Mechanism of activation of β-D-2′-Deoxy-2′-fluoro- 2′-C-methylcytidine and inhibition of hepatitis C virus NS5B RNA polymerase

Article abstract of DOI:10.1128/AAC.00400-06

β-D-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5′-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2′-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a K_m of 81 μM and a k_cat of 0.007 s ^-1, but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (K_i) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 μM. Similar results were obtained with 2′-C-methyladenosine triphosphate (K_i = 1.5 μM) and 2′-C-methylcytidine triphosphate (K_i = 1.6 μM). NS5B with the S282T mutation, which is known to confer resistance to 2′-C- methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination. Copyright

Full text of DOI:10.1128/AAC.00400-06

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 2007, p. 503–509
0066-4804/07/$08.00ϩ0 doi:10.1128/AAC.00400-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 51, No. 2
Mechanism of Activation of ␤-D-2Ј-Deoxy-2Ј-Fluoro-2Ј-C-Methylcytidine
and Inhibition of Hepatitis C Virus NS5B RNA Polymeraseᰔ
Eisuke Murakami,1* Haiying Bao,1 Mangala Ramesh,1 Tamara R. McBrayer,1† Tony Whitaker,1†
Holly M. Micolochick Steuer,1 Raymond F. Schinazi,2 Lieven J. Stuyver,1‡ Aleksandr Obikhod,1§
Michael J. Otto,1 and Phillip A. Furman1
Pharmasset, Inc., 303A College Road East, Princeton, New Jersey 08540,1 and Emory University VA Medical Center,
Medical Research-151, 1670 Clairmont Road, Decatur, Georgia 300332
Received 31 March 2006/Returned for modification 26 May 2006/Accepted 30 October 2006
␤-D-2؅-Deoxy-2؅-fluoro-2؅-C-methylcytidine (PSI-6130) is a potent specific inhibitor of hepatitis C virus
(HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be
phosphorylated to the 5؅-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B
were investigated. The phosphorylation of PSI-6130 by recombinant human 2؅-deoxycytidine kinase (dCK) and
uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130
was shown to be a substrate for purified dCK, with a Km of 81 ␮M and a kcat of 0.007 s؊1, but was not a
substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphos-
phate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphos-
phate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was
studied. The steady-state inhibition constant (Ki) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type
enzyme was 4.3 ␮M. Similar results were obtained with 2؅-C-methyladenosine triphosphate (Ki ؍ 1.5 ␮M) and
2؅-C-methylcytidine triphosphate (Ki ؍ 1.6 ␮M). NS5B with the S282T mutation, which is known to confer
resistance to 2؅-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation
of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.
Hepatitis C virus (HCV) is an RNA virus which possesses a
single-stranded positive-sense RNA as the viral genome. This
viral RNA plays important roles during viral replication, as it
serves as an mRNA for viral protein synthesis, a template for
RNA replication, and a nascent RNA genome for a newly
formed virus (17). HCV NS5B RNA-dependent RNA poly-
merase is a key enzyme in viral RNA replication. This enzyme,
which does not require a primer for initiation of RNA synthe-
sis, catalyzes de novo RNA synthesis (8, 11). Nucleoside ana-
logs have been used to treat viral infections, such as herpes
simplex virus, human immunodeficiency virus, and hepatitis B
virus infections (5, 6, 21). These drugs are designed to inhibit
viral polymerases by a process called chain termination, in which
DNA synthesis is quenched by incorporating the triphosphate
forms of these drugs, which lack the 3Ј-hydroxyl group on the
sugar moiety. In order for nucleoside analogs to inhibit a viral
polymerase, they must be transported into the cell and converted
to the active 5Ј-triphosphate form by cellular kinases. 2Ј-C-Meth-
ylnucleosides have been investigated as anti-HCV agents target-
ing HCV NS5B RNA polymerase (2, 20). 2Ј-C-Methyladenosine
(2Ј-C-Me-A) and 2Ј-C-methylguanosine (2Ј-C-Me-G) showed po-
tent anti-HCV activities in a cell-based replicon assay, and their
triphosphate forms inhibited replicase and NS5B RNA polymer-
ase in vitro (20). In addition, 2Ј-C-Me-A exhibited significant
activity against HCV in a cell culture system which involves com-
plete HCV replication and which produces infectious HCV (16).
A resistant replicon has been selected by passage of HCV in the
presence of 2Ј-C-Me-A or 2Ј-C-Me-G (20). Sequencing of the
replicon identified the S282T mutation in NS5B RNA polymer-
ase (20).
␤-D-2Ј-Deoxy-2Ј-fluoro-2Ј-C-methylcytidine (PSI-6130) is a
potent specific inhibitor of HCV RNA replication in Huh-7
replicon cells (4), and its structure is shown in Fig. 1. PSI-6130
has been shown to be phosphorylated to the 5Ј-triphosphate
(PSI-6130-TP), and a detailed study of the anabolism of PSI-
6130 will be presented elsewhere (unpublished data). Here, we
have studied the biochemical aspects of metabolism and the
antiviral activity of PSI-6130 and have specifically addressed
the following questions: (i) Which enzyme is responsible for
phosphorylating PSI-6130 to the monophosphate (PSI-6130-
MP)? (ii) Does UMP-CMP kinase (YMPK) phosphorylate
PSI-6130-MP? (iii) Which enzyme phosphorylates PSI-6130
diphosphate (PSI-6130-DP)? (iv) Does PSI-6130-TP inhibit
wild-type and mutant (S282T) HCV NS5B RNA polymerases?
(v) If it does inhibit wild-type and mutant (S282T) HCV NS5B
RNA polymerases, what is the molecular mechanism of the
inhibition? These studies should help provide an understand-
ing of the molecular mechanism of action of PSI-6130.
* Corresponding author. Mailing address: Pharmasset, Inc., 303A
College Road East, Princeton, NJ 08640. Phone: (609) 613-4100. Fax:
(609) 613-4150. E-mail: eisuke.murakami@pharmasset.com.
† Present address: RFS Pharma LLC., 1860 Montreal Road, Tucker,
GA 30084.
MATERIALS AND METHODS
‡ Present address: Virco BBA, Mechelen, Belgium.
§ Present address: Emory University VA Medical Center, Medical
Research-151, 1670 Clairmont Road, Decatur, GA 30033.
ᰔ Published ahead of print on 13 November 2006.
Enzymes. Human 2Ј-deoxycytidine kinase (dCK) was cloned from human
hepatoma (HepG2) cells. Total RNA was isolated from HepG2 cells grown to 75
to 85% confluence in 75-cm2 tissue culture flasks. Poly(A) RNA was isolated
from total RNA with a Poly(A) Purist MAG kit (Ambion, Austin, TX), and
503

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MURAKAMI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
was coupled with pyruvate kinase and lactate dehydrogenase, and oxidation of
NADH was monitored at 340 nm by using a Lambda 35 UV/VIS spectrometer
(Perkin-Elmer). The reaction temperature for dCK and UCK-1 was 25°C, and
the reaction temperature for YMPK and NDPK was 37°C. A 1-ml reaction
mixture contained 64 mM Tris-HCl (pH 7.5), 3.8 mM EDTA, 180 mM KCl, 12.8
mM MgCl2, 24 mM (NH4)2SO4, 1 mM ATP, 0.5 mM phosphoenolpyruvate, 0.1
mM NADH, 5 IU/ml pyruvate kinase, 13.8 IU/ml lactate dehydrogenase, nucle-
oside or nucleoside monophosphate substrate, and kinase. The final enzyme
concentrations for the dCK and UCK-1 reactions were 240 nM and 41 nM,
respectively, except for reactions with PSI-6130 and 2Ј-C-methylcytidine (2Ј-C-
Me-C), in which a 10-fold higher concentration of UCK-1 (410 nM) was used.
The enzyme concentrations in the YMPK reaction were 80 nM for natural
nucleoside monophosphates and 200 nM for PSI-6130-MP. In the NDPK reac-
tion, 216 nM enzyme was used in all the reactions. The reaction rates with
different concentrations of the nucleoside substrate were determined, and
steady-state parameters were determined by using the GraFit program (version
5; Erithacus Software, Horley, United Kingdom).
NS5B RNA polymerase assays. NS5B RNA polymerase reaction was studied
by monitoring the incorporation of 32P-labeled UMP into the newly synthesized
RNA strand by using minus IRES as the template. A steady-state reaction was
performed in a total volume of 140 ␮l containing 2.8 ␮g of minus IRES RNA
template, 140 units of anti-RNase (Ambion), 1.4 ␮g of NS5B, an appropriate
amount of [␣-32P]UTP, various concentrations of natural and modified nucleo-
tides, 1 mM MgCl2, 0.75 mM MnCl2, and 2 mM dithiothreitol in 50 mM HEPES
buffer (pH 7.5). The nucleotide concentration was changed depending on the
inhibitor. The reaction temperature was 27°C. At the desired times, 20-␮l ali-
quots were taken and the reaction was quenched by mixing the reaction mixture
with 80 ␮l of stop solution containing 12.5 mM EDTA, 2.25 M NaCl, and 225
mM sodium citrate. In order to determine steady-state parameters for a natural
nucleotide triphosphate (NTP) substrate, one NTP concentration was varied and
the concentrations of the other three NTPs were fixed at saturating concentra-
tions. For determination of the Ki for 2Ј-C-Me-ATP, the concentrations of UTP,
GTP, and CTP were fixed at 10, 100, and 100 ␮M, respectively, and the concen-
trations of ATP and 2Ј-C-Me-ATP were varied. For the 2Ј-C-Me-CTP and
PSI-6130-TP experiments, UTP, ATP, and GTP concentrations were fixed at 10,
100, and 100 ␮M, respectively, and the CTP and inhibitor concentrations were
varied.
The radioactive RNA products were separated from unreacted substrates by
passing the quenched reaction mixture through a Hybond Nϩ membrane (Am-
ersham Biosciences) by using a dot blot apparatus. The RNA products were
retained on the membrane and the free nucleotides were washed out. The
membrane was washed four times with a solution containing 0.6 M NaCl and 60
mM sodium citrate. After the membrane was rinsed with water followed by
rinsing with ethanol, the dots were cut out and the radioactivity was counted in
a Packard liquid scintillation counter.
The amount of product was calculated on the basis of the total radioactivity in the
reaction mixture. The rate of the reaction was determined from the slope of the time
course of product formation. To determine the inhibition constant (Ki), reaction
rates were determined with different concentrations of the substrate and the inhib-
itor and were fit to a competitive inhibition equation: v ϭ (Vmax ⅐ [s])/{Km ⅐ (1 ϩ
[I]/Ki) ϩ [S]}, where v is the observed rate, [S] is the substrate concentration, [I] is
the inhibitor concentration, and Vmax is the maximum rate. Km is the Michaelis
constant, and Ki is the inhibition constant. A nonlinear fit was performed by using
the GraFit program (version 5; Erithacus Software).
FIG. 1. Chemical structures of the nucleoside analogs used in this
study.
reverse transcription was performed with 1/10 of the poly(A) RNA by using a
First Strand cDNA kit (Roche Applied Science, Indianapolis, IN) and the re-
verse primer from the first round of PCR. The target gene was amplified by a
nested PCR procedure. The first round of PCR consisted of 25 cycles with the
outer primer pair. A further 35 cycles used 2 ␮l of the PCR product from the first
amplification as the template and the inner primer pair. The inner primer pair
also introduced restriction enzyme sites that allowed directional cloning. The
final PCR product was digested and cloned into the Escherichia coli expression
vector pQE-60 (QIAGEN, Valencia, CA), which introduced arginine and serine
residues followed by a 6-histidine tail at the carboxyl terminus of the protein for
affinity purification. The resulting product was verified by sequencing, and the
construct was cotransformed into XL1-Blue MRFЈ competent E. coli cells (Stra-
tagene, La Jolla, CA) along with plasmid pRep4, which codes for the Lac
repressor protein. The protein was purified by a metal affinity column with Talon
resin (Clontech, Mountain View, CA).
Human uridine-cytidine kinase 1 (UCK-1) was cloned from Huh-7 cells. We
preformed RNA isolation; cDNA preparation; and amplification, cloning, and
expression exactly as described above for dCK; however, UCK-1 was not ex-
pressed. We reasoned that this may have been due to codon usage problems and
thought that an easily expressible leader peptide might solve the problem, and so
a third amplification of the DNA was performed to allow directional cloning into
the BamHI and HindIII sites of pTrcHis A. The subsequent plasmid codes for a
36-amino-acid leader peptide, including a 6-His tag at the amino terminus, while
the carboxy terminus is identical to that of the wild-type enzyme. The amino acid
sequence of the leader sequence is GGSHHHHHHGMASMTGGNNMGRDL
YDDDDKDRWGS-GSAGG (the hyphen is where the wild-type protein se-
quence begins).
YMPK and nucleoside diphosphate kinase (NDPK) were cloned from Huh-7
and HepG2 cells, respectively. We followed the RNA isolation; cDNA prepara-
tion; and amplification, cloning, and expression procedures exactly as described
above for dCK.
Chain-termination study. An RNA polymerase reaction was performed in a
total volume of 20 ␮l containing 2.7 ␮M RNA 21-mer (5Ј-CCU UUU CUA
AUU CUC GUA UAC-3Ј); 10 ␮M UTP; 100 ␮M ATP and GTP; 5 ␮Ci of
[␣-32P]UTP; 20 units of anti-RNase (Ambion); 100 ␮M either CTP, 3Ј-dCTP,
2Ј-C-Me-CTP, or PSI-6130-TP; 400 ng of NS5B; 1 mM MgCl2; 0.75 mM MnCl2;
and 2 mM dithiothreitol in 50 mM HEPES buffer (pH 7.5). The reaction was
allowed to proceed for 1 h at 27°C and was quenched by adding 1 ␮l of 0.5 M
EDTA. After the reaction was quenched, 14 ␮l of a dye solution containing 95%
formamide and 0.1% each of bromophenol blue and xylene cyanol was added for
sequencing gel analyses. The samples (10 ␮l) were loaded onto a 20% poly-
acrylamide–12% formamide gel, and the electrophoresis was run at 60 W with a
sequencing gel apparatus. The gel was then exposed to a phosphorscreen and
visualized with a phosphorimager.
The 21-amino-acid C-terminal truncated HCV NS5B RNA polymerase was
expressed and purified as described previously (27). The S282T mutant en-
zyme was made by using a Quikchange mutagenesis kit (Stratagene). Pyruvate
kinase, lactate dehydrogenase, human creatine kinases (isoforms BB, MB,
and MM), and yeast 3-phosphoglycerate kinase were purchased from Sigma
(St. Louis, MO).
Other materials. 3Ј-Deoxycytidine triphosphate (3Ј-dCTP) was purchased
from Trilink Biotechnologies (San Diego, CA). All other nucleoside/nucleotide
analogs were synthesized by Pharmasset, Inc. (Princeton, NJ). The structures of
the compounds used in this study are shown in Fig. 1. The negative-strand
internal ribosomal entry site RNA template (referred to as minus IRES) was
prepared by using an in vitro transcription kit from Ambion (22). Natural nu-
cleoside triphosphates were purchased from Amersham Biosciences (Piscataway,
NJ). [␥-32P]UTP was purchased from Perkin-Elmer (Boston, MA). RNA 21-mer
(5Ј-CCU UUU CUA AUU CUC GUA UAC-3Ј) was synthesized and purified by
New England Biolabs (Ipswich, MA).
RESULTS
dCK, UCK-1, YMPK, and NDPK assays. The phosphorylation of nucleoside
and nucleoside monophosphates by human dCK, UCK-1, YMPK, and NDPK
was studied spectrophotometrically as described previously (13). The reaction
Phosphorylation of PSI-6130 by human dCK and UCK-1.
Other studies, presented elsewhere, have shown that incuba-

VOL. 51, 2007
Substrate
MECHANISM OF PSI-6130
505
TABLE 1. Steady-state parameters for dCK reactionsa
TABLE 3. Steady-state parameters for YMPKa
Relative
substrate
specificity
kcat/Km
Km
(␮M)
kcat
kcat/Km
Substrate
Km (␮M)
kcat (sϪ1
)
(␮MϪ1 sϪ1
)
(sϪ1
)
(␮MϪ1 sϪ1
)
UMP
151 Ϯ 31
56 Ϯ 10
837 Ϯ 69
282 Ϯ 88
81 Ϯ 5
30 Ϯ 2
30 Ϯ 1
0.54
2Ј-Deoxycytidine
Gemcitabine
2Ј-FdC
2Ј-C-Me-dC
Cytidine
PSI-6130
2Ј-C-Me-C
0.4 Ϯ 0.1
1.4 Ϯ 0.2
4.7 Ϯ 1.2
46.2 Ϯ 10.7
6.6 Ϯ 1.0
81.2 Ϯ 8.0
914 Ϯ 209
0.025
0.038
0.050
0.048
0.009
0.016
0.011
6.3 ϫ 10Ϫ2
2.7 ϫ 10Ϫ2
1.1 ϫ 10Ϫ2
1.0 ϫ 10Ϫ3
1.4 ϫ 10Ϫ3
1.9 ϫ 10Ϫ4
1.2 ϫ 10Ϫ5
1
CMP
0.54
0.43
dCMP
0.036
0.012
0.17
PSI-6130-MP
3.3 Ϯ 0.4
0.016
0.022
0.003
0.0002
a Values are reported as means Ϯ standard deviations.
a Values are reported as means Ϯ standard deviations.
tivity was observed for 2Ј-deoxycytidine up to 1 mM or for
2Ј-C-Me-C and PSI-6130 up to 3 mM when a 10-fold higher
concentration of enzyme compared to those used in the cyti-
dine and uridine experiments was used.
tion of cells with certain exogenous natural nucleosides can
reduce the antiviral activity of a nucleoside analog (7, 15). It
was shown that addition of exogenous 2Ј-deoxycytidine com-
pletely reversed the inhibition of HCV RNA replication by
PSI-6130 in replicon cells, whereas the addition of cytidine
resulted in only the partial reversal of antiviral activity (26).
These findings suggest that PSI-6130 is phosphorylated by dCK
and/or UCK. There are two isoforms of UCK (UCK-1 and
UCK-2). Northern blot analyses have shown that UCK-1 is
expressed in wide variety of tissues, including liver, kidney,
skeletal muscle, and heart tissues, whereas UCK-2 is expressed
only in the placenta (29). Therefore, UCK-1 was chosen for
use for the investigation of the phosphorylation of PSI-6130.
Cell-free phosphorylation assays were performed with PSI-
6130, 2Ј-deoxycytidine, cytidine, and other related compounds
(see Fig. 1 for the structures). Steady-state kinetic assays were
performed with an enzyme coupled system with pyruvate ki-
nase and lactate dehydrogenase, and the reaction was followed
by monitoring the oxidation of NADH at 340 nm (see the
description of the experimental procedure above).
The steady-state parameters for dCK are summarized in
Table 1. PSI-6130 and 2Ј-C-Me-C were phosphorylated by
human dCK with significantly lower affinities than 2Ј-deoxycy-
tidine, cytidine, 2Ј-deoxy-2Ј-fluorocytidine (2Ј-FdC), or gem-
citabine, with Km values of 81 and 914 ␮M, respectively. Com-
pared to the physiological substrate, 2Ј-deoxycytidine, PSI-6130
was phosphorylated approximately 300-fold less efficiently;
however, it was a 15-fold better substrate than 2Ј-C-Me-
cytidine. Addition of a methyl group at the 2Ј-arabinosyl (2Ј-
up) position to 2Ј-deoxycytidine decreased the efficiency by
62-fold. Gemcitabine and 2Ј-FdC were phosphorylated at ef-
ficiencies similar to that for 2Ј-deoxycytidine.
Phosphorylation of PSI-6130-MP by human YMPK. The
enzyme that catalyzes the phosphorylation of CMP, UMP, and
dCMP to their corresponding diphosphates is known as
YMPK. The enzyme also phosphorylates monophosphates of
cytidine analogs. Since PSI-6130 is a cytidine analog, YMPK
activity was examined with PSI-6130-MP. As shown in Table 3,
the Km value for PSI-6130-MP was comparable to those of the
natural nucleoside monophosphate substrates, with an approx-
imately twofold higher value than that for UMP and a three-
fold lower value than that for dCMP. The kcat value for PSI-
6130-MP was approximately 10-fold lower than those for CMP
and dCMP. Overall, the catalytic efficiency (kcat/Km) for PSI-
6130-MP was only threefold lower than that for dCMP, indi-
cating that PSI-6130-MP is a good substrate for YMPK.
Phosphorylation of PSI-6130-DP by human NDPK. In order
to study the phosphorylation of PSI-6130-DP to PSI-6130-TP,
human NDPK was cloned and purified. The steady-state pa-
rameters for CDP and PSI-6130-DP are summarized in Table
4. As described in the Materials and Methods section, our
kinase assay was coupled with pyuvate kinase and lactate de-
hydrogenase. It is known that pyruvate kinase phosphorylates
nucleoside diphosphates (9, 14). Therefore, the control reac-
tions were performed in the absence of NDPK and the rate was
subtracted from the overall reaction rate in the case of CDP
phosphorylation. Typically, the rate of reaction of pyuvate ki-
nase for CDP was less than 10% of the overall rate. In the
reactions with PSI-6130, the pyruvate kinase reaction rate was
negligible. Creatine kinase and 3-phosphoglycerate kinase are
also candidates for the enzyme responsible for phosphorylating
PSI-6130-DP, as these kinases are known to phosphorylate
nucleoside diphosphates (9, 14). Phosphorylation of PSI-
6130-DP was tested with different isoforms of creatine kinase
(the MM, MB, and BB isoforms) and 3-phosphoglycerate ki-
nase. No activity was observed with any of these enzymes up to
1 mM PSI-6130-DP (data not shown).
We evaluated UCK-1 with cytidine, uridine, 2Ј-deoxycyti-
dine, PSI-6130, and 2Ј-C-Me-C as phosphate acceptors. The
steady-state kinetic parameters are shown in Table 2. No ac-
Effects of PSI-6130-TP on HCV NS5B. The inhibition of
HCV NS5B RNA polymerase by PSI-6130-TP, 2Ј-C-Me-CTP,
TABLE 2. Steady-state parameters for UCK-1a
Substrate
Km (␮M)
kcat (sϪ1
)
kcat/Km (␮MϪ1 sϪ1
)
Cytidine
131 Ϯ 27
407 Ϯ 52
0.50 Ϯ 0.04
0.51 Ϯ 0.03
NA, 1 mM
NA, 3 mM
NA, 3 mM
3.8 ϫ 10Ϫ3
1.3 ϫ 10Ϫ3
NA, 1 mM
NA, 3 mM
NA, 3 mM
TABLE 4. Steady-state parameters for NDPKa
Uridine
2Ј-Deoxycytidine
2Ј-C-Me-C
PSI-6130
NA, 1 mMb
NA, 3 mMc
NA, 3 mM
kcat/Km
Substrate
Km (␮M)
kcat (sϪ1
)
(␮MϪ1 sϪ1
)
CDP
PSI-6130-DP
307 Ϯ 62
1380 Ϯ 310
183 Ϯ 14
21 Ϯ 3
0.60
0.015
a Values are reported as means Ϯ standard deviations.
b NA, 1 mM, no activity was observed up to 1 mM.
c NA, 3 mM, no activity was observed up to 3 mM.
a Values are reported means Ϯ standard deviations.

506
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ANTIMICROB. AGENTS CHEMOTHER.
TABLE 5. Steady-state parameters for wild-type and S282T mutant HCV NS5B RNA polymerasea
Wild type
S282T mutant
Nucleotide
Km (␮M)
kcat (minϪ1
)
kcat/Km (minϪ1 ␮MϪ1
)
Km (␮M)
kcat (minϪ1
)
kcat/Km (minϪ1 ␮MϪ1
)
UTP
CTP
ATP
GTP
0.4 Ϯ 0.15
13.7 Ϯ 2.59
2.7 Ϯ 0.66
9.6 Ϯ 1.73
(12.8 Ϯ 0.57) ϫ 10Ϫ4
(15.2 Ϯ 0.94) ϫ 10Ϫ4
(20.0 Ϯ 0.97) ϫ 10Ϫ4
(16.9 Ϯ 0.91) ϫ 10Ϫ4
32 ϫ 10Ϫ4
1.1 ϫ 10Ϫ4
7.4 ϫ 10Ϫ4
1.7 ϫ 10Ϫ4
0.18 Ϯ 0.05
2.7 Ϯ 0.92
0.26 Ϯ 0.14
5.0 Ϯ 1.5
(1.25 Ϯ 0.05) ϫ 10Ϫ4
(2.14 Ϯ 0.16) ϫ 10Ϫ4
(1.67 Ϯ 0.91) ϫ 10Ϫ4
(1.98 Ϯ 0.15) ϫ 10Ϫ4
6.9 ϫ 10Ϫ4
0.79 ϫ 10Ϫ4
6.4 ϫ 10Ϫ4
0.4 ϫ 10Ϫ4
a Values are reported means Ϯ standard deviations.
and 2Ј-C-Me-ATP was studied under steady-state kinetic con-
ditions by using purified RNA polymerase, 32P-labeled UTP,
and minus IRES as an RNA template. Since it has been shown
that the S282T mutation in NS5B RNA polymerase causes
resistance to 2Ј-C-Me-A and 2Ј-C-Me-G (20), experiments
were performed with both wild-type and S282T mutant en-
zymes to see if the resistance is common to all 2Ј-C-Me-nucleo-
sides. The Km and the maximum rate (kcat) were determined
for natural nucleoside triphosphates (Table 5). Overall, the
catalytic efficiencies (kcat/Km values) were similar for the wild
type and the S282T mutant; however, the affinity for the nat-
ural nucleotides was tighter (lower Km) and the rate was slower
(lower kcat) with the mutant than with the wild type. The Ki
values for 2Ј-C-Me-nucleoside triphosphates (2Ј-C-Me-nucle-
oside-TPs) were also determined (Table 6). The inhibition
constants for all the 2Ј-C-Me-nucleoside-TPs with the wild-
type enzyme were very similar, with a Ki range of 1.5 to 4.3
␮M. The Km values for natural nucleoside triphosphates
were 5- to 10-fold lower with the S282T mutant than with
the wild type. The drug resistance profile was analyzed by
product formation was stopped at the same position. The po-
sition of the band must be a 6-mer since 3Ј-dCTP is a known
chain terminator (24) and it stopped the RNA synthesis at the
same position (Fig. 2, lane 4). A minor band above the 6-mer
band is very likely due to a misincorporated product, since the
same band was seen with 3Ј-dCTP. These results indicate that
both 2Ј-C-Me-C-TP and PSI-6130-TP are chain terminators.
DISCUSSION
In order for a nucleoside analog to inhibit the viral polymer-
ase, it must to be activated to the 5Ј-triphosphate form by host
cell kinases. Typically, phosphorylation of a nucleoside to its
monophosphate is a rate-limiting step for the activation of
many cytidine analogs (1, 10). In order to understand the
phosphorylation of PSI-6130 to PSI-6130-MP, human dCK and
UCK-1 were cloned and purified. The kinetic results indicate
that PSI-6130 is a substrate for dCK, although it was an ap-
proximately 300-fold poorer substrate than the physiological
substrate, 2Ј-deoxycytidine. Similarly, some cytidine analogs,
such as zalcitabine (25) and lamivudine (3), are known to be
phosphorylated by dCK at low catalytic efficiencies; but this
enzyme still serves as the enzyme responsible for the phosphor-
ylation. On the other hand, UCK-1 was not able to phosphor-
ylate PSI-6130 or 2Ј-C-Me-C. These UCK-1 results are consis-
tent with the findings of previous structural and biochemical
studies, which showed that UCK has a high specificity for the
2Ј-hydroxyl group (28, 29). Clearly, the presence of the 2Ј-
methyl group also influences the affinities of these nucleosides
analogs for UCK.
comparing Ki(drug)/Km(natural
values between the
nucleotide)
wild-type enzymes and the S282T mutant enzymes. The
greatest resistance was noted with 2Ј-C-Me-ATP (150-fold).
Chain termination. We have shown that PSI-6130-TP inhib-
its HCV NS5B RNA polymerase. In order to understand the
molecular mechanism of inhibition, we examined whether or
not PSI-6130-TP acts as a chain terminator. An RNA 21-mer
oligonucleotide with only one guanine base in the strand, at the
sixth position from the 3Ј end, was used as a template. Thus,
during RNA synthesis, the RNA polymerase needs to incor-
porate cytidine only once at the sixth position from the 5Ј end.
The polymerase experiments were performed in the absence of
CTP but in the presence of CTP analogs, together with ATP,
GTP, and radiolabeled UTP. Therefore, if these analogs are
chain terminators, any products longer than 6-mers should not
be observed. As shown in Fig. 2, except for the sample con-
taining all four natural nucleoside triphosphates, the RNA
In previous cell-based studies, we have shown that the anti-
HCV activity of PSI-6130 was prevented only by the addition of
cytidine or 2Ј-deoxycytidine into the replicon cell culture (26).
PSI-6130’s anti-HCV replicon activity was completely reversed
by exogenously added 2Ј-deoxycytidine, probably because
phosphorylation of PSI-6130 by dCK was inhibited due to
competition with the physiological substrate. Since PSI-6130
TABLE 6. Inhibition of wild-type and S282T mutant HCV NS5B RNA polymerase by the nucleoside triphosphate analogsa
Fold resistance
(Ki/Km
Wild type
Ki (␮M)
S282T mutant
Nucleotide analog
)
/
mut
Km (␮M)b
Ki/Km
Km (␮M)c
Ki (␮M)
Ki/Km
(Ki/Km)WTc
PSI-6130-TP
2Ј-C-Me-CTP
2Ј-C-Me-ATP
13.7 Ϯ 2.59
13.7 Ϯ 2.59
2.7 Ϯ 0.66
4.3 Ϯ 0.7
1.6 Ϯ 0.3
1.5 Ϯ 0.4
0.31
0.12
0.56
2.7 Ϯ 0.92
2.7 Ϯ 0.92
0.26 Ϯ 0.14
2.5 Ϯ 0.9
3.8 Ϯ 1.0
21.8 Ϯ 4.7
0.93
1.4
84
3
12
150
a Values are reported means Ϯ standard deviations.
b The Km for CTP is shown for PSI-6130-TP and 2Ј-C-Me-CTP, and the Km for ATP is shown for 2Ј-C-Me-ATP.
c mut, mutant; WT, wild type.

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MECHANISM OF PSI-6130
507
more, PSI-6130 was only a 7.4-fold poorer substrate for dCK
than cytidine, whereas it was a 300-fold poorer substrate than
2Ј-deoxycytidine (Table 1). Therefore, there is a greater
chance for PSI-6130 to be phosphorylated by dCK in the pres-
ence of exogenously added cytidine than in the presence of
2Ј-deoxycytidine. This could explain why cytidine partially in-
hibited the antiviral activity of PSI-6130. Taken together, the
results of our enzyme and cell-based competition studies sug-
gest that dCK is likely responsible for the phosphorylation of
PSI-6130.
A structure-activity relationship (SAR) for 2Ј-modified
nucleosides with dCK is summarized in Fig. 3. Addition of
fluorine at the 2Ј position of 2Ј-deoxycytidine (2Ј-FdC) caused
an approximately 5-fold decrease in kcat/Km, whereas a hy-
droxyl group at this position (cytidine) led to a 50-fold de-
crease, indicating that the enzyme prefers fluorine over a hy-
droxyl group at the 2Ј-down position. A 2Ј-C-Me modification
of 2Ј-deoxycytidine, 2Ј-FdC, and cytidine (2Ј-OH) significantly
decreased the kcat/Km values (60- to 100-fold). Gemcitabine,
which possesses two fluorines at the 2Ј position, is a slightly
better substrate than 2Ј-FdC. A fluoro or hydroxyl group at the
2Ј-arabinosyl (2Ј-up) position is proposed to form a hydrogen
bond with the dCK active-site residue Arg 128, and the inter-
action could enhance the reaction rate (23).
FIG. 2. Evidence for chain termination of RNA synthesis by incor-
poration of PSI-6130-MP and 2Ј-C-Me-CMP.
was a much poorer (300-fold) substrate than 2Ј-deoxycytidine,
it is not surprising to see complete inhibition by deoxycytidine.
Interestingly, cytidine inhibited the antiviral activity of PSI-
6130 by 60% (26). Our enzyme studies have shown that the
kcat/Km values for cytidine were similar for dCK (1.4 ϫ 10Ϫ3
␮MϪ1 sϪ1) and UCK-1 (3.8 ϫ 10Ϫ3 ␮MϪ1 sϪ1); thus, cytidine
could compete with PSI-6130 for the dCK active site. Further-
The enzyme responsible for phosphorylating CMP, UMP,
and dCMP is YMPK. This enzyme also phosphorylates various
FIG. 3. Effect of 2Ј substitutions on dCK activity SAR.

508
MURAKAMI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
group, which is necessary to attack the alpha phosphate of the
next incoming nucleoside triphosphate for further RNA syn-
thesis. Migliaccio et al. (20) proposed for 2Ј-C-Me-A that the
2Ј-C-methyl group may sterically hinder the ribose ring of the
next incoming nucleoside triphosphate. Since PSI-6130 also
possesses a 2Ј-C-methyl group, it is possible that a similar
interaction may occur such that PSI-6130 also acts as a non-
obligate chain terminator.
FIG. 4. Proposed enzymatic phosphorylation pathway for PSI-6130.
In summary, we have investigated the mechanism of action
of PSI-6130, which is a potent HCV RNA replication inhibitor,
in a replicon system. Our enzyme kinetics and previous cell-
based competition assays suggested that dCK is responsible for
the first phosphorylation step. YMPK was able to phosphory-
late PSI-6130-MP to the diphosphate efficiently. The enzyme
that catalyzes the last step of the phosphorylation pathway is
likely to be NDPK, since other host kinases that are known to
phosphorylate nucleoside diphosphate were not capable of
phosphorylating PSI-6130-DP. The extent of inhibition of wild-
type NS5B RNA polymerase by PSI-6130-TP was similar to
those by other 2Ј-C-Me-nucleoside-TPs. However, our studies
with the S282T mutant enzyme suggested that this mutation
causes high levels of resistance to 2Ј-C-Me-nucleosides with
purine bases but not to the cytidine derivatives. Similar to
other 2Ј-C-Me-nucleoside-TPs, PSI-6130-TP acted as a nonob-
ligate chain terminator during RNA synthesis by NS5B RNA
polymerase. These results suggest that PSI-6130 is worthy of
further investigation as a treatment for HCV infection.
cytidine analog monophosphates (12, 18). PSI-6130-MP was
phosphorylated to the diphosphate with a catalytic efficiency
only threefold lower than that of one of the natural substrates,
dCMP. Previous SAR studies have shown that the most im-
portant determinant for the activity is the 3Ј-OH group (18).
Our results are consistent with the findings of those studies, as
PSI-6130 possesses a 3Ј-OH group.
The last step of the PSI-6130 activation pathway is the phos-
phorylation of PSI-6130-DP to PSI-6130-TP. Our kinetic study
results suggest that the enzyme responsible for this reaction is
likely to be NDPK, as creatine kinases and 3-phosphoglycerate
kinase were not able to phosphorylate PSI-6130-DP. There-
fore, we propose that the enzymes involved in PSI-6130 acti-
vation are dCK, YMPK, and NDPK. Our proposed scheme of
PSI-6130 activation is shown in Fig. 4.
Our steady-state parameters determined by using the 21-
amino-acid C-terminus-truncated NS5B were comparable to
previously reported values (19). The inhibition profile of wild-
type NS5B enzyme by PSI-6130-TP was similar to those of
other 2Ј-C-Me-nucleotides, with Ki values ranging from 1.5 to
4.3 ␮M and Ki/Km values ranging from 1.2 to 5.6 (Table 6).
Interestingly, with the S282T mutant enzyme, the inhibition
profiles were very similar for PSI-6130-TP and 2Ј-C-Me-CTP,
but 2Ј-C-Me-ATP showed much less of an inhibitory effect.
The Ki/Km value for 2Ј-C-Me-ATP was 60- and 90-fold higher
than those for 2Ј-C-Me-CTP and PSI-6130-TP, respectively.
When the fold resistances were compared, the value for 2Ј-C-
Me-ATP (150-fold) was significantly higher than those for PSI-
6130 (3-fold) and 2Ј-C-Me-CTP (12-fold). Previously, it was
shown that the S282T mutation showed resistance to both
2Ј-C-Me-A and 2Ј-C-Me-G (20). Therefore, these results sug-
gest that 2Ј-C-Me-nucleosides with purine bases show signifi-
cantly greater drug resistance by S282T mutation than the
cytidine derivatives. The Km value for ATP was 10-fold lower
with the S282T mutant than with the wild type. This may also
be contributing to the resistance with 2Ј-C-Me-ATP. A previ-
ous study showed that the Km for ATP increased by 3.8-fold
due to the S282T mutation when the 55-amino-acid truncated
enzyme was used (20). It is possible that ATP binding to the
active site of the mutant enzyme may be affected by the dif-
ferent number of amino acids truncated.
ACKNOWLEDGMENT
R. F. Schinazi is the principal founder and former director and
consultant of Pharmasset, Inc. His laboratory received no funding for
his participation in this work.
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