and the
fact that the analog acts as a functional chain terminator. Once the
nucleotide analog is incorporated into the replicon RNA, the resulting
truncated RNA chain, in the absence of a known proofreading activity,
is nonfunctional as a template for subsequent rounds of viral RNA
synthesis. However, in the enzyme assay, once the nucleotide analog has
been incorporated, the truncated RNA is still counted as the product.
Because the in vitro enzyme assay is performed under
conditions that differ from physiological, it is conceivable that the enzyme assay may not be truly representative of biological activity. Nonetheless, the use of in vitro enzyme assays is validated
by the demonstration of the inhibition of purified HCV NS5B by the triphosphates of nucleoside analogs that also are capable of inhibiting HCV replication in cell culture in the absence of cytotoxicity. A more
definitive correlation of enzyme inhibition with antiviral effect in
the replicon assay will be ascertained when resistant mutations are
identified that confer resistance both in vitro and in the
cell culture assay.
Whether the replicon assay will be a meaningful predictor of antiviral
activity in vivo has yet to be determined. Equally uncertain
at this point is the question of whether
2'-C-methyladenosine or 2'-O-methylcytidine
have pharmacokinetic and safety profiles that are sufficiently
attractive to warrant their development as HCV therapeutics. However,
the current work establishes the direct inhibition of HCV RNA
polymerase activity by 2'-modified nucleotides leading to inhibition of
HCV replication in cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2b and ribavirin, which results in a
sustained viral response in 40% of patients (7, 8). Investigational therapies using a combination of pegylated interferon and
ribavirin have lead to an sustained viral response in 54% of patients,
but the response rate (42%) of patients harboring HCV genotype 1 is lower (9, 10). Consequently, additional therapies for HCV infection are needed.
has been documented in
these lines (18).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
- or
-32P- and
-33P-labeled, were purchased from PerkinElmer Life
Sciences. Ultrapure nucleoside triphosphates were purchased from
Amersham 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 (19, 20).
The structures of the nucleoside analogs were verified by mass
spectrometry and gradient-enhanced homonuclear correlation NMR.
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).
was supplied by
T. Wang (Stanford University). DNA polymerase
was
purchased from AB Peptides (St. Louis, MO). DNA polymerase
was
supplied by W. Copeland (NIEHS, National Institutes of Health).
21
(GenBankTM accession number M58335) was expressed in
Escherichia coli BL21(DE3) harboring plasmid pT7(NS5B
21)
and purified as previously described (22). HCV (BK strain) NS5B
55
(23) was expressed in E. coli BL21(DE3) harboring plasmid
pT7(NS5B
55). Plasmid pT7(NS5B
55) was constructed from plasmid
pT7(NS5B
21) by introduction of a stop codon after Leu-537.
Protein expression and purification of HCV (BK strain) NS5B
55
followed the same protocol as that used for expression and purification
of HCV (BK strain) NS5B
21 (22). Protein concentration was determined
with use of quantitative amino acid analysis.
21 and NS5B
55 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 (22), 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 NS5B
21 or 25 nM NS5B
55 in a 50-µl
reaction containing 0.75 µg of t500, 20 mM Tris, pH 7.5, 80 mM KCl, 2 mM MgCl2, 5 mM DTT, 0.4 unit/µl RNasin (Promega), 0.2% polyethylene
glycol 8000, 50 µM EDTA, 1 µM NTPs (unless
otherwise noted), and ~1 µCi of either [
-32P]- or
[
-33P]GTP or -ATP. Reactions were initiated by the
addition of a mixture of NTPs, after preincubation of the other
reaction components for 30 min at RT. Reactions proceeded for 2 h
at RT and were quenched by addition of EDTA. Product formation was
determined by DE-81 filter binding (Whatman) as previously described
(22, 24). The inhibitor concentration at which the enzyme-catalyzed
rate is reduced by half (IC50) was determined by fitting
the relative rate data to the Hill equation,
where vi is the reaction velocity in the
presence of inhibitor, v0 is the reaction
velocity in the absence of inhibitor, and n is the Hill
coefficient. Data fitting was carried out with use of Kaleidagraph
(Synergy Software, Reading, PA).
(Eq. 1)
-32P]ATP in reactions catalyzed by T4 polynucleotide
kinase (US Biochemicals, Cleveland, OH or Invitrogen, Gaithersburg, MD)
as previously described (25). NS5B
55 (1 µM) was
preincubated with template 68N (600 nM) in reaction buffer
containing 20 mM Tris, pH 7.5, 80 mM KCl, 5 mM DTT, 50 µM EDTA, 2 mM
MgCl2, 0.4 unit/µl RNasin (Promega) for 30 min at room
temperature. Reactions were initiated by the addition of NTPs.
Reactions included 10 µM ATP and/or 2 µM
UTP or 1-50 µM 2'-C-methyladenosine
triphosphate with or without 2 µM UTP. Reactions were
allowed to proceed for 30 or 60 min, and then 5-µl aliquots were
removed and quenched with 15 µl of formamide gel load buffer. After
denaturing the RNA at 65 °C for 30 min, substrate and one or more
products were separated on 20% acrylamide-8 M urea gels
and analyzed using a PhosphorImager (Amersham Biosciences). In a
similar manner analogs of cytidine triphosphate were examined for
incorporation/extension in reactions with oligonucleotide 76N (sequence
5'-ACUGGGCCCGGUUUUCCGGGCC-3'). Oligoribonucleotides 68N and
76N were synthesized using 2'-acetate ester chemistry (Dharmacon,
Lafayette, CO), purified using denaturing PAGE gels, and deprotected
according to the manufacturer's instructions.
and
were assayed in reactions containing 1.6 µM deoxynucleoside triphosphates, including
[
-33P]dATP, 0.05 mg/ml gapped fish sperm DNA template,
0.005 unit/µl DNA polymerase
or 0.01 unit/µl
, 20 mM Tris-HCl, 50 µM EDTA, pH 7.5, 2 mM DTT, 10 mM MgCl2, and 200 µg/ml bovine serum albumin (26). Reactions catalyzed by DNA
polymerase
also contained 100 mM KCl. Reactions
proceeded for 1 h at 37 °C in a 50-µl reaction volume. Human
DNA polymerase
was assayed in reactions containing 20 mM Tris, pH 8, 2 mM
-mercaptoethanol, 10 mM MgCl2, 0.1 µg/µl bovine serum albumin,
0.4 µg/µl activated fish sperm DNA, 5 × 10
4
µg/µl polymerase
, and either 1 or 10 µM dNTPs,
including [
-33P]TTP. Reactions were quenched by the
addition of 20 µl of 0.5 M EDTA and analyzed for product
formation with use of DE-81 filter binding (22, 24).
-RAM scintillation detector (IN/US Systems).
The HPLC mobile phases consisted of (a) 10 mM
potassium phosphate with 2 mM tetrabutylammonium hydroxide
and (b) 50% methanol containing 10 mM potassium
phosphate with 2 mM tetrabutylammonium hydroxide. Peak
identification was made by comparison of retention times to standards.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
21 or NS5B
55 was reduced in the presence of either
2'-C-methyladenosine triphosphate or 2'-O-methylcytidine triphosphate (structures shown in Fig.
1), with IC50 values as shown
in Table I, as determined by monitoring the total incorporation of radiolabeled nucleotide as described under
"Experimental Procedures." The Hill coefficients did not significantly differ from unity. The potency of inhibition by either
nucleotide analog was not affected whether the radiolabeled nucleoside
triphosphate was GTP or ATP (data not shown), indicating that
replacement of the radiolabel by the nucleoside analog was not
responsible for the inhibition.
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Fig. 1.
Structures of (A)
2'-C-methyladenosine and (B)
2'-O-methylcytidine.
Inhibition of NS5B-catalyzed reactions in vitro by nucleoside analog
triphosphates
21, reactions were run in which the concentrations of ATP and
CTP were varied, respectively, holding the concentrations of the other
NTPs constant at concentrations above Km. Double-reciprocal plots of the data as shown in Fig.
2 indicated competitive inhibition of
activity by 2'-C-methyladenosine triphosphate and
2'-O-methylcytidine triphosphate with varying ATP and CTP, respectively. The Ki value as determined from a
replot of the slopes of the double-reciprocal plot was 0.9 µM for 2'-C-methyladenosine triphosphate and
0.3 µM for 2'-O-methylcytidine
triphosphate.
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Fig. 2.
Mode of inhibition by (A)
2'-C-methyladenosine triphosphate and
(B) 2'-O-methylcytidine triphosphate
of NS5B-catalyzed synthesis. Reactions included 250 nM
NS5B 21 and template t500 in reaction buffer as described under
"Experimental Procedures." A, reactions also included 20 µM of GTP, UTP, and CTP and from 1.5 to 30 µM ATP, and no (
), 0.75 (+), 1.5 (×), 3 (
), 4.5 (
), or 6 (
) µM 2'-C-methyladenosine
triphosphate. B, reactions included 20 µM of
GTP, UTP, and ATP and from 0.1 to 10 µM CTP, and 0 (
),
0.1 (
), 0.2 (+), 0.4 (×), 1 (
), 2 (
), or 4 (
)
µM 2'-O-methylcytidine triphosphate. Reactions
proceeded for 2 h at RT and were quenched by the addition of EDTA.
Product analysis was by DE-81 filter binding. Data were fit to a
competitive mechanism, with Ki values of 0.9 µM and 0.3 µM for
2'-C-methyladenosine triphosphate and
2'-O-methylcytidine triphosphate, respectively.
55
is capable of incorporating the nucleoside analogs into a growing RNA
strand, gel-based analyses of reactions using hairpin RNA templates
were carried out. The sequence of the RNA template (68N) was designed
such that an intramolecular hairpin could form, allowing the
incorporation of AMP followed by UMP. NS5B
55 showed a much greater
ability to incorporate nucleotides efficiently onto the hairpin RNA
substrates than did NS5B
21. The relatively low activity of NS5B
21
with the hairpin templates is likely a consequence of the low fraction
of catalytically competent NS5B
21 (~2%, Ref. 22) compared with
NS5B
55 (~40%, data not shown). The incorporation of AMP leads to
the appearance of a single product band (Fig.
3A, lane 3). In
reactions that included ATP and UTP, the product resulting from the
incorporation of AMP was completely extended by addition of UMP (Fig.
3A, lane 4). NS5B
55 was capable of
incorporating 2'-C-methyladenosine monophosphate onto the
3'-end of the RNA at the lowest nucleotide concentration tested, 1 µM. However, as shown in Fig. 3A (lanes
9-11), NS5B
55 could not add uridine monophosphate onto the
2'-C-methyladenosine-terminated template efficiently,
although a trace amount of extended product was evident. In a similar
manner, NS5B
55 was capable of incorporating 2'-O-methylcytidine monophosphate onto the 3'-end of RNA
hairpin 76N as shown in Fig. 3B. However, no detectable
extension product was visible when the next correct nucleoside
triphosphate, ATP, was added to the reaction.
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Fig. 3.
PhosphorImager results of the
incorporation/extension of nucleotide analogs. A,
2'-C-methyladenosine monophosphate. Lane 1 is a
control with no enzyme added. Reactions included 600 nM
5'-32P-end-labeled 68N (sequence shown as a hairpin
conformation), 1 µM NS5B 55 in reaction buffer as
described under "Experimental Procedures," and either no nucleoside
triphosphates (lane 2), 10 µM ATP (lane
3), 10 µM ATP and 2 µM UTP (lane
4), 2 µM UTP (lane 5), 1 µM
(lane 6), 10 µM (lane 7), or 50 µM (lane 8) 2'-C-methyladenosine
triphosphate. Lanes 9-11 contain the same concentrations of
2'-C-methyladenosine triphosphate as lanes 6-8
with 2 µM UTP. B,
2'-O-methylcytidine monophosphate. Lane 1 is a
control with no enzyme added. Reactions included 600 nM
5'-32P-end-labeled 76N (sequence shown as a hairpin
conformation), 1 µM NS5B
55 in reaction buffer, and
either no nucleoside triphosphates (lane 2), 10 µM CTP (lane 3), 10 µM CTP and 2 µM ATP (lane 4), 2 µM ATP
(lane 5), 1 µM (lane 6), 10 µM (lane 7), or 50 µM
(lane 8) 2'-O-methylcytidine triphosphate.
Lanes 9-11 contain the same concentrations of
2'-O-methylcytidine triphosphate as lanes 6-8
with 2 µM ATP. The results indicate that NS5B
55 is
capable of incorporating either 2'-C-methyladenosine
monophosphate or 2'-O-methylcytidine monophosphate onto the
3'-end of the synthetic RNA template but is not capable of efficiently
adding the next correct base onto the analog-terminated template.
,
, and
--
To determine
the specificity of inhibition the activity of human DNA
polymerases
,
, and
were monitored in vitro
in the presence of 2'-C-methyladenosine triphosphate and
2'-O-methylcytidine triphosphate. Less than 20% inhibition
of the activity of DNA polymerases
,
, or
was detected at 50 µM of either nucleoside triphosphate analog.
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Fig. 4.
Inhibition of the cell-based replicon
assay. HBI10A cells were incubated in the presence of the
indicated concentrations of 2'-C-methyladenosine ( ) or
2'-O-methylcytidine (
) for 24 h, and the level of
HCV RNA was determined by in situ ribonuclease protection
assay as described under "Experimental Procedures." The
curves represent the best fit of the data to Equation 1,
giving IC50 values of 0.31 µM for inhibition
by 2'-C-methyladenosine and 14 µM for
inhibition by 2'-O-methylcytidine. The signal:background
ratio in the assay was typically 25:1.
Inhibitory potency and toxicity of nucleoside analogs in the HCV
replicon assay in HB110A cells
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Fig. 5.
Analysis of intracellular nucleotides.
A, [8-3H]-2'-C-methyladenosine (2 µM) was incubated with Huh-7 cells in culture for 23 h, after which cells were collected and nucleotides were extracted and
chromatographed as described under "Experimental Procedures." The
elution time of a 2'-C-methyladenosine triphosphate standard
was the same as that of the last eluting peak on the
radiochromatograph. The two smaller peaks eluting at 22 and 10 min
probably correspond to the 2'-C-methyladenosine diphosphate
and monophosphate, respectively. Lysate corresponding to ~24,000
cells was analyzed per injection. B,
[5-3H]-2'-O-methylcytidine (2 µM) was incubated with Huh-7 cells in culture for 23 h, and radiolabeled nucleotides were extracted and analyzed by ion
pairing HPLC as described under "Experimental Procedures." Lysate
corresponding to ~2.4 × 106 cells was analyzed per
injection. Peak identification was made by comparing the retention
times of unlabeled compounds. Significant amounts of metabolic
derivatives of 2'-O-methylcytidine are present. Note the
difference in the scales of panels A and
B.
Intracellular metabolism of 2'-C-[3H]methyladenosine and
2'-O-[3H]methylcytidine in cell culture
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
55 having
~20-fold greater specific activity than NS5B
21. However,
IC50 values varied only slightly between the two enzyme
forms for the nucleoside analog triphosphates investigated. As expected
the two nucleoside analog triphosphates were competitive inhibitors
with varying nucleoside triphosphate, having Ki values that were submicromolar.
55 is capable of
incorporating both 2'-C-methyladenosine monophosphate and
2'-O-methylcytidine monophosphate onto the appropriate RNA template, implying that both triphosphates can bind to the enzyme in
the substrate NTP binding site and further implying there is some
additional room in the vicinity of the 2'-carbon and the 2'-oxygen when
bound in the active site that allows HCV NS5B to accommodate either the
2'-C-methyl or 2'-O-methyl substituent. The
presence of 2'-substituents likely confers specificity of inhibition of
the viral RNA polymerase over inhibition of the human DNA
polymerases tested. After incorporation of the nucleotide analog,
NS5B
55 is not capable of efficiently extending the incorporated analog by addition of the next correct nucleotide, suggesting that with
this template system, the nucleotide analogs act as functional chain
terminators, despite the presence in both cases of a 3'-OH. The results
suggest that after incorporation the 3'-OH is not able to perform
nucleophilic attack on the
-phosphorous of the incoming NTP. Further
investigation is necessary to understand the molecular details of the
apparent chain termination. Chain termination by incorporation of
nucleotide analogs that retain a 3'-hydroxyl has previously been
observed in the inhibition of DNA polymerase
by
arabinofuranosyladenine triphosphate (29) and arabinofuranosylcytidine
triphosphate (30).
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ACKNOWLEDGEMENTS |
---|
We thank V. Sardana and J. Zugay-Murphy for
the purification of HCV NS5B55 and M. Sardana for amino acid analysis.
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FOOTNOTES |
---|
* The use of the compounds 2'-C-methyladenosine and 2'-O-methylcytidine as therapies for HCV infection is disclosed in international patent applications WO 01/90121 A2 assigned to Idenix, Inc. and WO 02/57425 A2 assigned to Merck & Co., Inc. and Isis Pharmaceuticals, Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: WP26-265, Merck Research Laboratories, West Point, PA 19486. Tel.: 215-652-4488; Fax: 215-993-2330; E-mail: steve_carroll@merck.com.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M210914200
2 J. E. Tomassini, E. Boots, K. Getty, S. Shim, Z.-Q. Zhang, and G. Migliaccio (2002), submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are:
HCV, hepatitis C
virus;
NS5B, nonstructural protein 5B, the RNA-dependent
RNA polymerase;
NS5B21, NS5B
55, NS5B proteins with C-terminal
truncations of 21 and 55 amino acids, respectively;
t500, single-strand
heteromeric RNA of 500-nucleotide length;
RP, reverse phase;
HPLC, high-performance liquid chromatography;
LC/MS, liquid
chromatography/mass spectrometry;
DTT, dithiothreitol;
RT, room
temperature.
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