Laboratory of Virus Immunology, Institute for
Virus Research, Kyoto University, Kyoto
606-8507,1 Department of Applied
Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai
981-8555,2 Biochemicals Division, Yamasa
Corporation, Chiba 288-0056,3 Department
of Microbiology, Fukushima Medical University School of Medicine,
Fukushima 960-1295,5 and
Department of Immunopathophysiology and Internal
Medicine II, Kumamoto University School of Medicine, Kumamoto
860-0811,6 Japan, and Experimental
Retrovirology Section, Department of Developmental Therapeutics,
Medicine Branch, National Cancer Institute, Bethesda, Maryland
208924
Received 6 October 2000/Returned for modification 20 December
2000/Accepted 15 February 2001
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INTRODUCTION |
Combination chemotherapy or highly
active antiretroviral therapy (HAART) using two or more reverse
transcriptase (RT) inhibitors (RTIs) and protease inhibitors (PIs) has
dramatically improved the quality of life and survival of patients
infected with human immunodeficiency virus type 1 (HIV-1) (10,
23). However, the ability to provide effective long-term
antiretroviral therapy for HIV-1 infection has become a complex issue,
since a number of patients who initially achieved very favorable viral
suppression later experience treatment failure (40).
Moreover, 30 to 40% of HIV-1-infected individuals who had received no
prior antiviral therapy fail to achieve viral suppression to
undetectable levels (9, 17). In addition, 10 to 40% of
antiviral therapy-naive individuals infected with HIV-1 have persistent
viral replication (plasma HIV RNA >500 copies/ml) under HAART
(11, 12, 37), possibly due to transmission of
drug-resistant HIV-1 variants (40). Thus, the development
of novel compounds that are active against drug-resistant HIV-1
variants and that prevent or delay the emergence of resistant HIV-1
variants is urgently needed.
Certain 4'-substituted nucleosides have been described in the
literature. Maag et al. (19) reported that
4'-azido-2'-deoxythymidine (4'-AZT), the hydrogen atom at the 4'
position of which was substituted for with an azido group, exerted
potent activity against HIV-1 in vitro. Subsequently, Chen and
colleagues (4) reported that 4'-AZT was active against
HIV-1 through its DNA chain-terminating activity. More recently,
Sugimoto et al. have reported that 4'-substituted nucleosides including
4'-ethynylthymidine exhibited potent activity against not only HIV-1,
but also herpes simplex virus type 1 (38). We recently
designed and synthesized a series of 4'-ethynyl
(4'-E)-2'-deoxynucleosides and their analogs and identified
several highly potent anti-HIV-1 compounds, including
4'-E-2'-deoxycytidine (4'-E-dC),
4'-E-2'-deoxyadenosine (4'-E-dA),
4'-E-2'-deoxyribofuranosyl-2,6-diaminopurine
(4'-E-dDAP), and 4'-E-2'-deoxyguanosine
(4'-E-dG). These 4'-E compounds, unlike all of
the currently available nucleoside RTIs (NRTIs), lack the 2',3'-dideoxyribose configuration but have a 2'-deoxyribose
configuration (Fig. 1 and Table
1).
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FIG. 1.
Structures of 4'-substituted nucleosides. All nucleoside
analogs discussed here have substitutions at the 4' position of the
sugar moiety shown here except for the two compounds
4'-E-araT and 4'-E-araC, which have an
arabinofuranosyl sugar moiety. See Table 1.
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All of these 4'-E compounds blocked the replication of
a wide spectrum of laboratory and clinical HIV-1 strains in
vitro with low cellular toxicities. These compounds also
suppressed the replication of various drug-resistant HIV-1 clones,
including HIV-1M41L/T215Y, HIV-1L74V,
HIV-1K65R,
HIV-1M41L/T69S-S-G/T215Y,
HIV-1Y181C, and HIV-1A62V/V75I/F77L/F116Y/Q151M. These
4'-E compounds also suppressed various multidrug-resistant
clinical HIV-1 variants carrying a variety of drug resistance-related
amino acid substitutions, which were isolated from patients for whom
virtually all of the currently available antiviral regimens had failed.
Furthermore, we demonstrate in this study that these 4'-E
analogs most likely block HIV-1 by functioning as NRTIs.
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MATERIALS AND METHODS |
Antiviral agents.
3'-Azido-3'-deoxythymidine (AZT, or
zidovudine), 2',3'-dideoxyinosine (ddI, or didanosine), and
2',3'-dideoxycytidine (ddC, or zalcitabine) were purchased from Sigma
(St. Louis, Mo.). (
)-2',3'-Dideoxy-3'-thiacytidine (3TC, or
lamivudine) was a kind gift from R. F. Schinazi (Atlanta, Ga.). A
series of 4'-position-substituted nucleosides were designed and
synthesized as described elsewhere (16, 27, 28). Their basic formula is shown in Fig. 1. A non-NRTI (NNRTI), MKC-442, was a
gift from Mitsubishi Kasei Corporation (Yokohama, Japan) (2).
Cells.
MT-4 and H9 cells were grown in an RPMI 1640-based
culture medium, and Cos-7 cells were grown in Dulbecco's modified
Eagle medium (DMEM); each of these media was supplemented with 10%
fetal calf serum (FCS; HyClone Laboratories, Logan, Utah), 2 mM
L-glutamine, 50 U of penicillin per ml, and 50 µg of
streptomycin per ml. HeLa-CD4-LTR/
-gal cells (14) were
kindly provided by M. Emerman through the AIDS Research and Reference
Reagent Program, Division of AIDS, National Institute of Allergy and
Infectious Diseases (Bethesda, Md.). Prior to use, HeLa-CD4-LTR/-gal
cells were propagated in DMEM supplemented with 10% FCS, 0.1 ng of
hygromycin B per ml, and 200 µg of Geneticin per ml. In the
anti-HIV assay, cells were cultured in the DMEM-based culture medium
with addition of 50 U of penicillin per ml and 50 µg of streptomycin
per ml instead of hygromycin B and Geneticin. Peripheral blood
mononuclear cells (PBMCs) were obtained from healthy HIV-1-seronegative
donors by Ficoll-Hypaque gradient centrifugation and were stimulated
for 3 days with phytohemagglutinin M (PHA; 10 µg/ml; Sigma) prior to use.
Viruses and construction of recombinant HIV-1 clones.
Three
laboratory strains, HIV-1LAI,
HIV-2ROD, and HIV-2EHO,
were employed. Multidrug-resistant clinical HIV-1 strains were isolated
from patients with AIDS who had been treated with 9 to 11 anti-HIV-1
drugs for 39 to 64 months, as previously described (42).
These clinical HIV-1 isolates were passaged once or twice in
PHA-stimulated PBMCs (PHA-PBMCs), and titers of the culture supernatants obtained were determined for infectivity and stored at
70°C until use.
Recombinant infectious HIV-1 clones carrying various mutations in the
pol gene were generated as previously described (35, 39, 42). Briefly, the desired mutations were introduced into the
XmaI-NheI region (759 bp) of pTZNX1, which
encoded Gly-15 to Ala-267 of HIV-1 RT (strain BH 10), by the
oligonucleotide-based mutagenesis method (42). The
XmaI-NheI fragment was inserted into a pHXB2RIP7
(a kind gift from M. Reitz, Jr., National Institutes of Health,
Bethesda, Md.)-based plasmid, pSUM9, generating various molecular
clones with the desired mutations. Each molecular clone (10 µg/ml as
DNA) was transfected into Cos-7 cells (4 × 105 cells/100-mm-diameter dish) by the calcium
phosphate method (Promega, Madison, Wis.). After 24 h, MT-2 cells
(106 cells/dish) were added and cocultured with
Cos-7 cells for an additional 24 h. When an extensive cytopathic
effect was observed, cell supernatants were harvested, and the virus
was further propagated in H9 cells. The culture supernatant was
harvested, the titer was determined for infectivity, and the sample was
stored at 70°C until use. The presence of intended mutations and
the absence of unintended mutations in infectious clones were confirmed
by determination of the nucleotide sequence of proviral DNA isolated from the virus-producing H9 cells. HIV-1HXB2D was
generated by using pSUM9 (35) and served as a wild-type
infectious clone (HIV-1wt).
Determination of drug susceptibility of HIV-1.
The
inhibitory effects of test compounds on HIV-1 replication were
monitored by the inhibition of virally induced cytopathicity in MT-4
cells. Briefly, MT-4 cells were suspended at 105
cells/ml and exposed to HIV-1LAI at 100 50%
tissue culture infectious doses (TCID50s).
Immediately after viral exposure, the cell suspension (104 cells in 100 µl) was brought into each
well of a 96-well flat microtiter culture plate (Costar, Cambridge,
Mass.) containing various concentrations of test compounds. After
incubation for 5 days, the number of viable cells was determined by the
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
method as previously described (15, 30).
The sensitivity of infectious clones to various RTIs was determined in
the multinuclear activation of the galactosidase indicator (MAGI) assay
(14), with some modifications using the viral preparations titrated as previously described (20). Briefly, target
cells (HeLa CD4-LTR/-gal; 104/well) were
plated in 96-well flat microtiter culture plates. On the following day,
the medium was aspirated, and the cells were inoculated with HIV-1
clones (70 MAGI units/well, which gave 70 blue cells after 48 h of
incubation) and cultured in the presence of various concentrations of
drug in fresh medium. Forty-eight hours after viral exposure, all blue
cells in each well were counted. The cytotoxicity of the compound was
determined by the MTT method as previously described (15).
All experiments were performed in triplicate.
The drug susceptibility of HIV-1 clinical isolates was determined as
previously described (42). Briefly, PHA-PBMCs
(106 cells/ml) were exposed to each viral
preparation at a TCID50 of 50 and cultivated in
200 µl of culture medium containing various concentrations of the
drug in 10-fold serial dilutions in 96-well culture plates. All assays
were performed in triplicate, and the amounts of p24 antigen produced
by the cells into the culture medium were determined on day 7 in
culture with a commercially available radioimmunoassay kit (Du Pont,
Boston, Mass.).
The activity of test compounds is shown as the concentration that
blocks HIV-1 replication by 50% (EC50), and
cytotoxicity is shown as the concentration that suppresses the
viability of HIV-1-unexposed cells by 50%
(CC50). The window between
CC50 and EC50 is shown by
the CC50/EC50 ratios
(selectivity indices).
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RESULTS |
Activity of 4'-substituted nucleosides against HIV-1 in vitro.
We designed and synthesized more than 20 novel 4'-substituted
nucleoside analogs (16, 27, 28, 38) and tested them for in
vitro activity against HIV-1 by using the MTT colorimetric assay
employing MT-4 cells. A thymidine analog, 4'-ethynyl (E)-T, and a uracil analog, 4'-E-IdU, were found to be moderately
active against HIV-1LAI, with
EC50s of 0.83 and 0.29 µM, respectively (Table
1). Conversion of the 2'-deoxyribose of 4'-E-T to ribose, generating 4'-E-rT, nullified the antiviral activity of
4'-E-T. Other 4'-substituted thymidine and uridine analogs
had marginal or no activity. All four 4'-substituted cytidine analogs
examined were active, among which 4'-E-dC and
4'-fluoromethyl (FMe)-dC were most potent against HIV-1, although the
latter was substantially cytotoxic (Table 1). It was noted that
4'-E-araC was moderately active.
Among 4'-E purine analogs, 4'-E-dA,
4'-E-dDAP, and 4'-E-dG were highly potent against
HIV-1, with subnanomolar to nanomolar EC50s
(Table 1). 4'-E-dI was only moderately active against the virus. It is noteworthy that both 4'-E-dDAP and
4'-E-dA had a favorable toxicity profile, with selectivity
indices of 2,600 and 1,630, respectively (Table 1). All three
4'-E purine analogs with a ribose configuration
(4'-E-NM-A, 4'-E-A, and 4'-E-CldG) were inactive.
Activity of 4'-substituted nucleosides against HIV-1 variants
resistant to various RTIs.
We also evaluated whether
4'-substituted nucleosides were active against HIV-1 variants resistant
to various nucleoside RT inhibitors (NRTIs) by using the MAGI assay.
Four pyrimidine analogs (4'-E-dC, 4'-E-araC,
4'-Me-dC, and 4'-FMe-dC) that were potent against
HIV-1LAI were selected and tested further against
various infectious HIV-1 clones carrying resistance-conferring
amino acid substitutions (Table 2). It
was noteworthy that all four of these cytidine analogs examined
suppressed the replication of ddI- and ddC-resistant
HIV-1K65R and HIV-1L74V,
AZT-resistant HIV-1M41L/T215Y, and
multi-dideoxynucleoside-resistant (resistant to AZT, ddI, ddC and d4T)
variant HIV-1A62V/V75I/F77L/F116Y/Q151M
(35, 36). It was noted that among 4'-substituted
pyrimidine analogs, only 4'-E-dC remained active against
3TC-resistant variants HIV-1M184I and
HIV-1M184V, but was less active against the
multi-NRTI-resistant (resistant to AZT, ddI, ddC, d4T and 3TC) variant
HIV-1M41L/T69S-S-G/T215Y (41), while
4'-FMe-dC remained potent against
HIV-1M41L/T69S-S-G/T215Y. However, all three
4'-E purine compounds (4'-E-dA,
4'-E-dDAP, and 4'-E-dG) were active against the
infectious clones tested, including HIV-1M184V
and HIV-1M41L/T69S-S-G/T215Y. These purine analogs were also active against an NNRTI-resistant infectious clone,
HIV-1Y181C. When tested in HeLa-CD4-LTR/-gal
cells, the CC50s of the analogs examined were all
>100 or >200 µM, except for that of 4'-E-dG.
4'-Ethynyl group is required for activity against
HIV-1M184V and HIV-1M184I.
To investigate
whether and to what extent the 4'-E configuration was
crucial for activity against HIV-1, three thymidine analogs and two
2'-deoxycytidine analogs substituted at the 4'-position with different
groups (vinyl, ethyl, hydroxyethyl, methyl, or fluoromethyl) were
synthesized. 4'-ethylthymidine (4'-Et-T) was inert, but
4'-vinylthymidine (4'-V-T) and 4'-hydroxyethylthymidine (4'-HE-T) were
active against HIV-1LAI, although they were less so than to 4'-E-T (Table 1). These two thymidine analogs
were active against the control wild-type infectious clone
HIV-1HXB2 and a multidideoxynucleoside-resistant
infectious clone
(HIV-1A62V/V75I/F77L/F116Y/Q151M), but were
totally inert (>100 µM) against the 3TC-resistant clone HIV-1M184V (Table 2). Two 2'-deoxycytidine
analogs, 4'-Me-dC and 4'-FMe-dC, were as potent against
HIV-1LAI as 4'-E-dC. These analogs
were also active against various drug-resistant infectious clones, but
were less potent against HIV-1M184V and
HIV-1M184I (Table 2). Taken together, although
the 4'-E substitution is not essential for antiviral
activity against HIV-1, the 4'-E configuration appears to be
required for activity against 3TC-resistant
HIV-1M184V and HIV-1M184I.
Activity of selected 4'-substituted nucleosides against HIV-2
strains.
We also examined selected 4'-E analogs against
two HIV-2 strains, HIV-2ROD and
HIV-2EHO, in the MAGI assay (Table
3). Although the thymidine analog
4'-E-T showed only moderate activity, three cytidine
analogs, 4'-E-dC, 4'-E-araC, and 4'-Me-dC, were
highly active against HIV-2, and 4'-E-dC was the most potent
analog, with 50% inhibitory concentrations
(IC50s) of ~0.001 µM (Table 3). Four
4'-E purine analogs examined also suppressed the replication of both HIV-2 strains. AZT, tested as a control compound, was active
against both HIV-2 strains, as previously described (21). Considering that all of the currently known NNRTIs fail to suppress the
replication of HIV-2 (6), it appears that 4'-substituted nucleosides do not belong to NNRTIs.
Activity of 4'-ethynyl-2'-deoxynucleosides against HIV-1 isolated
from heavily drug-experienced patients.
4'-E analogs
were then evaluated for their activity against multidrug resistant
clinical HIV-1 isolates in vitro. In this study, we chose three most
potent 4'-E compounds, 4'-E-dC,
4'-E-dA, and 4'-E-dDAP. Four clinical HIV-1
strains were isolated from patients who received a variety of
anti-HIV-1 agents for 39 to 64 months and had failed to respond to any
existing regimens of antiviral therapy (42). As shown in
Table 4, all four clinical HIV-1 strains
contained a variety of drug resistance-conferring amino acid
substitutions in the RT- and protease-encoding regions of HIV-1 gene.
All three 4'-E compounds suppressed the replication of these
highly drug-resistant clinical strains as effectively as that of the
wild-type clinical strain HIV-1ERS104pre
(36) and two HIV-1 clinical strains
(HIV-1IVR205 and
HIV-1IVR207) isolated from drug-naive AIDS
patients. It was noted, however, that 4'-E-dC was moderately
active against HIV-1Pt6 and
HIV-1Pt9. There was no apparent association of
the observed reduced antiviral activity with amino acid substitutions
identified in these clinical isolates (Table 4).
Testing 4'-ethynyl nucleosides in the time-of-drug-addition
assay.
The triphosphate form of 4'-E nucleosides is not
presently available, and how 4'-E nucleosides block the
replication of HIV-1 and HIV-2 remains to be clarified. The
time-of-drug-addition assays have been used to approximately determine
what stage of the replication cycle of HIV-1 the compound in question
blocks (29). We therefore tested two potent
4'-E compounds, 4'-E-dC and 4'-E-araC,
in the assay together with three controls: a surface reactant, dextran sulfate 5000 (DS5000); NRTI AZT; and NNRTI MKC-442.
The concentrations of compounds tested were all fixed at their
EC90s. At indicated time points, compounds were
added to the HIV-1LAI-exposed MAGI cells. As
shown in Fig. 2, DS5000, which like
DS8000 (24) inhibits the absorption of HIV and formation of syncytia, showed anti-HIV-1 activity only when it was added early
after viral exposure. AZT, which requires triphosphorylation before it
becomes active (23), exerted its antiviral activity when
its addition was delayed for up to 4 h. MKC-442, which does not
require activation, was effective when its addition was delayed for up
to 6 h. The profiles of anti-HIV-1 activity of 4'-E-dC and 4'-E-araC were quite similar to that of AZT (Fig. 2).
These results suggest that 4'-E nucleosides suppress the
replication of HIV at or around the step of reverse transcription.
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FIG. 2.
Antiviral activity of 4'-ethynyl nucleosides in the
time-of-drug-addition assays. At indicated time points,
4'-E-dC (open circles) and 4'-E-araC
(closed circles) were added to the HIV-1LAI-exposed MAGI
(HeLa CD4/LTR--gal) cells, and the blue cells produced were counted
at the completion of the 48-h period of incubation. DS5000 (open
diamonds), AZT (open squares), and MKC-442 (open triangles) served as
controls.
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Reversal of antiviral activity of 4'-ethynyl nucleosides by natural
2'-deoxynucleosides.
It has been shown that biological effects
such as antitumor or antiviral activity of nucleoside analogs are
reversed by the addition of natural nucleosides (5, 8,
26). We first tested whether thymidine and 2'-deoxycytidine
reversed the antiviral activity of 4'-E-T and
4'-E-dC, respectively (Fig.
3). The concentrations of compounds
tested were all fixed at their EC90s. Antiviral
activity of 4'-E-T (2 µM) was suppressed by the addition
of thymidine in a dose-dependent manner, and the antiviral activity of
AZT (100 nM) was also reversed by the addition of thymidine, in
agreement with our previous published data (26), although
100 µM thymidine caused cytotoxicity (Fig. 3A). Activity of
4'-E-dC (8 nM) and 4'-E-dG (10 nM) was similarly
reversed by the addition of 2'-deoxycytidine and 2'-deoxyguanosine,
respectively (Fig. 3B and D). Likewise, the activity of ddC (2 µM)
and ddG (10 µM) was reversed by the addition of dC and dG,
respectively, in agreement with our previous data (25). In
contrast, the antiviral activity of 4'-E-dA was not reversed
by the addition of dA, a similar profile of the activity of ddI versus
dA (25) (Fig. 3C).
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FIG. 3.
Reversal of anti-HIV activity of
4'-ethynyl-2'-deoxynucleosides by 2'-deoxynucleosides. Antiviral
activities of 4'-E-thymidine (4'-E-T; 2 µM [open bars]) and AZT (100 nM [hatched bars]) were reversed by
the addition of their physiologic counterpart (thymidine) in a
dose-dependent manner (A). In the culture without antiviral agents
(solid bars), the activities of 4'-E-2'-deoxycytidine
(4'-E-dC; 8 nM) and ddC (2 µM [hatched]) were
similarly reversed by the addition of 2'-deoxycytidine (dC) (B). In
contrast, the anti-HIV-1 activities of
4'-E-2'-deoxyadenosine (4'-E-dA; 80 nM
[open bars]) and ddI (10 µM [hatched bars]) were not suppressed
by the addition of 2'-deoxyadenosine (C). The activities of
4'-E-2'-deoxyguanosine (4'-E-dG; 10 nM
[open bars]) and ddG (10 µM [hatched bars]) were moderately
reversed by the addition of 2'-deoxyguanosine (dG) (D).
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Acid stability of selected 4'-ethynyl nucleosides.
Certain
nucleoside analogs such as ddI are acid labile and require antacid upon
oral administration, generating adverse reactions, such as abdominal
discomfort and diarrhea (33). We therefore examined the
acid sensitivity of three selected 4'-E analogs
(4'-E-dC, 4'-E-dA, and 4'-E-dDAP).
These compounds were exposed to 1 N HCl for up to 20 min and then
neutralized with 1 N NaOH and added to target cells exposed to HIV-1.
As shown in Fig. 4, ddI lost all of its
antiviral activity following acid exposure for 20 min; however,
4'-E-dC, 4'-E-dA, 4'-E-dDAP, and the
control acid-resistant AZT did not lose their antiviral activity.
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FIG. 4.
Acid stability of 4'-ethynyl nucleosides. AZT, ddI,
4'-E-dC, 4'-E-dA, and
4'-E-dDAP were exposed to 1 N HCl for the indicated
periods of time, neutralized with 1 N NaOH, and added to the culture in
the MAGI assay at final concentrations of 100 nM, 20 µM, 8 nM, 80 nM,
and 8 nM, respectively, which approximately represent the
EC90 of each compound under the conditions used.
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DISCUSSION |
All of the currently available NRTIs have a
2',3'-dideoxyribofuranose configuration, and only after anabolic
intracellular phosphorylation do they exert their antiviral activity
against HIV by functioning as viral DNA chain terminators
(22). In this study, we identified 4'-E
nucleoside analogs potent against a variety of HIV strains, including
laboratory strains of HIV-1 and HIV-2, infectious clones, clinical
HIV-1 isolates, and multidrug-resistant clinical strains.
As of this writing, no 5'-triphosphate forms of the 4'-substituted
nucleoside analogs examined in this study have been available, and
enzymatic analyses with wild-type and mutant RT to elucidate the exact
mechanism of antiretroviral activity of the 4'-substituted nucleoside
analogs have not yet been carried out. Nevertheless, it is likely that
these 4'-substituted nucleoside analogs serve as NRTIs but not as
NNRTIs. First, the 4'-substituted nucleoside analogs were active
against the virus with any pyrimidines or purines, although their
antiviral potency varied (Table 1), a different profile from that of
HEPT and its analogs, which are active only with thymine as its base
component (1). Second, although all of the currently
available NNRTIs are incapable of inhibiting HIV-2, and this property
has been one of the salient features of NNRTIs, the selected
4'-substituted nucleoside analogs examined were active against both
HIV-1 and HIV-2 (Tables 1 and 3). Moreover, 4'-E-dC,
4'-E-dA, and 4'-E-dDAP inhibited an
NNRTI-resistant clone, HIV-1Y181C (Table 2).
Third, in the time-of-drug-addition assays (7), the
profiles of anti-HIV-1 activity of two 4'-E nucleosides
(4'-E-dC and 4'-E-araC) were quite similar to
that of AZT (Fig. 2), suggesting that 4'-E nucleosides
suppress the replication of HIV-1 at or around the step of reverse
transcription in the replication cycle. Fourth, antiviral activity of
4'-E-T, 4'-E-dC, and 4'-E-dG was
reversed by the addition of their corresponding physiologic
2'-deoxynucleosides (Fig. 3A, B, and D), strongly suggesting that
4'-E nucleosides serve as substrates for RT. In contrast,
the activity of 4'-E-dA was not reversed by its
corresponding physiologic nucleoside 2'-deoxyadenosine (dA)(Fig. 3C).
In this respect, the activity of ddA (and ddI) against HIV-1 is not
reversed by dA (26), which is possibly due to there being
no intracellular change in dATP levels regardless of extracellular dA
levels, since the ubiquitous adenosine deaminase (ADA) in the target
cells immediately converts dA to 2'-deoxyinosine (3, 34).
In fact, the addition of dA in the presence of an ADA inhibitor
(2'-deoxycoformycin) reversed the activity of 4'-E-dA
against HIV-1 (data not shown). Taken together, like the currently
available NRTIs, 4'-substituted nucleosides most likely block the
replication of HIV by functioning competitive inhibitors for RT.
It is noteworthy that there are a few previously reported
4'-substituted nucleoside analogs active against HIV. Maag et al. synthesized 4'-azidothymidine (4'-AZT), 4'-azido-2'-deoxyguanosine (4'-AZG), and 4'-azido-5-chloro-2'-deoxyuridine (4'-AZU) and found that
these compounds were active against HIV-1 (18, 19). With respect to their specific anti-HIV activity, Chen et al. reported that
following intracellular anabolic phosphorylation of 4'-AZT, HIV RT
efficiently incorporates two consecutive 4'-AZT molecules preventing RT
from further elongating the DNA chain (4). Incorporation of two 4'-AZT-monophosphate (MP) molecules separated by one
2'-deoxynucleoside 5'-MP (dAMP, dCMP, or dGMP) also abolishes DNA chain
elongation by HIV RT. In contrast, cellular DNA polymerases 
and incorporate a single 4'-AZT-MP molecule into nascent DNA at a very slow
rate, but do not incorporate a second consecutive 4'-AZT-MP and
continue to elongate cellular DNA (4).
It should be noted that 4'-E-dC, 4'-E-dA, and
4'-E-dDAP suppressed all infectious HIV-1 clones examined
which were resistant to all the currently available NRTIs, including
two multidrug-resistant HIV-1 variants,
HIV-1M41L/T69S-S-G/T215Y (41) and
HIV-1A62V/V75I/F77L/F116Y/Q151M (35,
36) (Table 2). It is also of note that 4'-E-araC,
4'-Me-dC, and 4'-FMe-dC were less potent against two 3TC-resistant
HIV-1 variants, HIV-1M184I and
HIV-1M184V (Table 2). This difference in drug
resistance profiles between the former three 4'-E
nucleosides and the latter two analogs should be intriguing from a
structure-activity relationship point of view. Recently, the extensive
structural analysis of RT has shed light on the mechanism of viral
resistance to NRTIs (13, 31, 32). Substitutions of amino
acids by mutations, which confer drug resistance, are accumulated
around the deoxynucleoside triphosphate (dNTP) binding sites.
Considering that all of the currently available NRTIs contain
3'-position modifications, HIV-1 is thought to alter the putative dNTP
binding site to develop drug resistance. Indeed, codons K65, L74, Q151,
and M184, which are associated with resistance to NRTIs, are located in
the neighborhood of the enzyme sites. These sites interact with
incoming nucleotides and are thought to be critical for the binding of
the active site of RT to NRTIs, which prevents the NRTIs from being
incorporated into the growing proviral DNA chain (32).
However, 4'-E nucleosides examined in this study retain
3'-OH moiety like natural substrates, which may enable 4'-E
nucleosides to interact with the mutated 3'-OH binding site of various
types of drug-resistant HIV. Isolation and characterization of
resistant mutants against 4'-E nucleosides should provide
more insights into nucleoside-enzyme interactions. Further development
of 4'-E analogs as potential therapeutics for infection with
multidrug-resistant HIV-1 is warranted.
We thank Shin-ichi Oka and Setsuko Ida for providing HIV-1
clinical strains and Miyuki Itoh and Takehiro Suzuki for excellent technical support.
This work was supported in part by a grant from a Research for the
Future Program of Japan Society for the Promotion of Science (JSPS-RFTF
97L00705; H.M.), a Grant-in-Aid for Scientific Research (Priority
Areas) from the Ministry of Education, Culture, Sports, Science, and
Technology of Japan ( E.K. and H.M.), and a Grant for Promotion of AIDS
Research from the Ministry of Health, Welfare, and Labor of Japan
(E.K., M.M., and H.M.).
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Abstract
Introduction
