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Antimicrobial Agents and Chemotherapy, August 1998, p. 2095-2102, Vol. 42, No. 8
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Mode of Action of
(1'S,2'R)-9-{[1',2'-Bis(hydroxymethyl) cycloprop-1'-yl]methyl}guanine
(A-5021) against Herpes Simplex Virus Type 1 and Type 2 and
Varicella-Zoster Virus
Nobukazu
Ono,1,2,*
Satoshi
Iwayama,1
Katsuya
Suzuki,1
Takaaki
Sekiyama,3
Harumi
Nakazawa,1
Takashi
Tsuji,3
Masahiko
Okunishi,1
Tohru
Daikoku,2 and
Yukihiro
Nishiyama2
Life Science Laboratories, Ajinomoto Co.,
Inc., Totsuka-ku, Yokohama 244,1
Central
Research Laboratories, Ajinomoto Co., Inc., Kawasaki-ku, Kawasaki
210,3 and
Laboratory of Virology,
Research Institute for Disease Mechanism and Control, Nagoya
University School of Medicine, Showa-ku, Nagoya
466,2 Japan
Received 31 December 1997/Returned for modification 31 March
1998/Accepted 5 June 1998
 |
ABSTRACT |
The mode of action of
(1'S,2'R)-9-{[1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl}guanine
(A-5021) against herpes simplex virus type 1 (HSV-1), HSV-2, and
varicella-zoster virus (VZV) was studied. A-5021 was
monophosphorylated at the 2' site by viral thymidine kinases (TKs).
The 50% inhibitory values for thymidine phosphorylation of
A-5021 by HSV-1 TK and HSV-2 TK were comparable to those for
penciclovir (PCV) and lower than those for acyclovir (ACV). Of these
three agents, A-5021 inhibited VZV TK most efficiently. A-5021 was
phosphorylated to a mono-, di-, and triphosphate in MRC-5 cells
infected with HSV-1, HSV-2, and VZV. A-5021 triphosphate accumulated
more than ACV triphosphate but less than PCV triphosphate in MRC-5
cells infected with HSV-1 or VZV, whereas HSV-2-infected MRC-5
cells had comparable levels of A-5021 and ACV triphosphates. The
intracellular half-life of A-5021 triphosphate was considerably longer
than that of ACV triphosphate and shorter than that of PCV
triphosphate. A-5021 triphosphate competitively inhibited HSV DNA
polymerases with respect to dGTP. Inhibition was strongest with ACV
triphosphate, followed by A-5021 triphosphate and then (R,S)-PCV triphosphate. A DNA chain elongation
experiment revealed that A-5021 triphosphate was
incorporated into DNA instead of dGTP and terminated elongation,
although limited chain extension was observed. Thus, the strong
antiviral activity of A-5021 appears to depend on a more rapid and
stable accumulation of its triphosphate in infected cells than that of
ACV and on stronger inhibition of viral DNA polymerase by its
triphosphate than that of PCV.
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INTRODUCTION |
A-5021, or
(1'S,2'R)-9-{[1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl}guanine,
is a novel guanosine analog with potent antiviral activity against
herpes simplex virus type 1 (HSV-1), HSV-2, varicella-zoster virus
(VZV), and human cytomegalovirus (14, 23). The in vitro antiviral activity of A-5021 is higher than that of acyclovir (ACV) or
penciclovir (PCV) against HSV-1, HSV-2, and VZV (14).
Nucleoside analogs having highly selective antiherpetic activity are
selectively phosphorylated by viral thymidine kinases (TKs) in
herpesvirus-infected cells (5). A-5021 shows reduced antiviral activity against TK-deficient strains of HSV, suggesting that
this agent requires selective phosphorylation by herpesvirus TK, as is
the case for ACV (11). The monophosphates of antiviral nucleosides are subsequently phosphorylated to triphosphates by cellular kinases (20, 21). These triphosphates accumulate in
virus-infected cells and inhibit viral DNA polymerases in a competitive
and chain-terminated fashion (7, 9, 10, 14, 18). Some
nucleoside analogs like PCV have prolonged antiviral activities derived
from the long intracellular half-lives of their triphosphates (2,
3, 7, 25). A-5021 also shows a prolonged in vitro antiviral
effect (14).
In this study, we sought to clarify the mechanism of the highly potent
and prolonged antiviral activity of A-5021 and examined its
phosphorylation by viral TKs, the inhibition of viral DNA polymerases
by A-5021 triphosphate, and the intracellular metabolism of this agent
in herpesvirus-infected cells compared with those of ACV and PCV
triphosphates.
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MATERIALS AND METHODS |
Cell culture and viruses.
MRC-5 cells (Dainippon
Pharmaceutical Co. Ltd., Osaka, Japan) and human embryo lung cells were
grown in Dulbecco's modified Eagle's medium supplemented with 10%
inactivated fetal bovine serum. Vero C1008 cells (Dainippon) and BU25
TK
cells (Dainippon) were grown in Eagle's minimum
essential medium supplemented with 10% fetal bovine serum.
Stocks of HSV (KOS), HSV-2 (UW-268), and VZV (Kawaguchi) were prepared
as described previously (14).
Compounds.
A-5021, ACV, and PCV were prepared at Ajinomoto
Co., Inc. (Kawasaki, Japan) (13, 23, 24).
(1'-Methylene-3H)-A-5021 was prepared by reduction of
(1'S,5'R)-9-[3'-oxa-2'-oxobicyclo(3.1.0)hex-1'-yl]methylguanine
as the lactone precursor of A-5021 (radiochemical purity, >97%;
chemical purity, >99%; specific activity, 15 Ci/mmol).
(4'-3H)-PCV was prepared by reduction of
9-[3-(diethoxycarbonyl)propyl]guanine (radiochemical purity, >99%;
chemical purity, >99%; specific activity, 63 Ci/mmol). ACV with a
2'-3H side chain (21.5 or 25.1 Ci/mmol) was
obtained from NEN Research Products (Stevenage, United Kingdom).
(1',2'-3H)-dGTP (32 Ci/mmol),
(methyl-[3H])thymidine (43 Ci/mmol),
[
-32P]dCTP, and [
-32P]ATP were
obtained from Amersham International plc (Little Chalfont, Buckinghamshire, United Kingdom).
A-5021-2'-monophosphate (Fig.
2b) was prepared by phosphorylation of a
regioselectively protected derivative. To 2.06 g (2.15
mmol) of
(1'
S,2'
R)-6-ben- zyloxy-2-(4,4'-dimethoxyltrityl)amino-9-({[1'-(4,4'-dimethoxyltrityl)oxymethyl-
2'-hydroxymethyl]cycloprop-1'-yl}methyl)purine
in dry tetrahydrofuran
(THF) (4.3 ml) and pyridine (21.5 mmol; 1.74 ml)
was added dibenzyl
phosphochloridate (21.5 mmol) in THF (4.3 ml). After
1 h of stirring
at room temperature, 20 ml of 50% aqueous
pyridine was added.
The mixture was concentrated in vacuo, and the
residue was purified
by silica gel column chromatography (0.5 to 1%
methanol in dichloromethane).
The dibenzyl phosphate was treated in
80% aqueous acetic acid
for 2 h to remove the trityl groups,
purified by silica gel column
chromatography (1.5 to 4% methanol in
dichloromethane), and then
reduced by catalytic hydrogenation with 10%
palladium/carbon in
80% aqueous acetic acid in a hydrogen atmosphere
(4 atm) for 40
h. After purification by C
18
reversed-phase chromatography, 230
mg (31%) of
A-5021-2'-monophosphate was obtained. The purity of
the compound was
96% by high-performance liquid chromatography
(HPLC);
1H
NMR (dimethyl sulfoxide [DMSO] d-6)
0.41 (t,
J = 4.8 Hz, 1H),
0.93 (dd,
J = 4.8, 8.7 Hz, 1H), 1.32 (m,
1H), 3.24 (d,
J = 12.6
Hz, 1H), 3.38 (d,
J = 12.6 Hz, 1H), 3.73 (d,
J = 14.1 Hz,
2H),
3.85 (m, 2H), 4.12 (d,
J = 14.1 Hz, 1H), 6.42 (bs,
2H), 7.67 (s,
1H), 10.54 (bs, 1H); high-resolution fast atom
bombardment-mass
spectrometry (FAB-MS) calculated
m/z
346.0916 (MH
+) for
C
11H
17O
6N
5P; found
m/z 346.0934 (MH
+).
As a reference, A-5021-1'-monophosphate (Fig.
2a) was prepared
starting with
(1'
S,2'
R)-6-benzyloxy-2-(4,4'-dimethoxyltrityl)amino-9-({[2'-(4,4'-dimeth-
oxyltrityl)oxymethyl-1'-hydroxymethyl]cycloprop-1'-yl}methyl)purine as de-
scribed
above. After purification by C
18 reversed-phase
chromatography,
A-5021-1'-monophosphate was obtained at
a 19% yield with 97% purity.
1H NMR (DMSO d-6)
0.44 (m, 1H), 0.90 (dd,
J = 5.1, 8.7 Hz, 1H),
1.28 (m, 1H),
3.38 (dd,
J = 8.1, 11.7 Hz, 1H), 3.51 (dd,
J = 6.3,
11.7 Hz, 1H), 3.78 (m, 2H), 3.88 (d,
J = 14.1 Hz, 1H), 3.99 (d,
J = 14.1 Hz,
1H), 6.38 (bs, 2H), 7.81 (s, 1H), 10.50 (bs, 1H);
FAB-MS
m/z
346 (MH
+).
A-5021-2'-triphosphate was prepared by phosphorylation of
A-5021-2'-monophosphate with pyrophosphate.
A-5021-2'-monophosphate
(34.56 mg; 0.1 mmol) suspended in 10 ml
of 50% methanol in ethanol
was mixed with an equimolar amount of
tri(
n-butyl)amine and heated
at 80°C for 4 h. After
concentration in vacuo, the residue was
dissolved in 1 ml of
N,
N-dimethylformamide (DMF) and 81.1 mg (0.50
mmol) of 1,1'-carbonyldiimidazole in 1 ml of DMF was added. After
the
mixture was allowed to stand for 20 h at room temperature,
34 ml
of methanol (0.80 mmol) was added, the mixture was stirred
for 30 min,
and then 181.7 mg (0.50 mmol) of tri(
n-butyl)ammonium
pyrophosphate in 5 ml of DMF was added. After 30 min of stirring,
the
mixture was filtered and 10 ml of methanol was added to the
filtrate,
which was concentrated in vacuo. The residue was desalted
by
C
18 reversed-phase chromatography, eluted with water,
purified
by ion-exchange HPLC (TSK gel DEAE-2SW), and eluted with 0.1 to
0.5 M potassium phosphate buffer (pH 3) containing 20%
acetonitrile.
Finally, the triphosphate was desalted by DEAE Sephadex
A-25 eluted
with ammonium bicarbonate buffer and freeze-dried to afford
31.6
mg of the ammonium salt (54% yield as determined by absorbance
at
255 nm [88% purity]).
1H NMR (D
2O)
0.71 (t,
J = 5.4 Hz, 1H), 1.11 (dd,
J = 5.4, 8.7
Hz, 1H), 1.67 (m, 1H), 3.56 (s, 2H), 3.93 (ddd,
J = 7.2, 8.7,
11.4 Hz, 1H), 4.11 (d,
J = 14.7 Hz, 1H), 4.19 (d,
J = 14.7 Hz,
1H), 4.23 (m, 1H), 8.18 (s, 1H);
31P NMR (D
2O, pD = 4) d
(H
3PO
4)
23.2 (t,
J = 45.2 Hz),
11.3 (d,
J = 52.8 Hz),
10.8 (d,
J = 45.6 Hz); FAB-MS
m/z 504 (M-H)
.
ACV triphosphate and (
R,
S)-PCV triphosphate were
prepared as described by Dixit and Poulter (
6) and Jarvest
et al. (
15),
respectively. The final products were purified
and desalted as
described above, and the purities of the triphosphates
were 95
and 70%, respectively.
Thymidine Sepharose was prepared as described by Kowal and Markus
(
16), except that ECH-Sepharose (12 to 16 mmol/ml;
Pharmacia)
was used as a support and galactosamine was used
for blocking.
The final loading of thymidine was 5.6 mmol/ml.
Purification and assay of viral TKs.
HSV-1 and HSV-2 TKs
were obtained from HSV-1 (KOS)- and HSV-2 (UW268)-infected BU25 cells,
respectively. Purification was performed as described by Cheng and
Ostrander (4) and Fyfe (12) with some
modifications. BU25 cells were infected with HSV-1 (KOS) or HSV-2
(UW-268). After 12 h of incubation, cells were harvested and
washed three times with phosphate-buffered saline (PBS). Cells were
disrupted with a Teflon homogenizer and sonicated in buffer A
containing 10 mM Tris-HCl (pH 7.5), 150 mM KCl, 1 mM
MgCl2, 1 mM 2-mercaptoethanol, 50 µM thymidine, 1 mM
phenylmethylsulfonyl fluoride (PMSF), 5 µg of leupeptin per ml, 5 µg of pepstatin A per ml, and 10% glycerol. Then the disrupted cell
suspension was centrifuged at 100,000 × g for 60 min
at 4°C. To the supernatant was added 20% streptomycin at a final
concentration of 1%, and the precipitate was removed by
centrifugation. The supernatant was fractionated with ammonium sulfate
in two steps (20 and 50%). The protein precipitated between 20 and
50% ammonium sulfate was suspended in buffer B, containing 20 mM
Tris-HCl (pH 7.5), 3 mM dithiothreitol (DTT), 5 µg of leupeptin per
ml, 5 µg of pepstatin A per ml, 1 mM PMSF, and 10% glycerol and was
applied to the thymidine Sepharose affinity column. HSV-1 and HSV-2 TKs were eluted with buffer containing 200 and 800 µM thymidine,
respectively. The enzymes were stored at 80°C and desalted with an
NAP-5 (Pharmacia) gel filtration column on the day of use.
VZV TK was obtained by expression in
Escherichia coli. The
tk gene from VZV (Kawaguchi) was cloned into the expression
vector
pET28a(+) (Novagen) with a fragment obtained by PCR. The PCR was
performed for 30 cycles with total DNA from MRC-5 cells infected
with
VZV (Kawaguchi) as the template, two 25-mer oligonucleotide
primers
(primer 1, 5'-TGTCTACAATACATATGTCAACGGA-3'; primer 2,
5'-AACACGTACACTCGAGTATGACAAT-3') (modified from the method
described
by Lacey et al. [
17]), and cloned
Pfu DNA polymerase (Stratagene).
The PCR fragment and
pET28a(+) were digested with restriction
enzymes
NdeI and
XhoI and ligated. The resultant plasmid (pETVZVTK)
was
used to transform
E. coli BL21 (DE3). VZV TK was expressed
by the induction of 100 µg of
isopropyl-
-
D-thiogalactopyranoside
(IPTG), purified by
His binding resin affinity chromatography
according to the method in
the Novagen manual, and further purified
by thymidine Sepharose
affinity chromatography. It was eluted
with buffer containing 800 mM
Tris-HCl (pH 7.5), 3 mM DTT, 0.4
mM thymidine, 1 mM ATP, 0.5 mg of
bovine serum albumin per ml,
and 10% glycerol. Then the enzyme was
stored and desalted as described
above.
A reaction mixture containing 50 mM Tris-HCl (pH 7.5), 2 mM ATP, 2 mM
MgCl
2, 9 mM NaF, 1 µM
[
methyl-
3H]thymidine, and the purified enzyme
was used to determine the
50% inhibitory concentration
(IC
50) of each nucleoside analog
for thymidine
phosphorylation. The reaction was performed at 37°C
for 15 min, and
then aliquots of 45 µl were spotted onto Whatman
DEAE-81 filter paper
disks. The filter was subsequently washed
three times (for 10 min each
time) in 1 mM ammonium formate and
finally rinsed once in methanol. The
disks were completely dried,
and radioactivity was determined by
scintillation counting.
For detection of the phosphorylation of A-5021 by viral TKs and
determination of the phosphorylation site of A-5021, a reaction
mixture
containing 50 mM Tris-HCl (pH 7.5), 2 mM ATP, 2 mM MgCl
2,
10 mM NaF, and the purified enzyme was used. The reaction mixture
was
analyzed by HPLC with a Whatman Partisil 10 SAX column (250
by 4.6 mm)
and UV detection at 254 nm. Elution was performed with
a linear
gradient of potassium phosphate (pH 3.5) from 0 to 1
M at a flow rate
of 1.5 ml/min.
Purification and assay of viral DNA polymerases.
HSV-1 and
HSV-2 DNA polymerases were partially purified from HSV-1 (KOS)- and
HSV-2 (UW-268)-infected Vero cells, respectively, by DEAE cellulose and
phosphocellulose ion-exchange chromatography as described previously
(22). VZV DNA polymerase was partially purified from VZV
(Kawaguchi)-infected human embryo lung cells as described above.
Standard assay mixtures of HSV DNA polymerases contained 50 mM
Tris-HCl (pH 8.0), 1 mM DTT, 80 µg of calf thymus-activated
DNA per
ml, 8 mM MgCl
2, 200 mM KCl, 0.5 mg of bovine serum albumin
per ml, 32 µM dATP, 32 µM dCTP, 32 µM TTP, 16 µM
[
3H]dGTP, and the purified enzyme. For kinetic
experiments, concentrations
of [
3H]dGTP and the inhibitor
were varied. Standard assay mixtures
of VZV DNA polymerases contained
50 mM Tris-HCl (pH 8.0), 1 mM
DTT, 20 µg of poly(dC)
oligo(dG)
12-18 per ml, 1 mM MgCl
2,
0.5 mg of
BSA per ml, 1 µM [
3H]dGTP, and the purified enzyme. The
reaction was performed at
37°C for 15 min and was terminated by
adding an excess amount
of ice-cold 10% trichloroacetic acid and 0.1%
NaPP
i. The precipitated
DNA was filtered with Whatman GF/C
filter disks. The filters were
washed twice with 5% trichloroacetic
acid and 0.02% NaPP
i, washed
twice with 50% EtOH, washed
once with 99% EtOH, and then dried.
Radioactivity was determined by
scintillation counting.
DNA elongation assay.
For the DNA elongation assay with
internal labeling (Fig. 8), M13mp18 single-stranded DNA was annealed to
a universal primer (17 mer) and used as a template primer. Reaction
mixtures (6 µl), comprising 30 mM Tris-HCl (pH 7.5), 1 mM DTT, 3 mM
MgCl2, 50 mM KCl, 33.3 µM dATP, 33.3 µM TTP, 33.3 µM
dGTP, 3.3 µM dCTP [-32P]dCTP, 38 µg of
template-primer per ml, various concentrations of inhibitor (A-5021
triphosphate or ACV triphosphate), and HSV-2 DNA polymerase, were
incubated at 37°C for 10 min. The reaction was stopped by adding
sequencing dye (4 µl) to the mixture, and then an aliquot was applied
to 6% sequencing gel.
For the DNA elongation assay with end labeling, M13mp18 single-stranded
DNA was annealed to a 5' end-labeled universal primer
(17 mer) and used
as a template primer. Reaction mixtures (6 µl),
comprising 30 mM
Tris-HCl (pH 7.5), 1 mM DTT, 3 mM MgCl
2, 50 mM
KCl,
33.3 µM dATP, 33.3 µM TTP, 33.3 µM dGTP, 33.3 µM
dCTP, 38
µg of template-primer per ml, 30 mM inhibitor [A-5021
triphosphate
or ACV triphosphate, and HSV-2 DNA polymerase, were
incubated
at 37°C for 10 min. Natural deoxynucleoside
triphosphates (dNTPs)
were omitted in accordance with the purpose of
the assay. The
reaction was stopped by adding sequencing dye (4 µl)
to the mixture,
and then an aliquot was applied to 15% sequencing gel.
The control sequence was confirmed by dideoxy sequencing with Sequenase
version 2.0 (United States Biochemical, Cleveland,
Ohio). The
radioactive bands were detected by an image analyzer
(model BAS2000;
Fuji Photo Film Co., Ltd., Tokyo, Japan).
Measurement of intracellular phosphate esters.
To study the
formation of intracellular phosphate ester, monolayers of MRC-5 cells
in 25-cm2 flasks were infected with HSV-1 (KOS) or HSV-2
(UW-268) at a multiplicity of infection of 0.01 PFU/cell and cultured
for 20 h as described by Vere Hodge and Perkins
(25), except that the virus solution was absorbed for
1 h. For VZV, monolayers of MRC-5 cells in 25-cm2
flasks were infected with VZV-infected MRC-5 cells (exhibiting a
minimum 70% cytopathic effect) at a ratio of 1:6 (infected/uninfected) and incubated for 48 h. At the start of drug treatment, the medium was replaced with 3 ml of fresh medium containing the
3H-labeled compound.
To study the intracellular stability of the phosphate esters,
monolayers of MRC-5 cells in 25-cm
2 flasks were infected
with HSV-1 (KOS) or HSV-2 (UW-268) at a
multiplicity of infection of 2 PFU/cell. After 1 h of absorption
of the virus solution, the cells
were treated with 3 ml of medium
containing radioactive compounds and
incubated for 5 h. VZV-infected
cells (which had been cultured for
48 h after infection) were
incubated with
3H-labeled
compounds for 6 h. After incubation, the cells were
washed three
times with ice-cold PBS and incubated with prewarmed
fresh drug-free
medium (50 ml).
At each designated time, the medium was removed, cells were washed
three times with ice-cold PBS, and the nucleotide analog
phosphates
were extracted with 3 ml of 50% ethanol containing
10% PBS
(
23). The extracts were evaporated under reduced pressure
and resolved in 300 µl of distilled water. Samples were stored
at
20°C until HPLC analysis with a Whatman Partisil 10 SAX column
(250 by 4.6 mm). Elution was performed with a linear gradient
of 0 to 1 M potassium phosphate (pH 3.5) at a flow rate of 1.5
ml/min.
Radioactivity was monitored by a
-RAM radiodetector (IN/US
Systems
Inc. Tampa, Fla.). The amounts of the phosphates were
calculated from
the peak area with SCINTIFLOW software (IN/US
Systems Inc.). A-5021
monophosphate, diphosphate, and triphosphate
were eluted at about 7.5, 16.5, and 28 min, respectively. ACV
monophosphate, diphosphate, and
triphosphate were eluted at about
8, 17.5, and 29.5 min, respectively.
PCV monophosphate, diphosphate,
and triphosphate were eluted at about
7.5, 15, and 27 min, respectively.
| |
RESULTS |
Phosphorylation of A-5021 by viral TK.
When 200 µM A-5021
was incubated with HSV-1 TK or VZV TK, an A-5021 monophosphate peak was
easily detected by HPLC (Fig. 1a and b),
but further phosphorylation of A-5021 monophosphate could not be
detected (data not shown). The phosphorylation site of A-5021 was also
revealed by HPLC. A-5021 has two free hydroxymethyl (-OH) groups on a
pseudosugar moiety in its chemical structure. Two regioisomers of
A-5021 monophosphate were synthesized in order to determine the site of
A-5021 phosphorylation by viral TK reaction (Fig.
2). The retention time of the
monophosphates was distinguished by HPLC with a SAX column. Because the
retention time of A-5021 monophosphate derived from the viral TK
reaction coincided with that of synthetic A-5021-2'-monophosphate
(Fig. 1c and d), the -OH group at position 2' was apparently
phosphorylated by the viral TKs.
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FIG. 1.
Phosphorylation of A-5021 by viral TKs and determination
of the site of phosphorylation. A-5021 was incubated with HSV-1 (a)
or VZV TK (b) at 37°C for 24 h. A-5021 monophosphate (A-5021MP)
was detected by HPLC. The reaction solution of VZV TK was mixed with
chemically synthesized A-5021-2'-monophosphate (MP) (C) or
A-5021-1'-MP (d) and was analyzed by HPLC. Peak number 1, peak
containing chemically synthesized A-5021-2'-MP and A-5021MP from viral
TK; peak number 2, A-5021-1'-MP; peak number 3, A-5021MP from viral
TK.
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FIG. 2.
Structure of chemically synthesized A-5021
monophosphates. (a) A-5021-1'-monophosphate. (b)
A-5021-2'-monophosphate.
|
|
Inhibitory effect of A-5021 on thymidine phosphorylation by viral
TKs.
The inhibitory effect of A-5021 on viral TKs was compared
with those of ACV and PCV. The IC50 of A-5021 for
phosphorylation of 1 µM thymidine by HSV-1 or HSV-2 TK was similar to
that of PCV and much lower than that of ACV (by HSV-1, 95-fold; by
HSV-2, 6.7-fold) (Table 1). In the case
of VZV TK, A-5021 was the most potent inhibitor of these three agents
(Table 1).
Formation of A-5021 phosphates in virus-infected MRC-5 cells.
The intracellular concentrations of phosphates in infected cells
were measured after incubation with A-5021, ACV, or PCV at 1 µM
(HSV-1) and 10 µM (HSV-2 and VZV). Formation of A-5021 monophosphate, diphosphate, and triphosphate was detected in the infected MRC-5 cells
(Fig. 3). In HSV-1- and VZV-infected
cells, the rate of formation and the amount of A-5021 triphosphate
exceeded those of ACV triphosphate (Fig.
4), while these parameters were
equivalent for the two compounds in HSV-2-infected MRC-5 cells.
However, more PCV triphosphate than A-5021 triphosphate formed and
accumulated in all virus-infected MRC-5 cells. A-5021 triphosphate
could not be detected in uninfected cells under the same conditions
(data not shown). After continuous exposure for 6 h, the
intracellular concentrations of the phosphates of these three agents in
HSV-1- and HSV-2-infected MRC-5 cells showed a linear
correlation with the drug concentrations from 0.1 to 30 µM (Fig.
5).
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FIG. 3.
HPLC analysis of A-5021 and its phosphate esters in
HSV-1-infected MRC-5 cells. HSV-1-infected MRC-5 cells were
treated with 1 mM [3H]A-5021 for 6 h, and the
extract was analyzed by HPLC. MP, monophosphate; DP, diphosphate; TP,
triphosphate.
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FIG. 4.
Formation of phosphate esters of nucleoside analogs in
virus-infected MRC-5 cells. MRC-5 cells infected with HSV-1
(1), HSV-2 (2), or VZV (3) were
treated with [3H]A-5021 (a), [3H]ACV
(b), or [3H]PCV (c) at the indicated
concentrations. At the designated times, cells were extracted, and the
extracts were analyzed by HPLC. Open circles, triphosphate; closed
triangles, diphosphate; closed squares, monophosphate.
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FIG. 5.
Concentration dependency of formation of triphosphate
esters of nucleoside analogs in virus-infected MRC-5 cells. MRC-5 cells
infected with HSV-1 (a) or HSV-2 (b) were treated with various
concentrations of [3H]A-5021, 3H[ACV],
or [3H]PCV for 6 h. Then cells were extracted, and
the extracts were analyzed by HPLC. Open circles, A-5021
triphosphate; closed triangles, ACV triphosphate; closed squares, PCV
triphosphate. Conc, concentration.
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Intracellular stability of A-5021 triphosphate in virus-infected
MRC-5 cells.
The changes in the intracellular concentrations of
A-5021 triphosphate, ACV triphosphate, and PCV triphosphate in infected cells were measured after incubation with these agents for 5 to 6 h at 1 µM (HSV-1) or 10 µM (HSV-2 and
VZV), followed by exposure to drug-free medium. A-5021 triphosphate was
more stable than ACV triphosphate and had a longer half-life in MRC-5
cells infected with HSV-1 and HSV-2 (Fig.
6). In VZV-infected MRC-5 cells, the half-life of ACV triphosphate could not be measured because of low
accumulation. The half-life of A-5021 triphosphate was shorter than
that of PCV triphosphate in HSV-1-, HSV-2-, and VZV-infected MRC-5 cells.
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FIG. 6.
Intracellular stability of phosphate esters of
nucleoside analogs in virus-infected MRC-5 cells. MRC-5 cells infected
with HSV-1 (1), HSV-2 (2), or VZV (3) were treated with
[3H]A-5021 (a) [3H]ACV (b), or
[3H]PCV (c) at the concentrations shown for 5 h
(HSV-1 and HSV-2) or 6 h (VZV). After extracellular
compounds were removed, the levels of intracellular phosphate esters
were analyzed by HPLC. Open circles, triphosphate; closed triangles,
diphosphate; closed squares, monophosphate. T1/2,
half-life.
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|
Inhibitory effect of A-5021 triphosphate on viral DNA
polymerases.
A-5021 triphosphate inhibited HSV-1 DNA
polymerase in a competitive manner with respect to dGTP (data not
shown). A-5021 showed strong inhibition of this viral DNA
polymerase, with the Ki value for
HSV-1 DNA polymerase being 12-fold lower than the
Km value for dGTP (Table
2). This Ki value
was 185-fold lower than that for (R,S)-PCV
triphosphate and 3.9-fold higher than that for ACV triphosphate
(Table 2). The same order of inhibitory effects was observed among
these three triphosphates in the case of HSV-2 (Table 2) and VZV
DNA polymerases [IC50s of A-5021TP, ACVTP, and
(R,S)-PCVTP were 0.014, 0.0053, and 3.6 µM,
respectively].
DNA extension assay.
The mechanism of DNA chain termination by
A-5021 triphosphate was studied with HSV-2 DNA polymerase. M13mp18
single-stranded DNA and universal primer were used as the template and
primer, respectively. Figure 7 shows the
inhibition of DNA synthesis by A-5021 triphosphate and ACV
triphosphate. DNA synthesis by HSV-2 DNA polymerase was inhibited
by both of these triphosphates in a concentration-dependent manner.
Chain-terminated bands were detected at G sites in both cases, but
the patterns of the gel bands were different. A-5021 triphosphate
caused strong termination at tandem G repeats and weak termination at
single G sites, but this tendency was marginal in the case of ACV
triphosphate. Figure 8 shows the pattern
of the DNA elongation adjacent to the primer. Both A-5021 triphosphate
and ACV triphosphate were incorporated at the first G site when natural
dNTP was omitted. A-5021-incorporated DNA had a slightly higher
migration rate on the gel. When dATP, dCTP, and dTTP were added, a
second chain-terminated band appeared at the site of the second G in
the case of A-5021 triphosphate, but not with ACV triphosphate.
This result was evidence that A-5021 triphosphate did not
cause absolute chain termination the way ACV triphosphate did.
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FIG. 7.
Chain termination by A-5021 triphosphate
(A-5021TP) or ACV triphosphate (ACVTP). HSV-2 DNA polymerase
was reacted with M13mp18 single-stranded DNA and specific oligomer (17 mer) as a template and a primer, respectively, in the presence of fixed
concentrations of natural dNTPs and various concentrations of
A-5021TP or ACVTP (lane 1, 1 mM; lane 2, 300 µM; lane 3, 100 µM; lane 4, 30 µM; lane 5, 10 µM; lane 6, 3 µM; lane 7, 1 µM). Cont., no chain terminator. Lanes ddG, ddA, ddT, and ddC,
dideoxy sequencing with ddGTP, ddATP, ddTTP, and ddCTP, respectively.
Samples were analyzed on a 6% sequencing gel, followed by
autoradiography.
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|
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FIG. 8.
DNA chain extension by HSV-2 DNA polymerase. The
reaction was performed with M13mp18 single-stranded DNA and a 5'
32P-end-labeled specific oligomer (17 mer) as the template
and the primer, respectively, in the presence or absence of 50 µM
dATP, dTTP, and/or inhibitor as shown. Lanes ddG, ddA, ddT, and ddC,
dideoxy sequencing with ddGTP, ddATP, ddTTP, and ddCTP, respectively.
Samples were analyzed on a 15% sequencing gel, followed by
autoradiography.
|
|
| |
DISCUSSION |
In this study, we characterized the mode of action of A-5021 and
obtained data possibly explaining its superior antiviral activity
relative to those of ACV and PCV against HSV-1, HSV-2, and VZV.
A-5021 has only weak antiviral activity against TK-deficient strains of
HSV-1 and HSV-2, suggesting that it is phosphorylated mainly by viral TKs (14). The present study clearly showed
that A-5021 was monophosphorylated by purified HSV-1 and VZV
TKs at the 2'-OH site (Fig. 1). However, A-5021 monophosphate was not phosphorylated to the diphosphate form by the viral kinases. In the
case of ACV monophosphate, there is phosphorylation to the triphosphate
forms by cellular kinases (20, 21). A-5021-2'-monophosphate was diphosphorylated by GMP kinase and triphosphorylated by nucleoside diphosphate kinase in a cell-free system (22a), so it is
thought to be phosphorylated to the triphosphate form by cellular
kinases in HSV-and VZV-infected cells.
The IC50s of A-5021 for HSV-1 and HSV-2 TK were
almost the same as those for PCV but were much lower than those for ACV
(Table 1). Of these three agents, A-5021 had the lowest
IC50 for VZV TK (Table 1). These results suggest that
A-5021 was phosphorylated far more efficiently than ACV.
Our data showed that A-5021 triphosphate acted as a potent inhibitor of
viral DNA polymerases. The apparent Ki of A-5021
triphosphate for HSV-1 DNA polymerase was lower than the
Km for dGTP (Table 2). The apparent
Ki of A-5021 triphosphate for HSV-1 DNA
polymerase was comparable to that for HSV-2 DNA polymerase and was
slightly higher than that of ACV triphosphate (Table 2). The
IC50 of A-5021 triphosphate for VZV DNA polymerase was also
slightly higher than that of ACV triphosphate, whereas the apparent
Kis (or IC50s) values of A-5021
triphosphate for viral DNA polymerases were much lower than those of
(R,S)-PCV triphosphate (Table 2), suggesting that
the strong affinity of A-5021 triphosphate for viral DNA polymerases
contributes to the potency of the antiviral activity of A-5021 compared
with that of PCV.
A-5021 was reported to show 25-fold-more-potent antiviral activity than
ACV against HSV-1 (KOS) and 5-fold-more-potent antiviral activity than ACV against VZV (Kawaguchi) but was only 2-fold more
potent than ACV against HSV-2 (UW-268) in MRC-5 cells
(14). In the present study, we found that the rate of A-5021
triphosphate formation and its level of accumulation were higher than
those of ACV triphosphate in HSV-1- and VZV-infected MRC-5 cells
but were similar in HSV-2-infected MRC-5 cells (Fig. 4 and 5). It seems likely that the high accumulation of A-5021 triphosphate in
HSV-1- and VZV-infected cells contributes to its more potent antiviral activity against HSV-1 and VZV compared with that of ACV.
However, it was not clear why A-5021 showed a stronger antiviral activity than did ACV against HSV-2 (UW-268). On the other hand, the accumulation of PCV triphosphate was much higher than that of
A-5021 triphosphate in all of the virus-infected cells that we studied
(Fig. 4 and 5). However, PCV triphosphate was a weak inhibitor of viral
DNA polymerases, while A-5021 and ACV triphosphates were strong
inhibitors (Table 2), so A-5021 was more potent than PCV against
HSV-1, HSV-2, and VZV.
We previously showed that A-5021 has prolonged antiviral activities
against HSV-1 and HSV-2 in in vitro virus yield experiments with short periods of drug treatment (14). The prolonged
antiviral activity of A-5021 is greater than that of ACV but less than
that of PCV. In this study, we found that A-5021 triphosphate was more stable than ACV triphosphate in HSV-1- and HSV-2-infected cells but less stable than PCV triphosphate (Fig. 6). This result correlates with the extent of prolonged antiviral effect. The intracellular stability of the triphosphate forms of PCV and
(R)-9-[hydroxy-2-(hydroxymethyl)-butyl]guanine (H2G) is
thought to contribute their prolonged antiviral effect (1, 7, 19,
25). The relatively high intracellular stability of A-5021
triphosphate may also contribute to its prolonged antiviral activity.
Lineweaver-Burk plots revealed that A-5021 triphosphate acted as a
competitive inhibitor of dGTP by HSV DNA polymerases. In the DNA
elongation study with HSV-2 DNA polymerase, highly processive DNA synthesis was inhibited by A-5021 triphosphate in a
dose-dependent manner, and this triphosphate was incorporated
into DNA at G sites and terminated elongation. However, the mode of
chain termination was different from that of ACV. There are two -OH
groups in the pseudosugar moiety of A-5021; the -OH at the 2' site
was phosphorylated by viral TKs (Fig. 1), so the other -OH at the 1'
site may act as the 3'-OH of dGTP. We found that further DNA elongation
could occur even after incorporation of A-5021 monophosphate at the 3' end of DNA (Fig. 8). These observations suggested that A-5021 was a pseudo-chain terminator like PCV. Interestingly, A-5021 triphosphate strongly terminated DNA elongation at tandem G repeats (Fig. 7). It is possible that tandem incorporation of A-5021
monophosphate into the 3' end of DNA causes greater conformational
distortion than incorporation of a single monophosphate, thereby
limiting further DNA elongation, particularly after the tandem G
repeat. Conformational distortion was shown to be actually induced in ganciclovir monophosphate-incorporated DNA by a study with
solution-state nuclear magnetic resonance (8). The base of
ganciclovir normally shows hydrogen binding to complementary dCMP, but
significant distortion is observed at or near the site of ganciclovir
incorporation.
The antiviral activity of nucleoside analogs is principally
dependent on both the intracellular concentration of the triphosphates achieved in infected cells and the inhibitory effect of the
triphosphates on viral DNA polymerases. Although A-5021 was not
always superior to ACV or PCV in these individual factors, A-5021
is the most effective of these three antiviral agents (14).
The superior antiviral activity of A-5021 against HSV-1 and VZV
may well be explained by the integrated effect of these two factors.
The prolonged antiviral effect of A-5021 could also be explained by
the stability of its triphosphate in virus-infected cells.
| |
ACKNOWLEDGMENT |
We thank T. Tsurumi for helpful discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Life Science
Laboratories, Ajinomoto Co., Inc., 214 Maeda-cho Totsuka-ku, Yokohama 244, Japan. Phone: 81-45-821-7496. Fax: 81-45-822-5211. E-mail: pld_ono{at}te3.ajinomoto.co.jp.
| |
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Antimicrobial Agents and Chemotherapy, August 1998, p. 2095-2102, Vol. 42, No. 8
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