Phosphorylation of ribavirin and viramidine by adenosine kinase and cytosolic 5′-nucleotidase II: Implications for ribavirin metabolism in erythrocytes

Article abstract of DOI:10.1128/AAC.49.6.2164-2171.2005

Many nucleoside analog drugs, such as ribavirin and viramidine, are activated or metabolized in vivo through 5′-phosphorylation. In this report, we determined the steady-state kinetic parameters for 5′-monophosphorylation of ribavirin and viramidine by adenosine kinase. The apparent K_m for ribavirin is 540 μM, and k_cat is 1.8 min^-1. Its catalytic efficiency of 3.3 × 10^-3 min^-1 is 1,200-fold lower than that of adenosine. In contrast to the common belief that ribavirin is exclusively phosphorylated by adenosine kinase, cytosolic 5′-nucleotidase II was found to catalyze ribavirin phosphorylation in vitro. The reaction is optimally stimulated by the physiological concentration of ATP or 2,3-bisphosphoglycerate. In phosphate-buffered saline plus ATP and 2,3-bisphosphoglycerate, the apparent K_m for ribavirin is 88 μM, and k_cat is 4.0 min ^-1. These findings suggest that cytosolic 5′-nucleotidase II may be involved in ribavirin phosphorylation in vivo. Like ribavirin, viramidine was found to be phosphorylated by either adenosine kinase or cytosolic 5′-nucleotidase II, albeit with a much lower activity. The catalytic efficiency for viramidine phosphorylation is 10- to 330-fold lower than that of ribavirin, suggesting that other nucleoside kinase(s) may be involved in viramidine phosphorylation in vivo. Both ribavirin and viramidine are not phosphorylated by deoxycytidine kinase and uridine-cytidine kinase. The coincidence of presence of high concentrated 2,3-bisphosphoglycerate in erythrocytes suggests that cytosolic 5′-nucleotidase II could play an important role in phosphorylating ribavirin and contribute to anabolism of ribavirin triphosphate in erythrocytes. Elucidation of ribavirin and viramidine phosphorylation mechanism should shed light on their in vivo metabolism, especially the ribavirin-induced hemolytic anemia in erythrocytes. Copyright

Full text of DOI:10.1128/AAC.49.6.2164-2171.2005

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2005, p. 2164–2171
Vol. 49, No. 6
0
066-4804/05/$08.00ϩ0 doi:10.1128/AAC.49.6.2164–2171.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Phosphorylation of Ribavirin and Viramidine by Adenosine Kinase and
Cytosolic 5Ј-Nucleotidase II: Implications for Ribavirin
Metabolism in Erythrocytes†
Jim Zhen Wu,* Gary Larson, Heli Walker, Jae Hoon Shim, and Zhi Hong
Drug Discovery, Valeant Pharmaceuticals International, 3300 Hyland Avenue, Costa Mesa, California 92626
Received 10 November 2004/Returned for modification 28 January 2005/Accepted 11 February 2005
Many nucleoside analog drugs, such as ribavirin and viramidine, are activated or metabolized in vivo
through 5؅-phosphorylation. In this report, we determined the steady-state kinetic parameters for 5؅-mono-
phosphorylation of ribavirin and viramidine by adenosine kinase. The apparent K for ribavirin is 540 ␮M,
m
؊1
؊3
؊1
؊1
and kcat is 1.8 min . Its catalytic efficiency of 3.3 ؋ 10 min · ␮M is 1,200-fold lower than that of
adenosine. In contrast to the common belief that ribavirin is exclusively phosphorylated by adenosine kinase,
cytosolic 5؅-nucleotidase II was found to catalyze ribavirin phosphorylation in vitro. The reaction is optimally
stimulated by the physiological concentration of ATP or 2,3-bisphosphoglycerate. In phosphate-buffered saline
؊1
plus ATP and 2,3-bisphosphoglycerate, the apparent Km for ribavirin is 88 ␮M, and kcat is 4.0 min . These
findings suggest that cytosolic 5؅-nucleotidase II may be involved in ribavirin phosphorylation in vivo. Like
ribavirin, viramidine was found to be phosphorylated by either adenosine kinase or cytosolic 5؅-nucleotidase
II, albeit with a much lower activity. The catalytic efficiency for viramidine phosphorylation is 10- to 330-fold
lower than that of ribavirin, suggesting that other nucleoside kinase(s) may be involved in viramidine
phosphorylation in vivo. Both ribavirin and viramidine are not phosphorylated by deoxycytidine kinase and
uridine-cytidine kinase. The coincidence of presence of high concentrated 2,3-bisphosphoglycerate in erythro-
cytes suggests that cytosolic 5؅-nucleotidase II could play an important role in phosphorylating ribavirin and
contribute to anabolism of ribavirin triphosphate in erythrocytes. Elucidation of ribavirin and viramidine
phosphorylation mechanism should shed light on their in vivo metabolism, especially the ribavirin-induced
hemolytic anemia in erythrocytes.
Although originally discovered more than 30 years ago, riba-
virin remains the only approved small molecule antiviral drug
that is active against both DNA and RNA viruses (28). Riba-
virin is approved by the U.S. Food and Drug Administration
for the treatment of pediatric respiratory syncytial virus infec-
tion and in combination with alpha-interferon for chronic hep-
atitis C virus (HCV) infection (26). More recently, therapeutic
benefits of ribavirin have been realized for the treatment of the
severe acute respiratory syndrome (16) and smallpox virus
infection, a potential bioterrorist threat (1). These clinical suc-
cesses have generated a renewed interest in understanding
ribavirin’s action mechanism. Structurally ribavirin is a purine
ribonucleoside analog capable of mimicking inosine, guano-
sine, or adenosine (Fig. 1). This unique propensity enables
ribavirin to concomitantly target multiple viral and host en-
zymes utilizing purine nucleoside or nucleotide as a substrate
or cofactor. The active form of ribavirin comprises its three
lular GTP pool for 5Ј-cap synthesis of RNA transcripts and
viral genome synthesis (33). Both ribavirin diphosphate (RDP)
and triphosphate (RTP) have been implicated as competitive
inhibitors with cognate nucleoside triphosphates for a number
of viral RNA polymerases, such as HCV RNA-dependent RNA
polymerase and influenza viral RNA polymerase (15, 36). More-
over, ribavirin can act as a viral mutagen by imitating adeno-
sine or guanosine to base pair with either thymidine or cytidine
in a viral genome. Because of this promiscuity, incorporation
of RTP into a viral genome could induce transitional mutation
and culminate virus error catastrophe (8). Additionally, as an
analog of guanosine nucleotide, RTP can be incorporated by
RNA guanylyl transferase in the place of GMP to produce
defected 5Ј-cap structure for viral RNA transcripts, attribut-
ing to a decrease of viral genome translation (6, 12). These
described mechanisms of action are interlinked, as the sup-
pression of intracellular GTP pool by inhibiting IMPDH po-
tentiates ribavirin’s effect as an alternative nucleotide for com-
petitive inhibition and incorporation. In terms of ribavirin’s
immunomodulatory activity, evidences suggest that ribavirin
can function as a small molecule immunomodulator and elicit
T-cell-mediated immunity that favors elimination of intracel-
lular viral pathogens (34). The fact that no ribavirin resistance
has ever been cogently identified in a clinical setting indirectly
supports the notion that ribavirin exerts antiviral effect through
pleiotropic action mechanisms.
5
Ј-phosphorylated derivatives. They exert antiviral activity by
interfering with several critical steps required for viral replica-
tion, including host nucleotide biosynthesis, viral genome tran-
scription, and translation. Ribavirin 5Ј-monophosphate (RMP)
is a potent inhibitor for host IMP dehydrogenase (IMPDH). It
disrupts the de novo synthesis of GMP and depletes intracel-
*
Corresponding author. Mailing address: Drug Discovery, Valeant
Pharmaceuticals International, 3300 Hyland Avenue, Costa Mesa, CA
2626. Phone: (714) 545-0100, ext. 3024. Fax: (714) 668-3142. E-mail:
jwu@valeant.com.
The paper is dedicated to Christopher Walsh on the occasion of
his 60th birthday.
9
Despite the well-documented antiviral activity, the clinical
application of ribavirin is somehow restricted due to some
serious adverse effects, especially the ribavirin-induced hemo-
†
2
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VOL. 49, 2005
RIBAVIRIN AND VIRAMIDINE 5Ј-MONOPHOSPHORYLATION
2165
FIG. 1. Chemical structures of adenosine, ribavirin, and viramidine.
lytic anemia (11). Accumulation of ribavirin phosphates in
erythrocytes or red blood cells (RBCs) attributes to hemolytic
anemia in a significant percentage of treated HCV patients,
many of whom require dose reduction or discontinuation of
therapy. Viramidine, a 3-carboxamidine derivative of ribavirin,
is a prodrug of ribavirin. It can be activated and converted to
ribavirin by adenosine deaminase in vitro (39). Pharmacoki-
netic analysis indicates that this prodrug is capable of deliver-
ing more ribavirin to the liver with a reduced propensity to be
trapped in RBCs (20, 38). In addition to the prodrug mecha-
nism, viramidine may concurrently act as a catabolic inhibitor
and slow down the catabolic rate of newly converted ribavirin
from viramidine (37). Because of these favorable pharmacoki-
netic properties, viramidine is currently in clinical trials for the
treatment of chronic HCV infection.
Ribavirin requires 5Ј-phosphorylation to be pharmacologi-
cally active. In the literature, it is generally believed that 5Ј-
monophosphorylation of ribavirin is exclusively catalyzed by
adenosine kinase. A partially purified rat liver lysate containing
both adenosine and 2Ј-deoxyadenosine kinase activities was
first observed to possess ribavirin phosphorylation activity in
vitro (32). Further studies with cell lines deficient in adenosine
kinase expression suggested that adenosine kinase is responsi-
ble for ribavirin phosphorylation (33, 35). However, it is un-
known whether the adenosine kinase deficiency has any effect
on the cellular concentration of ATP and thereby impairs
other nucleoside phosphorylation pathways. Interestingly, de-
spite the deficiency, a small amount of RTP is produced in
these cell lines (24). More recently, adenosine kinase purified
from Chinese hamster ovary (CHO) cells (13) and rabbit liver
the three 5Ј-phosphorylated forms. Although these viramidine
5Ј-phosphates are not known to associate with any antiviral
activities, it is important to comprehend its metabolic mecha-
nism. In this study, we investigated 5Ј-monophosphorylation of
ribavirin and viramidine by several nucleoside kinases and cy-
tosolic 5Ј-nucleotidase II (cN-II) and determined their kinetic
parameters. The implications of this study for in vivo drug
activation and metabolic mechanism, especially ribavirin phos-
phate anabolism in RBCs, are discussed.
MATERIALS AND METHODS
3
Materials. [2,8- H]inosine (30 Ci/mmol) was purchased from Amersham Bio-
3
sciences. [2,8- H]adenosine (25 Ci/mmol) was purchased from ICN Biomedicals.
1
4
14
[
5- C]ribavirin (54 mCi/mmol) and [5- C]viramidine (56 mCi/mmol) were cus-
tom synthesized by Moravek Biochemicals (Brea, CA). A purified recombinant
adenosine kinase from CHO cells was a gift from C. Cameron at Pennsylvania
State University. A human placental cDNA library and pCR4Blunt-Topo vector
were obtained from Invitrogen. 2,3-Diphospho-D-glyceric acid tris salt (or 2,3-
bisphosphoglycerate; BPG) was purchased from Sigma. Various nucleosides and
nucleotides were from Sigma or ICN Biochemicals. All other reagents were of
the highest grade available from Sigma or Fisher.
Adenosine kinase assay. A radiochemical-based thin-layer chromatography
(TLC) assay was developed to monitor 5Ј-phosphorylation of radiolabeled aden-
osine, ribavirin, or viramidine by adenosine kinase with ATP as a phosphate
donor. A typical assay was carried out at 37°C in 20 ␮l containing 1ϫ Dulbecco’s
phosphate-buffered saline (PBS), pH 7.3, 1 mM ATP, 1.5 mM MgCl . The final
2
concentration of adenosine kinase was 0.05, 2.5, or 5 ␮M, respectively, for assays
3
14
14
with [2,8- H]adenosine, [5- C]ribavirin, or [5- C]viramidine as a substrate. In
some experiments, the PBS was adjusted to pH 9.0 or replaced by 100 mM
HEPES, pH 7.3. An assay length was determined based on the activity of a
substrate. It lasted 4, 30, or 60 min, respectively, for adenosine, ribavirin, or
viramidine as a substrate. The reaction was quenched by addition of EDTA to
the final concentration of 80 mM. Six microliters of the quenched mixture was
spotted on a silica gel 60 TLC plate (Selecto Scientific, GA). A TLC plate was
developed in a solvent system of ammonium:isopropanol:water (3:5:2). After air
dry and exposure to a PhosphorImager overnight, products on the plate were
analyzed and quantified to calculate initial velocity. For steady-state kinetic
analysis, a substrate concentration was varied from 0.1 to 4 mM with ATP fixed
at 1.0 mM. Apparent kinetic parameters were calculated by applying a series of
initial velocity at different substrate concentrations into Michaelis-Menten equa-
tions using a nonlinear least-squares regression fit in KaleidaGraph (Synergy
Software). All the experiments were repeated at least three times to assure that
reproducible results were obtained. The reported kinetic parameters are pre-
sented as mean Ϯ standard deviation.
(22) were employed to demonstrate that it catalyzes ribavirin
phosphorylation in vitro. Yet the kinetics of the reaction has
not been fully characterized. Although adenosine kinase is
capable of catalyzing ribavirin phosphorylation, the observed
activity thus far is low. One study revealed that the reaction of
ribavirin phosphorylation by adenosine kinase is significantly
slower than that of adenosine (10).
In addition to the direct phosphorylation route catalyzed by
nucleoside kinases and related enzymes, alternative ribavirin
phosphorylation pathways have been explored. A nucleoside
could undergo phosphorolysis to nucleobase that is subse-
quently converted to nucleoside monophosphate by a phos-
phoribosyl transferase. Although ribavirin can undergo phos-
phorolysis by purine nucleoside phosphorylase, its triazole base
fails to convert to RMP by hypoxanthine-guanine phosphori-
bosyl transferase (31).
Cloning, expression, and purification of cytosolic 5؅-nucleotidase II. The cN-II
gene was cloned from a human placenta cDNA library using the published cN-II
sequence information (GenBank accession number NM_012229) (23). A for-
ward primer of 5Ј-TACATATGTCGACCTCCTGGAGTG-3Ј and a reverse an-
tiparallel primer of 5Ј-CGAATTCTTATTCTTCCTCCTCCTCC-3Ј were used to
amplify the 1,686-bp open reading frame of the cN-II gene. The PCR product
was cloned directly into a TOPO vector using the Zero Blunt TOPO PCR
cloning kit. The sequence of the cN-II insert was confirmed by DNA sequencing.
The insert was subsequently recloned to a pET22b(ϩ) vector using a Rapid DNA
Ligation kit (Roche) and transformed with Escherichia coli BL21(DE3) cells.
Fermentation for expression of recombinant cN-II was carried out in terrific
broth at 30°C. The cell culture was induced by 1 mM isopropyl-␤-D-1-thiogalac-
topyranoside when its optical density at 600 nm (OD600) reached 1.5. Cells were
Like ribavirin, viramidine undergoes 5Ј-phosphorylation in
vivo (19). A metabolic profile analysis of orally dosed virami-
dine in animals showed that about 40% of viramidine exists in

2
166
WU ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Apparent steady-state kinetic parameters of 5Ј-monophosphorylation of adenosine, ribavirin, and
a
viramidine by CHO cell-derived adenosine kinase
Substrate
Buffer
k
cat (minϪ1
)
K
m
(␮M)
k
cat/K
m
(minϪ1 · ␮MϪ1
)
Relative act.
5
Adenosine
Ribavirin
PBS, pH 7.3
PBS, pH 7.3
13 Ϯ 1
3.2 Ϯ 0.1
540 Ϯ 170
7,800 Ϯ 100
16,000 Ϯ 200
7,700 Ϯ 1,100
3.9
3.9 ϫ 10
Ϫ3
1.8 Ϯ 0.4
3.3 ϫ 10
7.9 ϫ 10
1.0 ϫ 10
3.4 ϫ 10
330
7.9
1
Ϫ5
Ϫ5
Ϫ5
1
00 mM Hepes, pH 7.3
0.62 Ϯ 0.01
0.16 Ϯ 0.02
0.26 Ϯ 0.08
Viramidine
PBS, pH 7.3
PBS, pH ϭ 9.0
3.4
a
The parameters were determined at 37°C in Dulbecco’s PBS, pH 7.3, with ATP concentration fixed at 1.0 mM, unless otherwise indicated.
harvested 3 h postinduction. The cell pellet was resuspended in a buffer contain-
ing 20 mM Tris, pH 7.5, 200 mM NaCl, 2 mM 2-mercaptoethanol in the presence
of a protease inhibitor cocktail. The suspension was passed through a microflu-
idizer to disrupt cells. The crude lysate was spun at 100,000 ϫ g for 1 h to obtain
supernatant. Recombinant cN-II was purified to apparent homogeneity using
two chromatography steps. Since cN-II contains a stretch of acidic amino acid
residues at the C terminus which enables it to bind to nickel-chelating resin (29),
the supernatant was first loaded onto a 1-ml nickel-chelating column equilibrated
with the suspension buffer. The column was washed with a buffer of 20 mM Tris,
pH 7.5, 200 mM NaCl, 2 mM 2-mercaptoethanol, and 5 mM imidazole. When a
step gradient of imidazole concentration raised to 25 mM, cN-II eluted from the
nickel column. The peak fractions containing cN-II were pooled, and its NaCl
concentration was adjusted to 50 mM through dilution. It was reloaded onto a
Mono Q column equilibrated with 20 mM Tris, pH 7.5, 50 mM NaCl, and 2 mM
dithiothreitol (DTT). Proteins were eluted over a 1 M NaCl gradient. cN-II
eluted at 500 mM NaCl with apparent homogeneity. The final protein yield was
about 1.2 mg per liter of culture.
concentration fixed at 1.0 mM, the apparent K for adenosine
m
Ϫ1
was 3.2 ␮M and kcat was 13 min . The catalytic efficiency
Ϫ1
Ϫ1
(
kcat/K ) of the reaction was 3.9 min
· ␮M
(Table 1).
m
These are consistent with the published results for CHO cell-
derived adenosine kinase (21).
5
Ј-Phosphorylation of ribavirin by adenosine kinase was ini-
tially investigated in a HEPES buffer with ATP fixed at 1 mM
as described in the literature (13). Kinetic analysis was per-
formed by determining initial velocity at various ribavirin con-
centrations. Saturation of Michaelis-Menten plot was not
reached at the highest concentration of ribavirin used (4 mM)
(
data not shown). From the available data, the apparent Km for
Ϫ1
ribavirin was estimated at 7.8 mM and kcat at 0.62 min
yielding a catalytic efficiency of 7.9 ϫ 10
,
Ϫ5
Ϫ1
Ϫ1
min
· ␮M
Phosphotransferase assay of cN-II. The phosphotransferase activity of cN-II
was assayed using a similar radiochemical TLC assay described for adenosine
kinase except that IMP was used as a phosphate donor. Briefly, an assay was
(Table 1). This catalytic efficiency is 50,000-fold lower than that
of adenosine. Ribavirin phosphorylation was also investigated
in PBS. Surprisingly, suppression of the enzyme activity in PBS
due to the presence of inorganic phosphate was not observed.
On the contrary, compared to HEPES buffer, PBS actually
performed at 37°C in 15 ␮l containing 20 mM Tris, pH 7.4, 5 mM MgCl , 1 mM
2
DTT, 4 mM IMP, and a radioactive nucleoside substrate. Small molecular ef-
fector(s) was included as indicated. The final concentration of cN-II was 20 nM
1
4
14
3
for all three substrates, [5- C]ribavirin, [5- C]viramidine, and [2,8- H]inosine.
The assay lasted for 1 h. Reaction products were analyzed by TLC and quantified
using a PhosphorImager. The effects of ATP, BPG, and Dulbecco’s PBS on cN-II
activity were investigated. In these studies, ribavirin concentration was fixed at
enhanced adenosine kinase’s activity. Further kinetic analysis
Ϫ1
yielded an apparent Km of 0.54 mM and a kcat of 1.8 min
.
These represent a 14- and 3-fold improvement of the respec-
tive kinetic parameters to the HEPES buffer. Overall, the cat-
alytic efficiency was increased by 42-fold after switching the
buffer from HEPES to PBS (Table 1). Regardless, ribavirin is
still a poor substrate for adenosine kinase with catalytic effi-
ciency about 1,200-fold lower than that of adenosine. This
conclusion is qualitatively consistent with a previous report
that the velocity of ribavirin phosphorylation catalyzed by
0
0
.125 mM or viramidine at 0.5 mM with an effector’s concentration varied from
to 5 mM for ATP and 0 to 1.6 mM for BPG. The BPG dependency of cN-II
activity was found hyperbolic and fitted to Michaelis-Menten equation for cal-
culating the apparent Km for stimulation.
Steady-state kinetic analysis of cN-II in the presence of a single effector was
performed in Tris buffer with IMP concentration fixed at 4.0 mM as described
above. An activator concentration was set at optimum with ATP at 2 mM or BPG
at 0.4 mM. The concentration of a nucleoside substrate was varied from Km/2
1
4
14
to 500 ␮M for [5- C]ribavirin and [5- C]viramidine and K
[
m
/2 to 10 mM for
5
3
CEM-cell adenosine kinase is 5 ϫ 10 -fold slower than that of
2,8- H]inosine. The apparent kinetic parameters were determined from
Michaelis-Menten equations. Likewise, the kinetic parameters for ribavirin phos-
phorylation were also determined in Dulbecco’s PBS in the presence of 2 mM
ATP with and without 3 mM BPG. All the experiments were repeated at least
three times to assure that reproducible results were obtained. The reported
kinetic parameters were presented as mean Ϯ standard deviation.
adenosine (10).
Likewise, phosphorylation of viramidine by adenosine ki-
nase was evaluated. In PBS, pH 7.3, viramidine phosphoryla-
tion was much slower than that of ribavirin. Kinetic analysis by
varying viramidine’s concentration from 0.1 to 4 mM with ATP
fixed at 1 mM gave an estimated K of 16 mM and a kcat of
m
RESULTS
Ϫ1
Ϫ5
0
.16 min . The resulting catalytic efficiency of 1.0 ϫ 10
Ϫ1
Ϫ1
Adenosine kinase catalyzed 5؅-phosphorylation. Adenosine
kinase is an abundant and ubiquitous enzyme that regulates
extracellular adenosine and intracellular adenine nucleotide
concentrations (30). Since it is widely accepted that adenosine
kinase is responsible for ribavirin 5Ј-monophosphorylation, we
obtained a recombinant mammalian adenosine kinase and de-
termined the kinetic parameters for the reaction. The adeno-
sine kinase is derived from CHO cells, and it was expressed
and purified to homogeneity from E. coli. This adenosine ki-
nase shares approximately 92% amino acid sequence identity
with the human form (21). We characterized the purified en-
zyme using adenosine as a substrate. In PBS, pH 7.3, with ATP
min · ␮M is 330-fold lower than that of ribavirin and 3.9 ϫ
10 -fold lower than that of adenosine (Table 1). The structures
5
of ribavirin and viramidine are very similar (Fig. 1). Both
compounds mimic the conformation of adenosine. But at
physiological pH, viramidine possesses an additional posi-
tive charge on the 3-carboxamidine group. This charge has
been shown to interfere with substrate recognition in virami-
dine deamination reaction catalyzed by adenosine deaminase
(39). When the assay pH was changed from 7.3 to 9.0, virami-
dine lost some of the positive charge and became partially
deprotonated. As a result, the deamination rate was increased
by 300-fold. To find out whether this positive charge exerts a

VOL. 49, 2005
RIBAVIRIN AND VIRAMIDINE 5Ј-MONOPHOSPHORYLATION
2167
with ribavirin concentration fixed at 125 ␮M or viramidine at
00 ␮M. As shown in Fig. 3A, the activation curves were bell
5
shaped, with the optimal ATP concentration at 2 mM for both
ribavirin and viramidine phosphorylation. The observed de-
crease of the stimulation effect at high ATP concentration (Ͼ2
mM) was not caused by depletion of MgCl , as elevated MgCl
2
2
concentration up to 20 mM yielded a similar bell-shaped curve
data not shown). Further experiments are required to address
(
the potential inhibitory effect observed at high ATP concen-
tration. The optimal ATP concentration (2 mM) is in good
agreement with reported value for inosine phosphorylation
FIG. 2. cN-II catalyzed ribavirin 5Ј-monophosphorylation and for-
mation of RMP. The assay was performed at 37°C for 1 h in 15 ␮l
containing 20 mM Tris, pH 7.4, 5 mM MgCl , 4 mM IMP, 1 mM DTT,
2
1
4
2
0 nM cN-II, 125 ␮M [5- C]ribavirin, and the following activator.
Lane 1: no enzyme control; lane 2: no activator; lane 3: 1 mM ATP;
lane 4: 0.4 mM BPG; lane 5: 1 mM ATP and 0.4 mM BPG; lane 6:
2
mM ATP; lane 7: 0.8 mM BPG; lane 8: 2 mM ATP and 0.8 mM BPG.
The reaction products were resolved on TLC and quantified using a
PhosphorImager.
similar effect on viramidine phosphorylation, we performed
adenosine kinase assay in PBS with pH adjusted to 9.0. At pH
9
.0, the phosphorylation reaction did accelerate. Compared to
pH 7.3, the apparent Km was declined by twofold to 7.7 mM
Ϫ1
and kcat elevated to 0.26 min , resulting in a 3.4-fold improve-
ment of catalytic efficiency (Table 1). Thus, deprotonation of
the 3-carboxamidine group improves viramidine’s reactivity,
but the effect is relatively small.
5
؅-Phosphorylation by other nucleoside kinases. In light of
the lower activity of adenosine kinase towards ribavirin and
viramidine, other nucleoside kinases were obtained and tested
for phosphorylation. We explored deoxycytidine kinase, a nu-
cleoside kinase notable for its broad substrate specificity, and
uridine-cytidine kinase, a major pyrimidine ribonucleoside ki-
nase. However, no ribavirin or viramidine phosphorylation was
detected for either of the enzymes in our radiochemical assay
(data not shown).
cN-II catalyzed phosphorylation. Cytosolic 5Ј-nucleotidase
II or cN-II is a ubiquitous enzyme capable of catalyzing 5Ј-
monophosphorylation of many inosine and guanosine nucleo-
side analogs, such as acyclovir, dideoxyinosine (18), tiazofurin
(
10), and carbocyclic 2Ј-deoxyguanosine (3). Since ribavirin
also resembles the conformation of inosine (Fig. 1), cN-II
could potentially catalyze ribavirin phosphorylation. To test
this hypothesis, we cloned the human cN-II gene from a human
placenta cDNA library and put it into an expression vector.
The recombinant cN-II was expressed in E. coli and purified to
homogeneity. The enzyme was fully active in both 5Ј-nucleoti-
dase assay and nucleoside phosphotransferase assay (data not
shown).
To investigate ribavirin phosphorylation by cN-II, we ini-
tially adapted the published assay buffer of 20 mM Tris, pH 7.4,
5
4
mM MgCl , and 1 mM DTT with IMP concentration fixed at
mM. In these conditions, ribavirin was phosphorylated by cN-
2
FIG. 3. Effects of ATP (A) and BPG (B) on 5Ј-phosphorylation of
ribavirin and viramidine. In the assay, the concentration of phosphate
1
4
II (Fig. 2). Interestingly, the presence of ATP or BPG drasti-
cally enhanced the activity. Similarly, viramidine was tested for
phosphorylation by cN-II. Like ribavirin, it was phosphorylated
by cN-II, albeit to a lesser degree (data not shown). The stim-
ulation effect of ATP on ribavirin and viramidine phosphory-
lation was further examined by titrating ATP from 0 to 5 mM
donor IMP was set at 4 mM with [5- C]ribavirin concentration fixed
14
at 125 ␮M or [5- C]viramidine at 500 ␮M. The hyperbolic curve of
enzyme activity-BPG relationship was fitted to Michaelis-Menten
equations for calculating the apparent Km of BPG for cN-II activation.
F: ribavirin as a substrate. ■: viramidine as a substrate. All the data
points are the average of at least three experiments and are presented
as mean Ϯ standard deviation.

2
168
WU ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
a
TABLE 2. Apparent kinetic parameters of 5Ј-monophosphorylation of inosine, ribavirin, and viramidine by cN-II
Substrate
Buffer
Activator
k
cat (minϪ1
)
K
m
(␮M)
k
cat/K
m
(minϪ1 · ␮MϪ1
Ϫ1
)
Relative act.
Inosine
Tris
Tris
Tris
Tris
PBS
PBS
2 mM ATP
0.4 mM BPG
2 mM ATP
0.4 mM BPG
2 mM ATP
2 mM ATP
1,310 Ϯ 100
1,160 Ϯ 30
9.4 Ϯ 1.5
1.6 Ϯ 0.1
1.4 Ϯ 0.1
4.0 Ϯ 0.8
2,170 Ϯ 130
1,840 Ϯ 20
95 Ϯ 17
6.1 ϫ 10
6.3 ϫ 10
9.9 ϫ 10
3.5 ϫ 10
5.8 ϫ 10
4.5 ϫ 10
240
250
40
Ϫ1
Ϫ2
Ϫ2
Ϫ2
Ϫ2
Ribavirin
47 Ϯ 13
14
24 Ϯ 8
23
88 Ϯ 11
18
0
.4 mM BPG
Ϫ3b
Ϫ3b
Viramidine
Tris
Tris
2 mM ATP
NA
NA
Ͼ500
Ͼ500
6.2 ϫ 10
2.5 ϫ 10
2.5
1
0.4 mM BPG
a
The apparent kinetic parameters were determined at 37°C in either Tris buffer or Dulbecco’s PBS with IMP concentration fixed at 4 mM.
The apparent kcat/K was estimated from the slope in the linear range of Michaelis-Menten plot.
m
b
(25). Compared to the reaction without ATP, 2 mM ATP
enhanced the phosphotransferase activity by 17-fold for riba-
virin and 10-fold for viramidine. The magnitude of activation
effect is a bit higher than reported for inosine phosphorylation,
which is 8.5-fold (25). The difference could be caused by the
distinct substrate, assay conditions, or enzyme source em-
ployed in these assays.
The stimulation effect of BPG on ribavirin and viramidine
phosphorylation was also assessed. For either substrate, titra-
tion of BPG against cN-II activity displayed a hyperbolic rela-
tionship (Fig. 3B). The Km of BPG for stimulation was 75 and
5
8 ␮M for ribavirin and viramidine phosphorylation, respec-
tively. These Km values are about 10-fold lower than reported
for inosine phosphorylation (25). Notably, they are also lower
than BPG’s physiological concentration in RBCs. Compared to
the reaction in the absence of BPG, the optimal BPG concen-
tration elevated ribavirin and viramidine phosphorylation by
eight- and fivefold, respectively. The magnitude of the stimu-
lation effect is in good agreement with that reported for inosine
phosphorylation (25). Moreover, the combination effect of
ATP and BPG on cN-II activity was examined by varying ATP
concentration from 1 to 2 mM and BPG from 0.4 to 0.8 mM.
Although significant activation effect was observed in all the
concentrations, the preliminary results shown in Fig. 2 suggest
that combination of the two activators is likely to be antago-
nistic. This result implies that ATP and BPG may act through
a similar mechanism and compete with each other.
Kinetic analysis of cN-II catalyzed 5؅-phosphorylation. To
further comprehend ribavirin and viramidine phosphorylation
by cN-II, we determined their steady-state kinetic parameters
in the presence of a single activator in Tris buffer with IMP
fixed at 4 mM. For comparison, we first determined the kinetic
3
parameters for [2,8- H]inosine. In the presence of 2 mM ATP,
Ϫ1
its kcat was 1,300 min and Km was 2.2 mM, resulting in a
Ϫ1
Ϫ1
catalytic efficiency of 0.61 min
literature, the reported Km for inosine ranges from 1.2 to 9 mM
3, 18). Our results are in good agreement with one of those
· ␮M
(Table 2). In the
(
studies (25). Similarly, the kinetics with inosine was deter-
mined in the presence of 0.4 mM BPG. In that condition, the
FIG. 4. Steady-state kinetics of ribavirin and viramidine 5Ј-mono-
Ϫ1
apparent kcat was 1,200 min and Km was 1.8 mM (Table 2).
phosphorylation catalyzed by cN-II. In the assay, the IMP concentra-
Ϫ1
Ϫ1
14
14
The resultant catalytic efficiency of 0.63 min · ␮M is sim-
ilar to that with ATP as an activator.
tion was set at 4 mM, with [5- C]ribavirin or [5- C]viramidine con-
centration varied from 12.5 to 500 ␮M. The initial velocity at various
substrate concentrations was fitted into Michaelis-Menten equations
for calculation of Km and kcat values. F: ribavirin as a substrate. ■:
viramidine as a substrate. All the data points are the average of at least
three experiments and are presented as mean Ϯ standard deviation.
(A) In the presence of 2 mM ATP; (B) in the presence of 0.4 mM BPG.
The steady-state kinetics of ribavirin phosphorylation by
cN-II was determined in Tris buffer. As illustrated in Fig. 4A,
in the presence of 2 mM of ATP, the apparent K for ribavirin
m
Ϫ1
was 95 ␮M and kcat was 9.4 min , yielding a catalytic efficiency

VOL. 49, 2005
of 0.099 min
RIBAVIRIN AND VIRAMIDINE 5Ј-MONOPHOSPHORYLATION
2169
Ϫ1
Ϫ1
· ␮M
(Table 2). When the activator was
ribavirin’s concentration in the plasma of treated HCV pa-
tients. These findings suggest ribavirin is not a good substrate
for adenosine kinase, which casts doubt whether ribavirin is
exclusively phosphorylated by adenosine kinase in vivo. Fur-
ther analyses show that the lower catalytic efficiency is attrib-
uted by both poor substrate binding and slower catalysis, which
can be explained structurally. Ribavirin’s triazole mimics ade-
nine nucleobase (Fig. 1); however, it misses the N1-C2-N3 seg-
ment of adenine. Apparently, the missing molecular moiety is
critical for substrate recognition and efficient catalysis by aden-
osine kinase. The lack of these structural elements in ribavirin
culminates in a 1,200-fold decrease of reactivity.
changed to 0.4 mM BPG, the apparent K was reduced to 47
m
Ϫ1
␮M and kcat declined to 1.6 min (Fig. 4B), resulting in a
Ϫ1
threefold reduction of catalytic efficiency (0.035 min
·
Ϫ1
␮M
) (Table 2). Likewise, the steady-state kinetics of vira-
midine phosphorylation by cN-II was analyzed. As illustrated
in Fig. 4, in the presence of either 2 mM ATP or 0.4 mM BPG
as an activator, saturation of Michaelis-Menten plot was not
reached even when [5- C]viramidine was at the highest con-
centration used (500 ␮M). We could not increase viramidine
concentration further due to its lower radioactive specific ac-
tivity. Judged from the available data, the Km for viramidine is
much higher than 500 ␮M. Although it is impossible to deduce
meaningful kcat and Km from the data set, we estimated its
catalytic efficiency from the slope in the linear range of
1
4
Viramidine phosphorylation by adenosine kinase was de-
tected in our radiochemical assay; however, its catalytic effi-
ciency is 330-fold lower than that of ribavirin. The extremely
Ϫ1
Ϫ1
Michaelis-Menten plot (Fig. 4). It was 0.0062 min · ␮M in
Ϫ1
high Km (16 mM) and low kcat (0.16 min ) make viramidine
Ϫ1
Ϫ1
the presence of 2 mM ATP and 0.0025 min · ␮M with 0.4
mM BPG (Table 2). Clearly, viramidine phosphorylation by
cN-II is about 10- to 15-fold slower than that of ribavirin.
To assess potential ribavirin phosphorylation by cN-II in the
physiological conditions, we determined its kinetic parameters
in PBS plus 2 mM ATP and 5 mM MgCl , with and without 3
mM BPG. This assay system contains all the important cN-II
effectors, including ATP, BPG, Mg , K , and inorganic phos-
phate. BPG is a bisphosphorylated metabolite that acts as an
allosteric modulator for oxygen binding to hemoglobin in
RBCs (7). Its concentration in mammalian RBCs is as high as
an unlikely substrate for adenosine kinase in vivo. Structurally,
viramidine should mimic adenosine better than ribavirin, as its
two nitrogen atoms in 3-carboxamidine could imitate both
6
-amino and N1 nitrogen of adenosine (Fig. 1). But our kinetic
analysis suggests otherwise. Whereas the N1 atom in adenosine
is a hydrogen bond acceptor, the nitrogen atoms in the 3-car-
boxamidine moiety are positively charged and are a hydrogen
bond donor. The electronic property of 3-carboxamidine is
likely responsible for the lower viramidine phosphorylation by
adenosine kinase.
2
2
ϩ
ϩ
Structurally related to ribavirin, tiazofurin is another purine
nucleoside analog with a five-membered ring nucleobase. It is
known to be phosphorylated by three enzymes, including aden-
osine kinase, nicotinamide ribonucleoside kinase, and a 5Ј-
nucleotidase (27). cN-II is known as high-Km 5Ј-nucleotidase
or GMP-IMP-specific 5Ј-nucleotidase (4). It possesses both
5Ј-nucleotidase and nucleoside phosphotransferase activities.
Depending on the availability of phosphate acceptors, cN-II
catalyzes phosphotransfer from a purine nucleoside mono-
phosphate, such as IMP, to either a water molecule (5Ј-nucle-
otidase activity) or a purine nucleoside (phosphotransferase
activity) (25). Although it has not been thoroughly character-
ized, the interplay of these two enzymatic activities is highly
influenced by the reaction pH as well as the presence of suit-
able nucleoside phosphate acceptors, nucleoside triphosphate
activators, and other small molecular effectors. Studies showed
that pH 6.5 favors nucleotidase activity and pH 7.3 is optimal
for phosphotransferase activity (2). Moreover, cN-II activity is
very sensitive to the presence of ATP, BPG, and potassium
salt, which are the known activators, and inorganic phosphate,
which is an inhibitor.
4
to 13 mM; however, its concentration in other tissues is at
least 2 to 3 magnitudes lower and can be neglected. To mimic
the physiological conditions in RBCs and other tissues, we
determined the kinetic parameters in the absence and pres-
ence of 3 mM BPG. As summarized in Table 2, in the absence
of BPG, the apparent K for ribavirin was 24 ␮M and kcat was
1
ence of 9.3 mM inorganic phosphate, 2.7 mM KCl, and 140
mM NaCl in PBS collectively contributes to a decrease of kcat
by 6.7-fold and Km by 4-fold. Overall, the resulting catalytic
efficiency of 0.058 min · ␮M is comparable to that in Tris
buffer. These results indicate that switching buffer from Tris to
PBS has no significant effect on ribavirin phosphorylation by
cN-II. Further kinetic analysis was performed in the presence
of BPG. Compared to the reaction without BPG, the presence
m
Ϫ1
.4 min . Compared to the reaction in Tris buffer, the pres-
Ϫ1
Ϫ1
Ϫ1
of 3 mM BPG increased kcat by 3-fold to 4.0 min and Km by
3
.7-fold to 88 ␮M (Table 2). These results confirm the assess-
ment in the literature that BPG can enhance nucleoside phos-
phorylation rate in the physiological conditions when both
ATP and inorganic phosphate are present. However, the pres-
ence of BPG does have a negative impact on substrate affinity,
yielding a similar catalytic efficiency in the absence and pres-
ence of 3 mM BPG. Nevertheless, when ribavirin concentra-
tion is below the Km value as in the plasma of treated HCV
patients, we anticipate a positive effect of BPG on ribavirin
phosphorylation.
Our studies indicate that like tiazofurin, ribavirin phosphor-
ylation can be catalyzed by a 5Ј-nucleotidase, cN-II, with a
catalytic efficiency much better than that by adenosine kinase.
Judged from the kinetic parameters obtained in PBS, its kcat is
Ϫ1
2.2-fold higher (4.0 versus 1.8 min ) and K is 6.1-fold lower
m
(
88 versus 540 ␮M) (Table 2). Remarkably, in contrast to most
cN-II substrates whose Km values are in the millimolar range
18), the Km for ribavirin is in the micromolar range, suggesting
DISCUSSION
(
Our steady-state kinetic studies indicate that although aden-
osine kinase can catalyze ribavirin phosphorylation in vitro, the
that the observed ribavirin phosphorylation by cN-II may be
physiologically relevant. The concomitant 5Ј-nucleotidase ac-
tivity of cN-II should have little effect on ribavirin phosphor-
ylation in vivo, as RMP is only an intermediate in conversion to
activity is unexpectedly low, with a K of 0.54 mM and a kcat of
m
Ϫ1
1
.8 min in PBS. Notably, the Km value is much higher than

2
170
WU ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
RTP. RMP is not known to accumulate to a high concentra-
tion, and it should not be susceptible to cN-II’s hydrolysis.
As phosphotransferase activity of cN-II is highly influenced
by the presence of various small molecular effectors, we made
an effort to include most notable cN-II effectors in our studies.
Interestingly, the overall catalytic efficiency of ribavirin phos-
phorylation in Tris buffer is comparable to that in PBS (Table
catabolic rate of RTP is a lot slower in RBCs than in either of
the nucleated cell lines, resulting in a very long half-time of
RTP in RBCs (24). If it turns out to be true in vivo, the slow
dephosphorylation of RTP or the lack of RTP-specific nucle-
otidase and/or phosphatase should play a key role in accumu-
lating RTP. On the other hand, the relatively fast anabolic rate
in RBCs should also contribute to the accumulation. Our stud-
ies reveal that cN-II is able to catalyze ribavirin phosphor-
ylation in vitro. Coincidently, cN-II is activated by BPG, a
bisphosphate metabolite that only amasses to a high concen-
tration in RBCs. It has been described that a high concentra-
tion of BPG can maximally stimulate cN-II’s activity and ren-
ders it almost insensitive to physiological concentrations of
ATP and inorganic phosphate (5). Our kinetic analysis sup-
ports BPG being a partial stimulator for ribavirin phosphory-
lation in the presence of ATP and inorganic phosphate. Al-
though it is hard to predict the exact role of BPG in ribavirin
phosphorylation based on in vitro kinetic analysis, it is possible
that BPG is a major stimulator for cN-II to drive the fast
anabolism of RTP in RBCs that contributes to hemolytic ane-
mia in ribavirin-treated patients.
2
). In addition to phosphate donor IMP and nucleotide acti-
vator ATP included in our assays, other small molecular effec-
tors, such as GMP, dGMP, dATP, and GTP are significantly
present in vivo (25). They will certainly influence cN-II’s ac-
tivity for ribavirin phosphorylation. Therefore, it is imperative
to conduct in vivo experiments to assess whether our in vitro
conclusions are plausible and determine how much each of
cN-II and adenosine kinase contributes to ribavirin phosphor-
ylation. The catalytic efficiency values reported in this study
reflect the intrinsic catalytic power of adenosine kinase and
cN-II towards ribavirin and viramidine phosphorylation. In
physiological conditions, the respective contribution of cN-II
and adenosine kinase for ribavirin and viramidine phosphory-
lation is likely determined by the concentrations of individual
enzyme as well as various effectors present in vivo. Our studies
do not exclude any other nucleoside kinase and related enzyme
that may be involved in ribavirin phosphorylation, nor do they
exclude the possibility of RMP formation from deamination of
VMP.
Like ribavirin, viramidine can be phosphorylated by cN-II in
vitro, albeit to a lesser degree. From our kinetic analysis, its
high Km (Ͼ0.5 mM) would make it unlikely to be efficiently
phosphorylated by cN-II in vivo. Because of the lower activity
of both adenosine kinase and cN-II towards viramidine, it is
possible that other nucleoside kinases or related enzymes are
involved in its phosphorylation in vivo.
Elucidation of ribavirin phosphorylation mechanism has im-
portant implications in understanding ribavirin metabolism in
vivo, especially in RBCs. Notably, among the HCV patients
treated with ribavirin, significant numbers of them suffer from
hemolytic anemia due to ribavirin phosphate accumulation in
RBCs. The ribavirin phosphate concentration can reach a level
that is 60- to 100-fold higher than its plasma concentration
ACKNOWLEDGMENTS
We are grateful to S. Eriksson for helpful discussion and suggestion
and for supplying us with purified deoxycytidine kinase, C. Cameron
for supplying purified adenosine kinase, and C.-C. Lin and L.-T. Yeh
for helpful discussion.
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