Inhibition of dengue virus RNA synthesis by an adenosine nucleoside

Article abstract of DOI:10.1128/AAC.00140-10

We recently reported that (2R,3R,4R,5R)-2-(4-amino-pyrrolo[2,3-d]pyrimidin- 7-yl)-3-ethynyl-5-hydroxymethyl-tetrahydro-furan-3,4-diol is a potent inhibitor of dengue virus (DENV), with 50% effective concentration (EC₅₀) and cytotoxic concentration (CC₅₀) values of 0.7 μM and >100 μM, respectively. Here we describe the synthesis, structure-activity relationship, and antiviral characterization of the inhibitor. In an AG129 mouse model, a single-dose treatment of DENV-infected mice with the compound suppressed peak viremia and completely prevented death. Mode-of-action analysis using a DENV replicon indicated that the compound blocks viral RNA synthesis. Recombinant adenosine kinase could convert the compound to a monophosphate form. Suppression of host adenosine kinase, using a specific inhibitor (iodotubercidin) or small interfering RNA (siRNA), abolished or reduced the compound's antiviral activity in cell culture. Studies of rats showed that ¹⁴C-labeled compound was converted to mono-, di-, and triphosphate metabolites in vivo. Collectively, the results suggest that this adenosine inhibitor is phosphorylated to an active (triphosphate) form which functions as a chain terminator for viral RNA synthesis. Copyright

Full text of DOI:10.1128/AAC.00140-10

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2010, p. 2932–2939
0066-4804/10/$12.00 doi:10.1128/AAC.00140-10
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 54, No. 7
Inhibition of Dengue Virus RNA Synthesis by an Adenosine Nucleosideᰔ†
Yen-Liang Chen,1§ Zheng Yin,1¶§ Jeyaraj Duraiswamy,1 Wouter Schul,1 Chin Chin Lim,1 Boping Liu,1
Hao Ying Xu,1 Min Qing,1 Andy Yip,1 Gang Wang,1 Wai Ling Chan,1 Hui Pen Tan,1 Melissa Lo,1
Sarah Liung,1 Ravinder Reddy Kondreddi,1 Ranga Rao,1 Helen Gu,2 Handan He,2
Thomas H. Keller,1* and Pei-Yong Shi1*
Novartis Institute for Tropical Diseases, 10 Biopolis Road, No. 05-01 Chromos, Singapore 138670, Singapore,1 and
Novartis Pharmaceuticals Corporation, East Hanover, New Jersey2
Received 1 February 2010/Returned for modification 26 April 2010/Accepted 30 April 2010
We recently reported that (2R,3R,4R,5R)-2-(4-amino-pyrrolo[2,3-d]pyrimidin-7-yl)-3-ethynyl-5-hydroxy-
methyl-tetrahydro-furan-3,4-diol is a potent inhibitor of dengue virus (DENV), with 50% effective concentration
(EC50) and cytotoxic concentration (CC50) values of 0.7 ␮M and >100 ␮M, respectively. Here we describe the
synthesis, structure-activity relationship, and antiviral characterization of the inhibitor. In an AG129 mouse
model, a single-dose treatment of DENV-infected mice with the compound suppressed peak viremia and
completely prevented death. Mode-of-action analysis using a DENV replicon indicated that the compound
blocks viral RNA synthesis. Recombinant adenosine kinase could convert the compound to a monophosphate
form. Suppression of host adenosine kinase, using a specific inhibitor (iodotubercidin) or small interfering
RNA (siRNA), abolished or reduced the compound’s antiviral activity in cell culture. Studies of rats showed
that 14C-labeled compound was converted to mono-, di-, and triphosphate metabolites in vivo. Collectively, the
results suggest that this adenosine inhibitor is phosphorylated to an active (triphosphate) form which func-
tions as a chain terminator for viral RNA synthesis.
The family Flaviviridae consists of three genera, Flavivirus,
Pestivirus, and Hepacivirus. Many viruses from the genus Fla-
vivirus are arthropod-borne and cause significant human dis-
eases. The four serotypes of dengue virus (DENV) alone pose
a health threat to 2.5 billion people worldwide, leading to 50 to
100 million human infections and 500,000 cases of dengue
hemorrhagic fever (DHF) and dengue shock syndrome (DSS)
each year (11). Besides DENV, other flaviviruses, such as West
Nile virus (WNV), Japanese encephalitis virus (JEV), yellow
fever virus (YFV), and tick-borne encephalitis virus (TBEV),
also cause frequent outbreaks throughout the world. No clin-
ically approved antiviral therapy is currently available for treat-
ment of flavivirus infections. Human vaccines are available
only for YFV, JEV, and TBEV. The development of a suc-
cessful DENV vaccine has been challenging due to the facts
that (i) the vaccine should simultaneously induce a long-lasting
protection against all four DENV serotypes and (ii) an incom-
pletely immunized individual may be sensitized to the life-
threatening DHF or DSS. The challenge associated with the
development of a vaccine underlines the importance of antivi-
ral development for DENV and other flaviviruses.
The flavivirus genome is a single-strand RNA of plus-sense
polarity. The single open reading frame from the 11-kb viral
genome encodes three structural proteins (capsid [C], pre-
membrane [prM], and envelope [E]) and seven nonstructural
proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).
Nonstructural proteins are responsible for viral replication. Of
those, NS3 and NS5 have enzymatic activities. The N-terminal
domain of NS3 (with NS2B as a cofactor) functions as a serine
protease, and the C-terminal domain acts as an RNA helicase,
an RNA triphosphatase, and an NTPase (10, 34, 35). The
N-terminal domain of NS5 serves as a methyltransferase, and
the C-terminal domain functions as an RNA-dependent RNA
polymerase (RdRp) (1, 8, 27, 32). Both viral and host proteins
are potential targets for antiviral development (22).
Nucleoside inhibitors represent the largest class of antiviral
drugs. These inhibitors act as chain terminators during viral
DNA (retroviruses and DNA viruses) or RNA (hepatitis C
virus [HCV]) synthesis (7). Upon entering cells, most nucleo-
side inhibitors need to be converted into their corresponding
triphosphates by viral or host kinases. The 5Ј-triphosphate
metabolites compete with the natural substrate in the viral
DNA or RNA polymerization reaction (4). Nucleoside analogs
with ribose 2Ј-C-methyl (3, 5) or 4Ј-C-azido modifications (13,
14) are potent inhibitors of HCV, a virus closely related to the
flaviviruses. Interestingly, the 7-deaza-2Ј-C-methyl-adenosine
also inhibits DENV in cell culture and in mouse models (29).
Although these nucleoside analogs possess an intact 3Ј-OH at
the ribose moiety, the 2Ј-C-methyl and 4Ј-C-azido modifica-
tions may impose steric hindrance for subsequent chain elon-
gation. We recently found that 7-deaza-2Ј-C-acetylene-adeno-
sine (Fig. 1, compound 1) is a potent inhibitor of flaviviruses
(36); however, a more detailed characterization is needed to
further understand the antiviral activity of this compound.
* Corresponding author. Mailing address: Novartis Institute for
Tropical Diseases, 10 Biopolis Road, No. 05-01 Chromos, 138670,
Singapore. Phone for Thomas H. Keller: 65 67222908. Fax: 65
67222910. E-mail: thomas.keller@novartis.com. Phone for Pei-Yong
Shi: 65 67222909. Fax: 65 67222916. E-mail: pei_yong.shi@novartis
.com.
¶ Present address: College of Pharmacy and the State Key Labora-
tory of Elemento-Organic Chemistry, Nankai University, Tianjin
300071, People’s Republic of China.
§ Y.-L.C. and Z.Y. contributed equally to this work.
† Supplemental material for this article may be found at http://aac
.asm.org/.
ᰔ Published ahead of print on 10 May 2010.
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AN ADENOSINE INHIBITOR OF DENGUE VIRUS RNA SYNTHESIS
2933
topyranoside (IPTG; 0.4 ␮M) was added to induce protein expression. After the
cells were cultured at 18°C for another 16 h, the cell pellet was harvested for
protein purification. The recombinant proteins, containing an N-terminal His6
tag, were purified through a nickel column. The protein was eluted using an
imidazole gradient of 0 to 500 mM. Fractions containing the recombinant protein
were pooled and further purified through a HiLoad 16/60 Superdex 75 gel
filtration column (GE Healthcare). The purified recombinant protein was stored
in aliquots at Ϫ80°C.
Transient DENV-2 replicon assay. DENV-2 replicon RNA (10 ␮g) was elec-
troporated into baby hamster kidney cells (BHK-21) cells (8 ϫ 106) as described
previously (30). The transfected cells were seeded into 12-well plates (3.2 ϫ 105
cells per well) and immediately incubated with 1 ␮M and 3 ␮M compound 1. As
a control, 0.9% dimethyl sulfoxide (DMSO) was added to the cells (because the
compound stock was dissolved in DMSO and assayed at a final concentration of
0.9% DMSO). Luciferase activity was measured as reported previously (25) at
time points indicated below. For viral protein analysis, the cells were lysed at 24 h
and 35 h posttransfection (p.t.) and assayed for NS3 expression using Western
blotting. The blotting used an NS3 antibody (1:5,000 dilution; generated in-house
in rabbit) and a mouse monoclonal anti-␥-tubulin antibody (1:1,000 dilution;
Sigma) as primary antibodies; horseradish peroxidase-labeled anti-rabbit and
anti-mouse IgG (1:8,000 dilution), respectively, were used as secondary an-
tibodies.
FIG. 1. Nucleoside flavivirus-specific inhibitor general structure A
and compounds 1 to 4.
Adenosine kinase assay. The adenosine kinase assay was performed in 20 mM
Tris-HCl, pH 7.5, 20 mM potassium phosphate, 100 ␮M MgCl2, 10 ␮M ATP, 10
␮Ci [␥-33P]ATP, 100 nM recombinant adenosine kinase, and 100 ␮M different
nucleosides or compound 1 in a total volume of 10 ␮l. After incubation at 23°C
for 1 h, the reactions were stopped by adding 10 ␮l of stop buffer (25 mM
Tris-HCl, pH 7.5, 75 mM NaCl, and 20 mM EDTA). The resulting mixture (l ␮l)
was spotted onto a polyethyleneimine (PEI)-cellulose thin-layer chromatography
(TLC) plate (Merck). The TLC plate was developed with solvent containing 0.4
M ammonium sulfate for 2 h. The plate was then dried and analyzed by auto-
radiograph.
siRNA transfection. Two sets of siRNAs were purchased from Dharmacon to
target human adenosine or deoxycytidine kinase. Each siRNA set contained a
mixture of four siRNAs, designed to maximize the chance of hitting the target
gene while minimizing hits of off-target genes. The siRNAs (10 pmol) were
mixed with 0.15 ␮l RNAiMax (Invitrogen) in 20 ␮l of Opti-MEM medium
(Gibco BRL); the mixture was incubated at 23°C for 20 min in a 96-well tissue
culture plate (Nunc). HEK-293 cells (2 ϫ 104 in 100 ␮l) containing DENV-2
luciferase replicons were then added to the wells. After the transfected cells were
incubated at 37°C for 24 h, various concentrations of compound 1 were added to
the cells. The cells were assayed for luciferase activity 3 days after treatment with
the compound using a Renilla luciferase kit (Promega).
Here we report the synthesis, structure-activity relationship
(SAR), and biological characterization of a number of nucleo-
sides with the general structure A which led to the identifica-
tion of compound 1 (Fig. 1). The results of biochemical exper-
iments showed that compound 1 could be converted to the
monophosphate by a recombinant human adenosine kinase.
Suppression of the adenosine kinase expression by small inter-
fering RNAs (siRNAs) or inhibition of the kinase activity by
iodotubercidin decreased the cellular activity of compound 1.
A
14C-labeled compound 1 was converted to mono-, di-, and
triphosphate derivatives in rats. Furthermore, we show that
compound 1 inhibits the viral RNA synthesis of DENV-1.
Remarkably, a single-dose treatment of DENV-infected mice
with compound 1 suppressed viremia and fully protected the
infected mice from death.
MATERIALS AND METHODS
In vivo phosphorylation of compound 1. Plasma or blood pellet samples were
obtained at different time points after a single intravenous (5 mg/kg of body
weight) or oral (25 mg/kg) dose of 14C-labeled compound 1 to male Han-Wistar
rats. Samples were extracted with ice-cold acetonitrile at a ratio of 3:1 (aceto-
nitrile/sample [vol/vol]) to precipitate proteins. The resulting samples were then
mixed by sonication using a Covaris sonicator (S2 series, SonoLab single-sample
preparation system) and centrifuged at 1,000 ϫ g for 10 min. The supernatants
were dried and reconstituted in 50 ␮l of solvent {10 mM ammonium formate
with 0.1% formic acid/methanol [98:2 (vol/vol)]} for sample analysis. The blood
pellet sample was reconstituted with 2 volumes of 10 mM ammonium formate
buffer containing 0.1% formic acid (pH 2.5) and then subjected to the extraction
method described above. The samples were analyzed by high-performance liquid
chromatography (HPLC) with off-line radioactive detection to determine the
metabolite profiles. Following HPLC separation, the structural identification of
metabolites was carried out using a Finnigan hybrid LTQ linear ion trap/orbitrap
or linear ion trap LTQ mass spectrometer (Thermo Electron).
Antiviral assays. Four types of antiviral assays were performed. (i) A cell-
based flavivirus immunodetection (CFI) assay (33) was used to study SAR.
Briefly, 2 ϫ 104 A549 cells were seeded per well of a 96-well plate. On the
following day, the cells were infected with DENV-2 at a multiplicity of infection
(MOI) of 0.3 for 1 h. During the 1-h incubation, the compound was added to the
viral inoculums. The culture fluid was then replenished with fresh medium
containing the compound. On day 2 p.i., the cells were fixed with methanol and
viral E protein detected by enzyme-linked immunosorbent assay (ELISA). The
ELISA used mouse monoclonal antibody 4G2 and goat anti-mouse IgG conju-
gated with horseradish peroxidase as the primary and secondary antibodies,
respectively. The 50% effective concentrations (EC50s) were calculated by non-
linear regression analysis using software Prism (Graphpad) or ActivityBase (ID
Business Solutions). (ii) A viral titer reduction assay was performed to test the
antiviral activities of compound 1 and other compounds (36). (iii) A luciferase-
reporting replicon of DENV-2 was used to estimate the effects of the compound
on viral translation and RNA synthesis (26). (iv) HEK-293 cells harboring the
DENV-2 luciferase replicon (21) were used to examine the effect of siRNAs
(targeting the human adenosine kinase or deoxycytidine kinase) on the efficacy
of compound 1. The procedures for the second to fourth antiviral assays de-
scribed above were described in detail in the references cited. The cytotoxic
concentration (CC50) values were estimated by measuring the intracellular level
of ATP (CellTiter-Glo luminescent cell viability assay; Promega) as previously
described (33).
RESULTS
Chemistry. The synthesis of the compounds is described in
detail in the supplemental material. Nucleoside analogs 2 to 4
were prepared as described in the literature (9, 12), and the
synthesis of compounds 11 to 14 is summarized in schemes 1 to
3 in the supplemental material. Dess-Martin oxidation of ␣-D-
1,3,5-tri-O-benzoyl-ribofuranose (compound 5) followed by reac-
tion of the ketone compound 6 with ethynylmagnesium bromide
or 1-propynyl magnesium bromide afforded the desired modifi-
Cloning, expression, and purification of human adenosine kinase. The cDNA
fragment of human adenosine kinase was PCR amplified from an IMAGE clone
(Invitrogen) and inserted into pET28a at the NdeI and BamHI restriction sites.
Deoxycytidine kinase was similarly cloned into the pET28a vector at the NheI
and XhoI restriction sites. The Escherichia coli BL21 RIL strain containing the
plasmid was cultured at 37°C to an optical density at 600 nm (OD600) of 0.4 to
0.5. The entire culture was cooled at 4°C for 1 h before isopropyl-␤-D-thiogalac-

2934
CHEN ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Results of 2Ј-C substitutions in the adenosine analogsa
TABLE 2. Selectivity between adenine and other basesa
Compound
Base
EC50 (␮M)b
CC50 (␮M)c
11
13
14
Adenine
Cytosine
Guanosine
1.41
Ͼ100
Ͼ100
40
Ͼ100
Ͼ100
Compound
2Ј-C substitutionb (R)
EC50 (␮M)c
CC50 (␮M)d
2
Me
1.12
Ͼ100
Ͼ50
Ͼ50
Ͼ100
Ͼ59
40
a All values are the means of the results of at least three independent exper-
iments.
b The EC50s were determined by cellular flavivirus immunodetection (CFI)
3
Et
4
11
12
CHACH2
C'C-H
C'C-Me
1.41
assay.
Ͼ100
Ͼ100
c The CC50s were estimated by measuring the intracellular level of ATP as
previously described (33).
a All values are the means of the results of at least three independent exper-
iments.
b Me, methyl; Et, ethyl.
c The EC50s were determined by cellular flavivirus immunodetection (CFI)
assay (see Materials and Methods for details).
d The CC50s were estimated by measuring the intracellular level of ATP as
previously described (33).
bition, we synthesized a panel of adenosine nucleosides con-
taining various 2Ј-C-alkyl substitutions (Table 1). For each
compound, the antiviral activity was tested by quantifying viral
E protein in a DENV-2 infection assay (CFI assay; see Mate-
rials and Methods for details); the cytotoxicity was monitored
by measuring the intracellular level of ATP (33). The CFI
assay consistently showed a signal-to-background ratio of 6- to
7-fold, with a ZЈ factor of about 0.75; when studying DENV
entry inhibitors, we previously showed that the EC50s derived
from the CFI assay were comparable to the EC50s derived
from the plaque reduction assays (33). The results (Table 1)
showed that compound 2 with a 2Ј-C-methyl substitution in-
hibited DENV, confirming the results of the previous studies
(20, 29). Substitution with 2Ј-C-ethyl (compound 3) and 2Ј-C-
vinyl (compound 4) abolished the antiviral activity. In contrast,
a 2Ј-C-acetylene substitution led to a potent nucleoside (com-
pound 11) with an EC50 of 1.41 ␮M; however, the compound
showed some cytotoxicity, with a CC50 of 40 ␮M. Further
replacement of the 2Ј-C-acetylene proton with a methyl group
(compound 15) led to a loss of activity. The results demon-
strate that adenosine with a 2Ј-C-acetylene substitution is a
potent inhibitor of DENV.
cation of ribose. Further benzoylation of the tertiary alcohol gave
the intermediate compounds 7 and 8, respectively (scheme 1 in
the supplemental material). The bases were then introduced us-
ing Vorbruggen methodology followed by aminolysis, providing
access to nucleoside analogs 11 to 13 (9, 12). Reaction of
6-chloro-9H-purin-2-ylamine with compound 7 and hydrolysis of
the resulting chloro intermediate with NaOH led to compound 14
(schemes 2 and 3 in the supplemental material).
The synthesis of compounds with a substituent at the C-7
position of the base is depicted in scheme 4 in the supplemen-
tal material. Ketone compound 15 was prepared as described
in the literature (2). A Grignard reaction with ethynylmagne-
sium bromide followed by protection of the tertiary alcohol by
a 2,5-dichlorobenzyl group and deprotection of the acetonide
furnished the diol compound 16. Protection of the diol with
toluoylchloride followed by removal of the acetonide led to com-
pound 17. Oxidation of compound 17 with periodic acid followed
by treatment with triethylamine and BCl3 cleavage of the benzyl-
protecting group gave the key intermediate compound 18. Epoxi-
dation followed by treatment with the modified nucleobases in
the presence of NaH yielded the corresponding protected
nucleoside analogs 19a to 19c. Deprotection of the toluoyl-
protecting groups followed by aminolysis provided the mod-
ified nucleoside analogs 1 and 20 to 22.
To examine whether other base types with a 2Ј-acetylene
ribose would also inhibit DENV, we prepared a panel of 2Ј-
C-acetylene derivatives containing cytosine (compound 13)
and guanosine (compound 14) (Table 2). None of the base
substitutions yielded active compounds. The results suggest
that only adenosine with a 2Ј-C-acetylene ribose substitution is
active against DENV.
The required modified nucleobases for the above synthesis
were prepared as shown in scheme 5 in the supplemental
material. Commercially available 4-chloro-7H-pyrrolo[2,3-d]
pyrimidine (compound 23) was converted to 4-chloro-5-fluoro-
7H-pyrrolo[2,3-d]pyrimidine (compound 24) with Selectfluor.
The nitrile derivative compound 26 was prepared from com-
pound 23 in a two-step process using an iodination-cyanation
sequence. Finally, compound 23 was converted to compound
27 with N-bromosuccinimide, followed by lithium exchange
and quenching with methylchloroformate to give the required
carboxylic acid methyl ester compound 28.
It was previously shown that adenosine with a ribose 2Ј-C-
alkyl substitution could be deaminated by cellular adenosine
deaminase to an inosine derivative (6) which was not active
against DENV (data not shown). Since this metabolic pathway
is also potentially relevant for compound 11, we replaced the
adenine C-7 nitrogen with a carbon (compound 1; Table 3).
This modification slightly improved the anti-DENV activity but
significantly reduced the cytotoxicity (CC50 Ͼ 100 ␮M). We
further explored the possible substitutions at the adenine C-7
position (Table 3). When the C-7 proton was replaced with F,
the compound (compound 20) became slightly more potent
(EC50, 0.4 ␮M) but more cytotoxic (CC50, 44 ␮M). CN (com-
pound 21) and CONH2 substitutions (compound 22) led to less
potent compounds, with EC50s of 3.1 ␮M and 2.0 ␮M, respec-
SAR analysis. Nucleoside analogs with the general structure
A (Fig. 1) are potent inhibitors of various members of the
genus Flaviviridae (20). To explore the SAR for DENV inhi-

VOL. 54, 2010
AN ADENOSINE INHIBITOR OF DENGUE VIRUS RNA SYNTHESIS
2935
TABLE 3. Results of C-7 substitutions in 2Ј-acetylene-7-
deaza-adenosinea
Adenine C-7
substitution (R)
Compound
EC50 (␮M)b
CC50 (␮M)c
1
H
0.7
0.42
3.1
Ͼ100
44
Ͼ100
62
20
21
22
F
CN
CONH2
2.0
a All results are the means of the results of at least three independent exper-
iments.
b The EC50s were determined by cellular flavivirus immunodetection (CFI)
assay.
c The CC50s were estimated by measuring the intracellular level of ATP as
previously described (33).
tively. Overall, the SAR study identified 7-deaza-2Ј-C-acety-
lene-adenosine (compound 1) as a potent DENV inhibitor,
which was selected for subsequent biological characterization.
Inhibition of DENV RNA synthesis. We confirmed the an-
tiviral activity of compound 1 using a viral titer reduction assay
(36). Baby hamster kidney cells (BHK-21) were infected with
DENV-2 at an MOI of 0.3; viral titers in culture fluid were
measured at 48 h postinfection (p.i.). Treatment of the infected
cells with compound 1 suppressed viral yield in a dose-respon-
sive manner, with an EC50 of 0.7 ␮M; incubation of the culture
with 6 ␮M compound reduced the viral titer by Ͼ160-fold (Fig.
2A). Changes in MOI from 0.01 to 1 did not significantly affect
the EC50s of compound 1 (data not shown). A cell viability
assay showed that the compound was not cytotoxic up to 100
␮M, the highest concentration tested (Fig. 2B).
Next, we used a DENV-2 luciferase-reporting replicon to
analyze the mechanism of inhibition. Transfection of BHK-21
cells with the replicon revealed two luciferase peaks. The first
peak occurred at 2 to 4 h p.t., indicative of input replicon
translation; the second peak occurred at Ͼ24 h p.t., indicative of
RNA synthesis (16, 26). Treatment of the replicon-transfected
cells with compound 1 did not affect the luciferase signals at 2 to
4 h but significantly suppressed the luciferase activities at 24 h and
35 h p.t. (Fig. 2C). Western blotting results showed that less viral
NS3 protein was expressed when the transfected cells were
treated with the compound (Fig. 2D). Collectively, the results
demonstrate that the compound inhibits DENV through suppres-
sion of viral RNA synthesis. This conclusion is further supported
by our previous results showing that the triphosphate form of
compound 1 could directly inhibit RNA synthesis in a recombi-
nant RdRp assay (36).
Phosphorylation of compound 1 by recombinant adenosine
kinase. To exhibit antiviral activity, compound 1 should be
converted to the triphosphate form. Adenosine kinase is pos-
tulated to be the first enzyme in this intracellular sequence,
converting compound 1 to a monophosphate form (18). To test
this hypothesis, we cloned and expressed the human adenosine
kinase using an E. coli system. The recombinant adenosine
kinase, with an N-terminal His6 tag, was purified through a
FIG. 2. Inhibition of DENV RNA synthesis by compound 1. (A) Re-
sults of viral titer reduction assay. BHK-21 cells were infected with
DENV-2 (MOI of 0.3) and immediately treated with compound 1 at the
indicated concentrations. Viral titers in culture fluid at 48 h p.i. were
quantified by plaque assays. Average results and standard deviations (n ϭ
3) are presented. (B) Results of cytotoxicity assay. BHK-21 cells were
incubated with compound 1 for 48 h. A CellTiter-Glo luminescent assay
was used to measure the intracellular level of ATP (to indicate cell via-
bility) according to the manufacturer’s protocol (Promega). Cell viability
is presented as the percentage of luminescence derived from the com-
pound-treated cells compared with the luminescence from the mock-
treated cells. Average results and standard deviations (n ϭ 3) are pre-
sented. (C) Results of transient DENV-2 replicon assay. A DENV-2
replicon containing a luciferase reporter was used to measure the effects
of the compound on viral translation and RNA synthesis. The effects of
compound 1 (1 ␮M and 3 ␮M) on viral translation and RNA synthesis
were examined by measuring the luciferase signals at 2 to 6 h p.t. and at
Ն24 h p.t., respectively. Average results and standard deviations (n ϭ 3)
are presented. (D) Results of Western blotting of viral NS3 protein. The
expression levels of viral NS3 protein from the cells at 24 h and 35 h p.t.,
described for panel C, were analyzed using Western blotting. Host protein
␥-tubulin was used as loading control (see Materials and Methods for details).

2936
CHEN ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 3. Conversion of compound 1 to its monophosphate form by
recombinant adenosine kinase. (A) Purified recombinant human adeno-
sine kinase. See Materials and Methods for cloning, expression, and pu-
rification of the adenosine kinase. Fractions from the gel filtration column
(the final purification step) were analyzed on a 10% SDS–PAGE gel. The
recombinant adenosine kinase is indicated by an arrow. (B) Thin-layer
chromatography analysis of monophosphate nucleosides. Compound 1,
adenosine, uridine, guanosine, and cytidine were incubated with
[␥-33P]ATP in the presence of adenosine kinase. One microliter of the
reaction mixture was spotted on the original and analyzed on a TLC plate.
The plate was then dried and exposed to a film. Cold markers, ATP, AMP,
and compound 1 monophosphate (indicated as NMP on the side of the TLC
image), were analyzed on the TLC plate and detected under UV light.
FIG. 4. Effect of adenosine kinase on antiviral activity. (A) Ef-
fect of iodotubercidin on antiviral efficacy. A CFI antiviral assay
(see Materials and Methods for details) was performed using com-
pound 1 in the presence or absence of iodotubercidin (0.2 ␮M). The
antiviral activity is presented as the percent inhibition. (B) Effect of
siRNAs on antiviral activity. HEK-293 cells harboring the DENV-2
luciferase replicon were cotransfected with a set of four siRNAs
targeting human adenosine kinase or deoxycytidine kinase. The
target sequences of each interfering RNA are shown (top panel).
The cells were treated with compound 1 at the indicated concen-
trations at 24 h p.t. and assayed for luciferase activities at 72 h
after treatment with the compound. The antiviral activity is pre-
sented as the percent luciferase activity. The averages of the results
of two independent experiments are shown.
Ni2ϩ column, followed by gel filtration chromatography (Fig.
3A). A sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE) analysis showed a protein of the ex-
pected size of 39 kDa in fractions 5 to 10 from the gel filtration
column; the protein purity was Ͼ90%. The recombinant pro-
tein phosphorylated both compound 1 and adenosine to their
monophosphate forms, although the phosphorylation of com-
pound 1 was less efficient than that of the natural substrate
adenosine, as judged by the intensities of their monophosphor-
ylated derivatives analyzed on a thin-layer chromatograph
(TLC) (Fig. 3B). The adenosine kinase did not phosphory-
late uridine, guanosine, or cytidine. Since deoxycytidine ki-
nase was reported to be a promiscuous kinase capable of
phosphorylating both purine and pyrimidine nucleosides
(28), we also prepared recombinant human deoxycytidine
kinase. However, the recombinant deoxycytidine kinase did
not phosphorylate compound 1 (data not shown). The re-
sults suggest that adenosine kinase is responsible for the
phosphorylation of compound 1.
Effects of adenosine kinase on antiviral activity in cell cul-
ture. We took two approaches to demonstrate the biological
relevance of the cellular adenosine kinase to the antiviral ac-
tivity of compound 1. If the adenosine kinase is essential for
the antiviral activity, inactivation of the kinase activity (using
an inhibitor, first approach) or suppression of the kinase ex-
pression (using siRNAs, second approach) should reduce the
potency of the compound. For the first approach, DENV-2-
infected human alveolar basal epithelial cells (A549) were
treated with compound 1 in the presence or absence of iodo-
tubercidin, a known inhibitor of adenosine kinase (17). As
shown in Fig. 4A, the addition of iodotubercidin (0.2 ␮M)
completely abolished the antiviral activity of compound 1 (up
to 20 ␮M, the highest concentration tested). No cytotoxicity of
iodotubercidin was detected at the concentrations tested (data
not shown). For the second approach, a set of four siRNAs
(Fig. 4B) targeting different regions of the human adenosine
kinase mRNA were cotransfected into human embryonic kidney
cells (HEK-293) harboring the DENV-2 luciferase replicon (21).
The replicon cells were then treated with various concentrations
of compound 1. As shown in Fig. 4B, transfection of the siRNAs
targeting the adenosine kinase shifted the EC50 from 0.79 ␮M to
9.2 ␮M, resulting in an 11-fold decrease in antiviral efficacy. The
adenosine kinase siRNAs did not completely abolish the antiviral
activity of compound 1, possibly due to residual expression of
adenosine kinase. As a negative control, cotransfection of a set of
four siRNAs targeting the deoxycytidine kinase did not affect the
potency of compound 1. No observable cytotoxicity was observed
when cells were transfected with the siRNAs alone (data not

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AN ADENOSINE INHIBITOR OF DENGUE VIRUS RNA SYNTHESIS
2937
FIG. 5. Conversion of compound 1 to mono-, di-, and triphosphate derivatives in vivo. (A) Analysis of metabolite in rat plasma. The 14C-labeled
compound 1 was either intravenously dosed at 5 mg/kg or orally dosed at 25 mg/kg in rats. Plasma was collected from 4 to 24 h after dosing.
Metabolites of compound 1 were extracted from the pooled plasma and analyzed using FPLC (see Materials and Methods for details). (B) Analysis
of metabolites in blood cells. Total blood cells were harvested at 1 to 12 h after dosing. The cell pellets collected at different time points were pooled
together, and compound metabolites extracted. The extracts were then subjected to HPLC analysis.
shown). These results demonstrate that adenosine kinase plays an
important role in activating the antiviral activity of compound 1 in
cell culture.
Conversion of compound 1 to mono-, di-, and triphosphate
forms in rats. We examined the phosphorylation profile of
compound 1 in rats using a 14C-labeled compound. The com-
pound was 14C labeled at the C-2 position of the adenine base.
After the rats were dosed with 5 mg/kg of compound 1 intrave-
nously or with 25 mg/kg orally, nucleoside metabolites were ex-
tracted from plasma and blood cells, respectively. Fast-protein
liquid chromatography (FPLC) analysis of these extracts
showed that only the parent compound was detected in the
plasma (Fig. 5A), whereas the mono-, di-, and triphosphate
derivatives, together with the parent compound, were de-
tected in the blood cells (Fig. 5B). The results demonstrate
that compound 1 can be converted into the corresponding
triphosphate form in vivo.
Single-dose efficacy of compound 1 in dengue mouse models.
We recently reported the potent antiviral activity of compound
1 in repeat-dose regimens in DENV-infected mice (36). How-
ever, the single-dose efficacy of compound 1 remains to be
determined in vivo. We examined this possibility in both
DENV viremia and lethal mouse models. For the viremia
model, AG129 mice (defective in alpha/beta interferon [IFN-
FIG. 6. A single-dose efficacy of compound 1 was seen in mouse
␣/␤] and IFN-␥ receptors) were intraperitoneally infected with
DENV models. (A) Results for a mouse model of viremia (29). AG129
mice were intraperitoneally inoculated with 2 ϫ 106 PFU of DENV-2
(strain TSV01). The mice (8 animals per group) were orally dosed once
with the indicated amount of compound 1 at 12 h p.i. The peak viremia on
day 3 p.i. was measured by plaque assay. An asterisk indicates that the
difference between the compound-treated and the vehicle-treated group
is statistically significant (P value Ͻ 0.05 using Student’s t test). Error bars
show standard deviations. (B) Results for lethal mouse model. AG129
mice (6 mice per group) were intravenously inoculated with 3 ϫ 107 PFU
of DENV-2 strain D2S10 (31), treated with a single dose of 75 mg/kg of
compound 1 at 12 h p.i., and monitored for mortality. The detailed
protocols for the mouse experiments were reported previously (36). The
difference between the compound-treated and the vehicle-treated groups
is statistically significant at a P value of Ͻ0.01.
DENV-2 (strain TSVO1) and treated with a single oral dose of
compound 1 (from 25 to 300 mg/kg) at 12 h postinfection. The
animals were sacrificed at the peak of viremia on day 3 p.i., and
plasma viremia was quantified by plaque assay. As shown in
Fig. 6A, the viremia level was reduced in a dose-dependent
manner from 25 to 75 mg/kg, achieving a Ͼ10-fold reduction
after a single-dose treatment of 75 mg/kg. Higher dosing at 150
and 300 mg/kg did not further lower the viremia, indicating
that the uptake of compound 1 reached saturation at 75 mg/kg.
For comparison, we also included the results from the optimal
multiple-dose regimen (twice daily dosing at 25 mg/kg per dose

2938
CHEN ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
for 3 days starting immediately after infection, equating to a
total exposure of 150 mg/kg).
the antiviral outcome. An inactive nucleoside may be the result
of an inefficient conversion to the triphosphate form. For this
reason, nucleoside inhibitors are often discovered serendipi-
tously. To determine the intrinsic potency against viral poly-
merase, one needs to synthesize the triphosphate compound
and test it directly in a viral polymerase assay. Different cell
lines may have different expression levels of nucleoside ki-
nases, leading to discrepant EC50s of the same compound (15).
It is therefore useful to evaluate nucleoside inhibitors in more
than one clinically relevant cell line.
Since the conversion of a nucleotide to its monophosphate
form is often the rate-limiting step in triphosphorylation (19),
we examined the monophosphorylation of compound 1. Re-
combinant human adenosine kinase could efficiently convert
the compound to its monophosphate form (Fig. 3). Inhibition
of adenosine kinase using a specific inhibitor (iodotubercidin)
or siRNAs abolished or reduced the compound’s antiviral ac-
tivity (Fig. 4). In contrast, recombinant deoxycytidine kinase
did not phosphorylate compound 1; treatment of cells with
siRNAs targeting the deoxycytidine kinase did not affect the
potency of compound 1. These results indicate that adenosine
kinase may be responsible for intracellular monophosphoryla-
tion of the compound. For nucleosides that could not be effi-
ciently converted to their monophosphate forms, one could
synthesize the monophosphate or phosphonate prodrugs. The
phosphoramidate approach was successfully used to convert
inactive nucleoside analogs to potent inhibitors of HCV (23,
24). Other examples of nucleotide phosphonates are adefovir
disoproxil and tenofovir disoproxil, which are in clinical use for
treatment of hepatitis B virus and HIV, respectively.
Another challenge for the development of nucleoside anti-
virals is the unpredictable toxicity. This was experienced during
the development of compound 1. The compound did not show
any negative signs in in vitro assays, including a mitochondrial
toxicity assay, the Ames test for genotoxicity, the hERG chan-
nel test for cardiovascular toxicity, and a micronucleus assay
for mutagenicity, as well as various receptor, ion channel, and
kinase assays. However, when rats (10 mg/kg/day) and dogs (1
mg/kg/day) were dosed with the compound daily for 2 weeks, a
no-observed-adverse-effect level (NOAEL) could not be
achieved; NOAEL was achieved when rats were orally dosed with
compound 1 at 50 mg/kg/day for 1 week (36). Experiments are
ongoing to determine the nature of the toxicity of compound 1.
Two major issues remain to be addressed for further devel-
opment of compound 1. One issue is related to the in vivo
efficacy. Although the AG129 mouse model could be used to
estimate the antiviral efficacy (29), caution should be taken
when interpreting the results from such experiments. Since the
AG129 mouse lacks IFN-␣/␤ and IFN-␥ receptors, the mouse
is defective in an innate immune response that plays a critical
role in host defense when encountering a pathogen. Thus, this
model may underestimate the real efficacy of the compound.
Another issue is related to the length of therapeutic treatment
versus the 2-week NOAEL result. Dengue is an acute disease
with a fever duration of less than a week (11), so the length of
treatment is expected to be less than a week. This is in contrast to
the treatment of chronic diseases which require long-term treat-
ment (such as HCV and HIV). The difference between the acute
and chronic diseases should be considered during preclinical de-
velopment of DENV inhibitors. Notably, the above-described
Next, we used a lethal mouse model to determine the anti-
viral effect of a single-dose treatment on disease severity.
AG129 mice were intravenously infected with a lethal dose
(3 ϫ 107 PFU) of the mouse-adapted DENV-2 strain D2S10
(31) and treated with a single 75-mg/kg dose of compound 1 at
12 h postinfection. The single-dose treatment provided full
protection against the lethal virus challenge (Fig. 6B). The
single-dose efficacy is similar to the results when mice were
treated with a multiple-dose regimen (twice-daily dosing at 10
mg/kg per dose for 3 days, which was previously shown to be
the lowest dose to give full protection [36]). Collectively, the
results demonstrated that a single-dose treatment of DENV-
infected mice with compound 1 suppressed peak viremia and
completely protected the infected mice from death.
DISCUSSION
A number of strategies have been pursued to develop flavi-
virus inhibitors (22). Since nucleoside analogs have been used
successfully as therapy for other viral diseases, we took this
approach to develop DENV inhibitors. Our chemistry efforts
led to the discovery of compound 1 as a potent inhibitor of
DENV. We recently showed that this compound was active in
vivo; a multiple-dose treatment suppressed peak viremia, re-
duced cytokine elevation, and completely protected the in-
fected mice from death (36). In this study, we demonstrated
that a single-dose treatment of DENV-infected mice with com-
pound 1 could also suppress peak viremia by Ͼ10-fold and
completely protected the infected mice from death. To our
knowledge, compound 1 represents the first small-molecule
inhibitor that shows a potent in vivo efficacy against a flavivirus
after a single-dose treatment.
Our results strongly indicate that compound 1 exerts its
antiviral activity through RNA chain termination. This conclu-
sion is supported by three lines of evidences. First, using a
DENV replicon, we showed that the compound suppressed
viral RNA synthesis; it did not inhibit viral translation (Fig. 2).
Second, we previously showed that the triphosphate form of
compound 1 was able to directly inhibit RNA elongation cat-
alyzed by a recombinant DENV NS5 (36). Third, cocrystalli-
zation of DENV RdRp in complex with the triphosphate form
of compound 1 showed that the triphosphate moiety was lo-
cated at the active site of the RdRp (S. S. Yeong, T. L. Yap,
Y.-L. Chen, D. Luo, P. Niyomrattanakit, H. Dong, C. C. Lim,
P.-Y. Shi, and J. Lescar, unpublished results). The results also
lead to the inference that upon entering cells, the compound is
phosphorylated to its triphosphate form before it can be uti-
lized by viral RdRp to terminate RNA synthesis. This notion is
further supported by the results showing that after oral dosing
of rats with compound 1, the compound was converted to the
mono-, di-, and triphosphate forms in blood cells (Fig. 5).
The nucleoside-based approach for antiviral development
faces several challenges. Because multiple cellular enzymes are
required to convert a nucleoside (inactive) into its triphosphate
form (active), the antiviral efficacy is a combined outcome
derived from multiple enzymatic reactions. Besides the intrin-
sic potency against the viral polymerase, the efficiency of ki-
nase-mediated phosphorylation may contribute significantly to

VOL. 54, 2010
AN ADENOSINE INHIBITOR OF DENGUE VIRUS RNA SYNTHESIS
2939
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issues are related to each other. An improved animal model that
allows a better estimation of in vivo potency will be critical to set
up an appropriate level for a preclinical toxicity study.
This study has extended our previous finding that an aden-
osine nucleoside is a potent inhibitor of DENV. A single-dose
treatment with the compound completely protected the
DENV-infected mice from death. Upon entering cells, the
compound is likely converted to its monophosphate form by
adenosine kinase and further metabolized to its active triphos-
phate forms. The compound functions as a chain terminator to
inhibit viral RNA synthesis.
ACKNOWLEDGMENT
We thank Julien Lescar for help and discussion during the course of
the project.
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