Received 9 April 1999/Returned for modification 15 July
1999/Accepted 6 August 1999
 |
INTRODUCTION |
Reverse transcriptase (RT)
inhibitors play a cornerstone role in the therapy for human
immunodeficiency virus type 1 (HIV-1) infection. Based on structure and
mechanism of action, these inhibitors can be classified into two major
groups, nucleoside RT inhibitors (NRTIs) and nonnucleoside RT
inhibitors (NNRTIs). NRTIs are usually 2',3'-dideoxy derivatives of
natural substrates of DNA polymerases. All NRTIs are believed to act in
a similar fashion to inhibit the RT activity (9, 15, 20, 34,
39); i.e., following intracellular conversion to their
5'-triphosphate derivatives, they bind to RT in competition with
natural substrates and subsequently cause chain termination through
incorporation into the nascent DNA strand. Chain termination is caused
by the lack of a 3'-hydroxyl motif, which is needed to form a 3'-5'
phosphodiester bond with the next nucleoside substrate in the
elongating DNA strand. NNRTIs are a group of compounds which
specifically inhibit HIV-1 RT by binding to a hydrophobic pocket close
to the polymerase active site, which results in a direct inactivation
of RT (8, 14, 18, 26, 33, 38).
Currently, the appearance of drug-resistant virus is an inevitable
consequence of prolonged exposure of HIV-1 to antiretroviral agents.
This is believed to be caused by both a high turnover of HIV-1 in
patients (16, 37) and low fidelity of the viral RT
(7). To achieve efficient inhibition of HIV-1 replication in
patients, and to delay or prevent the appearance of drug-resistant virus, drug combinations have been used effectively in treating HIV-1
infection (2, 22). However, recent studies have suggested that HIV-1 can become multidrug resistant under combination therapy, albeit requiring a longer time to develop than in a single-drug regime
(4, 31). Therefore, it is still necessary to develop alternate drug combinations for the long-term successful treatment of
HIV-1 infection. We are focusing our efforts on developing new agents
which are effective against existing drug-resistant virus and can be
rationally incorporated into combination drug therapy.
In this regard, several groups have recently reported the synthesis of
pyrimidine- and purine-derived nucleoside analogues containing a
dioxolane sugar derivative, in which an oxygen atom is found at the 3'
position of the sugar ring (3, 6, 21). The
-D
analogues of purine bases have been reported to be potent anti-HIV-1
agents (17, 32). In particular,
(
)-
-D-1',3'-dioxolane guanosine (DXG) and
(
)-
-D-2,6-diaminopurine dioxolane (DAPD) have been
reported to inhibit HIV-1 in vitro (17, 32). Following oral
administration of DAPD to woodchucks or rhesus monkeys, plasma concentrations of DXG are significantly higher than those of DAPD (23, 24). These data suggest that DAPD is quickly converted into DXG in vivo and should be considered a prodrug of DXG. None of the
currently approved anti-HIV-1 nucleoside analogues contains a dioxolane
sugar motif. Therefore, DXG and its putative prodrug analogue represent
a novel class of nucleosides with potential utility in anti-HIV-1
therapy. The present report describes our results in further
elucidating the mechanism of action of DXG and DAPD. In addition, we
have examined the effect of these compounds on wild-type (wt) and
drug-resistant clinical isolates of HIV-1, alone and in combination
with other NRTIs or NNRTIs.
 |
MATERIALS AND METHODS |
Reagents.
DXG, DAPD, DXG 5'-triphosphate (DXG-TP),
(+)-
-D-1',3'-dioxolane guanosine, and
2',3'-dideoxy-3'-thiacytidine (3TC) were synthesized at BioChem Pharma
as previously described (3, 32). All of the dioxolanyl
nucleosides were enantiomerically pure. 3'-Azido-2',3'-dideoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC) were purchased from Sigma (Oakville, Ontario, Canada). Ultrapure nucleoside 5'-triphosphates, 2'-deoxynucleoside 5'-triphosphates (dNTPs), 2',3'-dideoxynucleoside 5'-triphosphates (ddNTPs), and polynucleotides poly(rA) · oligo(dT)12-18 and poly(rC) · oligo(dG)12-18 were purchased from Pharmacia Biotech Inc.
(Montreal, Quebec, Canada). AZT triphosphate (AZT-TP) and 3TC
triphosphate (3TC-TP) were purchased from Moravek Biochemicals. [3H]dGTP, [3H]TTP, and
[
-32P]ATP were obtained from Du Pont NEN (Montreal,
Quebec, Canada). [3H]thymidine was obtained from Amersham
(Oakville, Ontario, Canada). Nevirapine was generously provided by
Boehringer-Ingelheim Inc. (Burlington, Ontario, Canada).
Cells and viruses.
Human cord blood mononuclear cells
(CBMCs) and peripheral blood mononuclear cells (PBMCs) were obtained
from HIV-1-negative and hepatitis B virus-negative donors (Department
of Obstetrics, Jewish General Hospital, Montreal, Quebec, Canada) and
isolated by Ficoll-Hypaque (Pharmacia) density gradient centrifugation. Cells were cultured in RPMI 1640 medium (Gibco BRL Laboratories, Mississauga, Ontario, Canada) containing 0.1% (vol/vol) (5 µg/ml) phytohemagglutinin (Boehringer Mannheim, Montreal, Quebec, Canada), 10% fetal calf serum (Flow Laboratories, Toronto, Ontario, Canada), 2 mM glutamine, 100 U of penicillin per ml, 100 µg of streptomycin per
ml, and 15 U of interleukin 2 (Boehringer Mannheim) per ml. Cells were
incubated at 37°C, in an atmosphere of 5% CO2, for 3 to
4 days prior to being used for antiviral assays (27).
The T-cell lines MT-2, MT-4, H9, and Jurkat were obtained from either
the National Institutes of Health AIDS Research and Reference Reagents
(Rockville, Md.) or the American Type Culture Collection (Manassas,
Va.). These cells were used for antiviral and cytotoxicity studies and
were maintained as suspension cultures in RPMI 1640 medium containing
10% fetal calf serum, 2 mM glutamine, 100 U of penicillin per ml, and
100 µg of streptomycin per ml. Other tumor cell lines, Molt-4,
HT-1080, DU145, and HepG2, were also obtained from the American Type
Culture Collection. One normal cell line, human skin fibroblasts (HSF),
was obtained from M. Chevrette (McGill University, Montreal, Quebec,
Canada). These cells were used for cytotoxicity assays. HSF, HT-1080,
DU145, and HepG-2 cells were cultured in minimal essential medium.
Molt-4 cells were cultured in RPMI 1640 medium.
HIV-1IIIB and the recombinant HIV-1 clone HXB2-D were
kindly supplied by R. C. Gallo (Institute of Human Virology,
Baltimore, Md.). Recombinant mutated HIV-1 variants were prepared by
site-directed mutagenesis as previously described (10, 12).
HIV-1 clinical isolates were obtained by coculture of peripheral blood
lymphocytes from HIV-1-infected individuals with CBMCs and then
propagated on CBMCs in the absence of drugs as previously described
(28).
Antiviral assays.
The anti-HIV-1 activities of DXG and DAPD
were assessed by employing HIV-1IIIB in a variety of cell
types as previously described (10, 12, 25, 28). A number of
recombinant drug-resistant variants and low-passage clinical isolates
from individuals who had received long-term anti-HIV therapy were also
used to evaluate the effects of these two compounds. Briefly, cells
were incubated for 2 to 3 h with virus at a multiplicity of
infection of 0.005 for T-cell assays or 0.5 for monocytic-cell assays.
The infected cells were cultured in the presence of the test compound
for 5 to 7 days. The anti-HIV-1 efficacy was determined by testing for HIV-1 RT activity in the cell culture supernatants. All assays were
performed in duplicate, and at least two independent experiments were
performed. AZT and/or 3TC was used as a control in each experiment. The
data are expressed as the means of the 50% effective concentrations (EC50s) as calculated from the linear portion of the
dose-response curve.
Effects of combining DXG and standard anti-HIV-1 agents were assessed
in CBMCs by using HIV-1IIIB. The combinations were
performed by using a checker board cross pattern of drug
concentrations. The antiviral effects were determined by monitoring RT
activity in the culture supernatants at day 7. The data were analyzed
according to the method described by Chou and Talalay (5).
The combination indices (CIs) of DXG with other anti-HIV-1 agents were
calculated by using CalcuSyn software (Biosoft, Cambridge, United
Kingdom). Theoretically, a CI value of 1 indicates an additive effect,
a CI value of >1 indicates antagonism, and a CI value of <1 indicates synergism.
Cytotoxicity analysis.
Cellular toxicity was assessed by
[3H]thymidine uptake and WST-1 staining. In the
[3H]thymidine uptake experiments, Molt-4, HT1080, DU-145,
HepG2, and HSF were plated at a concentration of 1 × 103 to 2 × 103 cells per well (96-well
plates). Phytohemagglutinin-stimulated PBMCs were cultured at a
concentration of 4 × 104 per well. Following a 24-h
preincubation period, test compounds (at 10
4 to
10
10 M concentrations) were added and the cells were
incubated for an additional 72 h. [3H]thymidine was
added during the final 18-h incubation period. The cells were then
washed once with phosphate-buffered saline, treated with trypsin if the
cells were adherent, and resuspended in water (hypotonic lysing of
cells). The cellular extract was applied directly to a Tomtec Harvester
96 apparatus. The 50% cytotoxic concentration (CC50) was
determined by comparing the radioactive counts per minute obtained from
drug-tested samples to those obtained from the control (untreated) cells.
In the WST-1 staining experiments, cell lines were cultured in RPMI
medium in 96-well plates at a density of 5 × 104
cells/well. CBMCs were plated at a concentration of 0.5 × 106/well. Compounds (at 10
4 to
10
7 M concentrations) were added at day zero. Cell
viability was assessed on day 7 by using the WST-1 reagent (Boehringer
Mannheim) in accordance with the protocol provided by the supplier.
RT inhibition assay.
wt recombinant HIV-1 RT was expressed
as a histidine-tagged protein in Escherichia coli and
purified to 95% homogeneity as previously described (11,
13). Inhibition of HIV-1 RT RNA-dependent DNA polymerase activity
by DXG-TP was assessed by employing both homopolymeric and
heteropolymeric RNA templates/DNA primers (T/P). The heteropolymeric
RNA template (HIV-PBS)/18-mer oligodeoxynucleotide primer (dPR) was
prepared as described previously (11). The reverse
transcription reaction mixture contained final concentrations of 50 mM
Tris-HCl (pH 7.8), 60 mM KCl, 10 mM MgCl2, 0.1 U of
homopolymeric T/P per ml, 5 µM dNTP substrate or 25 nM HIV-PBS/dPR,
and 5 µM each dATP, dCTP, dGTP, and dTTP in 100 µl. Reaction
mixtures were incubated for 30 min at 37°C in the presence or absence
of ddNTP inhibitors as described previously (11).
The effect of DXG-TP on RT activity was also assessed by using a chain
termination/dNTP incorporation assay in which inhibition of nascent DNA
synthesis (chain termination) was monitored based on cDNA synthesis as
previously described (1, 13).
Determination of HIV-1 RT genotype.
To determine the RT
genotypes of the HIV-1 clinical isolates, proviral DNA of each isolate
was extracted from infected CD4+ T cells or CBMCs and the
complete RT coding regions were amplified by PCR as previously reported
(10). The PCR product was purified and then directly
sequenced by using primer RTS (5'-CCAAAAGTTAAACAATGGC-3'), which corresponds to the 5' portion of the RT coding region
(nucleotides 2603 to 2621 of HXB2-D coordinates).
 |
RESULTS |
Inhibition of HIV-1 RT polymerase activity by DXG-TP.
The
chemical structures of 1',3'-dioxolanylpurine nucleosides DXG and DAPD
are shown in Fig. 1. DXG-TP is the active
antiviral form of DAPD in vivo (23, 24). To better define
the molecular mechanism by which these nucleoside analogues inhibit
HIV-1, DXG was chemically converted to its triphosphate derivative
(DXG-TP) and tested for its direct effect on HIV-1 RT. The inhibitory
effect of DXG-TP on HIV-1 RT activity was assessed by using various
homopolymeric and heteropolymeric T/P (Table
1). DXG-TP was a potent HIV-1 RT
inhibitor, with a 50% inhibitory concentration (IC50) of
0.012 µM, when using wt HIV-1 RT, complementary T/P poly(rC) · oligo(dG), and substrate dGTP (Table 1). This value is similar to that
obtained for ddGTP. Similarly, DXG-TP and ddGTP were observed to have
the same inhibitory effect on HIV-1 RT when the heteropolymeric T/P (HIV-PBS/dPR) was used (Table 1). The inhibition of HIV-1 RT by DXG-TP
was observed to occur via competition with the natural substrate; i.e.,
the higher the concentration of dGTP, the lower the inhibitory effect
of DXG-TP (data not shown). In addition, as expected, DXG-TP did not
show any inhibition of HIV-1 RT activity at concentrations up to 10 µM when the noncomplementary T/P poly(rA) · oligo(dT) was used
along with dTTP as the substrate (Table 1).
We also analyzed the effect of DXG-TP on HIV-1 RT activity by a chain
elongation/termination assay which provides a method to directly
visualize the incorporation of dideoxynucleotide monophosphates into
nascent DNA by monitoring the reaction products through polyacrylamide gel electrophoresis. It was showed that DXG monophosphate was incorporated into the nascent DNA strands and resulted in chain termination (results not shown). The pattern of chain termination generated by incorporation of DXG-TP into elongating DNA strands was
exactly the same as that for ddGMP. In general, the inhibitory effect
of DXG-TP on RT activity in this cell-free assay was approximately equivalent to those observed for ddGTP and AZT-TP but stronger than the
chain termination observed for 3TC-TP (results not shown).
Cellular toxicity.
DXG and DAPD, along with 3TC and AZT, were
also tested for their effect on cell proliferation by measuring
[3H]thymidine uptake and cell toxicity by WST-1 cell
viability assays. DXG and DAPD did not inhibit cell proliferation at
concentrations up to 500 µM in various cells (Table
2). In the same experiments, CC50s for AZT and ddC were found to be less than 10 µM.
DXG was not toxic to human CBMCs (Table 2), MT-2, H9, or Jurkat cell lines (data not shown) at concentrations up to 100 µM by a WST-1 cell
viability assay. In contrast, the CC50s obtained for AZT and ddC in CBMCs were 74 and 29 µM, respectively (Table 2).
Anti-HIV-1 efficacy in different cell types.
The anabolism of
nucleoside analogues can be greatly influenced by cell type. Therefore,
we assessed the anti-HIV-1 activity of DXG and DAPD in human CBMCs and
a variety of human T-cell lines. A dose-response curve showing the
inhibition of HIV-1IIIB in MT-2 cells is presented in Fig.
2. The results indicate that the activity of DXG is approximately equivalent to that of 3TC but is 5- to 10-fold
lower than that of AZT. DAPD was approximately 10-fold less active than
DXG. The EC50s obtained for the test compounds in primary
cells and cell lines are compiled in Table
3.

View larger version (22K):
[in this window]
[in a new window]
|
FIG. 2.
Dose-response curve of inhibition of HIV-1 replication.
MT-2 cells were infected with HIV-1IIIB at a multiplicity
of infection of 0.005. The infected cells were cultured in the presence
of various concentrations of antiviral compounds as indicated. Viral
susceptibility to the compounds was assayed by measurement of HIV-1 RT
activity in the culture supernatants as described in Materials and
Methods. Data are expressed as means ± standard deviations of
data from at least five separate experiments, each performed in
duplicate.
|
|
We also compared the antiviral activities of DXG and
(+)-
-D-1',3'-dioxolane guanosine. Our results show that
the (+) enantiomer (EC50, 0.7 µM) has less activity
against HIV-1IIIB in MT-2 cells than the (
) enantiomer
(EC50, 0.085 µM).
Susceptibility of recombinant drug-resistant HIV-1 variants.
Recombinant HIV-1 variants carrying a drug resistance mutation(s) were
employed to test the cross-resistance phenotypes of DXG and DAPD in
CBMCs and MT-2 cells. All of the recombinant viral strains were derived
from HXB2-D. A summary of the genetic backgrounds of the variants and
their sensitivities to the dioxolanylpurine compounds, 3TC and AZT, in
CBMCs is shown in Table 4. These viruses contain mutations seen for the most common RT inhibitor- and protease inhibitor-resistant HIV-1 variants (summarized in reference
29). The variants of HIV-1 carrying
2',3'-dideoxyinosine (ddI), ddC, or 3TC resistance mutations (i.e.,
65K, 74V, and 184V substitutions, respectively) in the RT gene had
minimally (two- to fivefold) decreased sensitivity to DXG and DAPD
compared to the wt HXB2-D in CBMCs (Table 4). Similar results were
obtained when the recombinant viruses were tested in MT-2 cells (data
not shown). In addition, the variant bearing mutations 41L, 215Y, and
184V had approximately a twofold-decreased sensitivity to DXG, which
was similar to that of the 184V single-mutant recombinant. This variant
had high-level resistance to 3TC but increased sensitivity to AZT
(36).
In contrast, AZT-resistant virus, carrying multiple substitutions (41L,
70R, 215Y, and 219Q) in its RT gene, remained completely sensitive to
DXG and DAPD in CBMCs (Table 4) and MT-2 cells (data not shown). In
addition, the dioxolanyl nucleoside analogues were effective against
NNRTI-resistant (EC50 = 0.05 µM) and protease inhibitor-resistant (EC50 = 0.12 to 1.37 µM)
variants (Table 4).
Susceptibility of HIV-1 clinical isolates.
The sensitivity of
viruses found in clinical isolates to antiviral chemotherapy might be
quite variable due to the presence of quasispecies. In addition, HIV-1
isolates obtained from patients receiving long-term antiretroviral
therapy might behave differently from cloned viruses containing
genetically engineered mutations in the RT gene. For these reasons,
clinical isolates of HIV-1 from antiviral-agent-naive and drug-treated
patients were assayed in CBMCs for their sensitivity to DXG and DAPD. A
summary of the recent therapy histories of the patients from which the
HIV-1 isolates were obtained, the RT genotypes of the isolates, and their sensitivities to the various anti-HIV agents is presented in
Table 5.
Four isolates (no. 3887, 4246, 4877, and 4526) were sensitive to AZT
and/or 3TC or had marginally decreased sensitivity to one of these two
drugs compared with recombinant variants (Table 4). Using these four
isolates, the EC50s obtained for both DXG and DAPD (Table
5) were comparable to those observed with the wt strains
HIV-1IIIB and HXB2-D assessed in CBMCs (Tables 3 and 4).
Isolates 3350 and 4205, obtained from patients who had received 3TC
therapy and carried the 184V mutation, were 3TC resistant and AZT
sensitive. These 184V mutant isolates had an approximately fivefold-decreased susceptibility to DXG compared to the 3TC- and
AZT-sensitive isolates (Table 5). This was consistent with the results
obtained with the recombinant variants (Table 4).
Clinical isolate 4242, obtained from a patient treated with AZT,
exhibited decreased sensitivity to AZT but remained sensitive to DXG,
DAPD, and 3TC. The NNRTI-resistant strain 4924, isolated from an
individual undergoing AZT therapy, had an EC50 for
nevirapine of >10 µM but was sensitive to the dioxolane nucleoside
analogues (Table 5). The protease inhibitor-resistant isolate 4833 was obtained from an individual who had received 48 weeks of saquinavir therapy. This isolate exhibited a 20-fold-decreased sensitivity (EC50 = 0.11 µM) to the protease inhibitor compared
with the baseline isolate, 4526 (EC50 = 0.0063 µM).
The 4833 isolate remained sensitive to the dioxolanyl compounds
(EC50 = 0.17 µM for DXG and 0.63 µM for DAPD)
(Table 5).
Anti-HIV drug combination effects.
The antiviral efficacy of
DXG against HIV-1IIIB was assessed in combination with AZT,
3TC, or nevirapine in CBMCs. The CIs of DXG in combination with the
approved anti-HIV-1 agents are summarized in Table
6. The CIs were calculated at several
different effective concentrations (EC50, EC75,
and EC90) and in different molar ratios of the combined
drugs. The CIs obtained were between 0.4 and 0.9 in the case of DXG
combined with either 3TC or nevirapine, which suggests that DXG had
moderate synergy with these two agents. However, DXG demonstrated
greater synergy with the thymidine analogue AZT, with CIs between 0.3 to 0.8 at the EC50 and less than 0.3 at higher effective
concentrations, which indicates a strong synergy.
 |
DISCUSSION |
The dioxolanyl guanosine analogues DXG and DAPD were previously
reported to possess anti-HIV and anti-hepatitis B virus activities (17, 30, 32). In this article, we present an expanded and detailed evaluation of antiviral and biochemical characteristics of DXG
and DAPD.
In vitro antiviral assays demonstrated that both DXG and DAPD had very
promising anti-HIV-1 activity in the various types of cells tested.
Comparison of the (
) and (+) enantiomers of
-1',3'-dioxolane
guanosine showed that both were effective inhibitors of HIV-1, but the
(
) enantiomer, DXG, displayed approximately 10-fold higher activity.
Generally, the anti-HIV-1 activity of DXG was at the same level as that
observed for 3TC in our assays but was 5- to 10-fold less than that of
AZT. In vivo, DAPD is quickly and efficiently converted into DXG after
either oral or intravenous administration to woodchucks or rhesus
monkeys (23, 24). This biotransformation is believed to be a
deamination process catalyzed by a ubiquitous enzyme, adenosine
deaminase. In our experiments, DAPD had consistently lower anti-HIV
activity than DXG. These results were consistent with previous reports for the antiretroviral effects of these two dioxolanylpurine nucleoside analogues (17, 32). The relatively low anti-HIV-1 activity of the putative prodrug, DAPD, in cell culture may reflect
less-efficient metabolic conversion into the active form, DXG. This
does not necessarily indicate that this compound has less anti-HIV-1
activity in vivo. From a pharmaceutical point of view, DAPD might be
equipotent to DXG in vivo. The ultimate choice of compound to advance
into human therapy would be dependent on other parameters, such as oral
bioavailability (23, 24).
The appearance of drug-resistant virus following prolonged
administration of antiviral agents is a major obstacle for the therapy
of HIV-1 infection. Therefore, we conducted antiviral assays to
elucidate the cross-resistance profiles of the novel dioxolanylpurine
analogues. Using recombinant and clinical drug-resistant HIV-1 variants
carrying the most common RT inhibitor resistance-related mutations, it
was demonstrated that DXG and DAPD possess marginal cross-resistance
with 3TC (Tables 4 and 5). Our results show that HIV-1 strains carrying
65R and 74V substitutions in their RT genes had approximately
fivefold-reduced sensitivity to the dioxolane compounds, which is
consistent with previously reported values (19). However,
both DXG and the DAPD retained their potency to AZT-resistant,
NNRTI-resistant, and protease inhibitor-resistant HIV-1 variants. These
data suggest that DXG or DAPD could be used as an alternative drug for
the treatment of HIV-1-infected individuals who have developed
tolerance to currently approved drug regimens.
Combination therapy has proven to be an exciting approach to combat
HIV-1 infection. Combination therapy increases the therapeutic efficiency of anti-HIV agents and delays or prevents selection of
drug-resistant virus. Combination profiles with approved anti-HIV-1 agents have become an essential prerequisite for new drug candidates. DXG showed synergy with the NRTIs AZT and 3TC and the NNRTI nevirapine (Table 6). These data suggest that the combination of DXG with approved
anti-HIV agents in the treatment of HIV-1 infection could have
increased clinical benefits. Previous reports have demonstrated that
the mutations 65R, 74V, and 184V in HIV-1 RT, which confer minimal
cross-resistance to DXG and DAPD, can revert AZT-resistant virus to AZT
sensitivity (19, 35, 36). The strong in vitro synergy of DXG
and AZT, combined with the increased sensitivity of the AZT drug
resistance phenotype, indicates a potential benefit for a DXG and AZT combination.
The guanosine analogue dideoxyguanosine displays excellent anti-HIV
activity in vitro. However, it was never developed into an
antiretroviral agent because of its high cellular toxicity. DXG and
DAPD had relatively low cellular toxicity to human primary cells and a
variety of established normal and tumor cell lines. Neither compound
showed significant inhibition of cellular DNA synthesis or cell
proliferation, and both were found to be much less toxic than AZT or
ddC (Table 2).
Our RT enzymatic analysis elucidated that DXG-TP, the putative active
intracellular metabolite of DXG and DAPD, is a competitor of the
natural substrate deoxyguanosine (Table 1) and a DNA synthesis chain
terminator (data not shown). Similar to other nucleoside analogues
(9, 11, 13), DXG-TP inhibits HIV-1 in vivo by competitively
inhibiting the binding of the natural substrate dGTP to the HIV-1 RT.
DXG-TP is incorporated into the nascent DNA strand, resulting in chain termination.
In summary, the dioxolanylpurine analogues DXG and DAPD are distinct
from the related nucleoside analogue dideoxyguanosine in that they are
selective inhibitors of the viral polymerase and have relatively low
cellular toxicity, yielding a large selective index. These novel
guanosine analogues have potent antiretroviral activity against both
wild-type and drug-resistant HIV-1 variants, as well as synergistic
activity when tested in combination with approved anti-HIV-1 agents.
Thus, these observations suggest that the heterosubstituted guanosine
analogues have potential as anti-HIV-1 drug candidates.
| 1.
|
Arts, E. J.,
X. Li,
Z. Gu,
L. Kleiman,
M. A. Parniak, and M. A. Wainberg.
1994.
Comparison of deoxynucleotide and tRNA3lys as primers in an endogenous human immunodeficiency virus type-1 in vitro reverse transcription/template-switching reaction.
J. Biol. Chem.
269:14672-14680[Abstract/Free Full Text].
|
| 2.
|
Autran, B.,
G. Li,
T. S. Blanc,
D. Matthez,
R. Tubiana,
C. Katlama,
P. Debre, and J. Leibowitch.
1997.
Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease.
Science
277:112-116[Abstract/Free Full Text].
|
| 3.
| Belleau, B., D. Dixit, N. Nguyen-Ba, and J. Krause. 1989. Design and activity of a novel class of nucleoside
analogs effective against HIV-1. Presented at the 5th International
Conference on AIDS, Montreal, Quebec, Canada, 4 to 9 June 1989.
|
| 4.
|
Carpenter, C. C. J.,
M. A. Fishl,
S. M. Hammer,
M. S. Hirsch,
D. M. Jacobson,
D. A. Katzenstein,
J. S. G. Montaner,
D. D. Richman,
M. S. Saag,
R. T. Schooley,
M. A. Thompson,
S. Valla,
P. G. Yani, and P. A. Volberding.
1997.
Antiretroviral therapy for HIV infection in 1997. Update recommendations of the International AIDS Society-USA panel.
JAMA
277:1962-1969[Abstract].
|
| 5.
|
Chou, T.-C., and P. Talalay.
1984.
Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors.
Adv. Enzyme Regul.
22:27-55[Medline].
|
| 6.
|
Chu, C.,
S. Ahn,
H. Kim,
A. J. Alves,
J. W. Beach,
L. S. Jeong,
Q. Islam, and R. F. Schinazi.
1991.
Asymmetric synthesis of enantiomerically pure ( )-(1'R, 4'R-dioxolane-thymidine) and its anti-HIV activity.
Tetrahedron Lett.
32:3791-3794.
|
| 7.
|
Coffin, J. M.
1995.
HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy.
Science
267:483-489.
|
| 8.
|
Ding, J.,
K. Das,
H. Moereels,
L. Koymans,
K. Andries,
P. A. L. Janssen,
S. H. Hughes, and E. Arnold.
1995.
Structure of HIV-1 RT/TIBO R 86183 complex reveals similarity in the binding of diverse nonnucleoside inhibitors.
Nat. Struct. Biol.
2:407-415[Medline].
|
| 9.
|
Furman, P. A.,
J. A. Fyfe,
M. H. St. Clair,
K. Weinhold,
J. L. Rideout,
G. A. Freeman,
S. N. Lehrman,
D. P. Bolognesi,
S. Broder,
H. Mitsuya, and D. W. Barry.
1986.
Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase.
Proc. Natl. Acad. Sci. USA
83:8333-8337[Abstract/Free Full Text].
|
| 10.
|
Gu, Z.,
Q. Gao,
M. A. Parniak, and M. A. Wainberg.
1992.
Novel mutation in the human immunodeficiency virus type 1 reverse transcriptase gene that encodes cross-resistance to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine.
J. Virol.
66:12-19[Abstract/Free Full Text].
|
| 11.
|
Gu, Z.,
R. S. Flether,
E. J. Arts,
M. A. Wainberg, and M. A. Parniak.
1994.
The K65R mutant reverse transcriptase of HIV-1 cross-resistance to 2',3'-dideoxycytidine, 2',3'-dideoxy-3'-thiacytidine, and 2',3'-dideoxyinosine shows reduced sensitivity to specific dideoxynucleoside triphosphate inhibitors in vitro.
J. Biol. Chem.
269:28118-28122[Abstract/Free Full Text].
|
| 12.
|
Gu, Z.,
Q. Gao,
H. Fang,
H. Salomon,
M. A. Parniak,
E. Goldberg,
J. Cameron, and M. A. Wainberg.
1994.
Identification of a mutation at codon 65 in the IKKK motif of reverse transcriptase that encode human immunodeficiency virus resistance to 2',3'-dideoxycytidine and 2',3'-dideoxy-3'-thiacytidine.
Antimicrob. Agents Chemother.
38:275-281[Abstract/Free Full Text].
|
| 13.
|
Gu, Z.,
E. J. Arts,
M. A. Parniak, and M. A. Wainberg.
1995.
Mutated K65R recombinant HIV-1 reverse transcriptase shows diminished chain termination in the presence of 2',3'-dideoxycytidine-5'-triphosphate and other drugs.
Proc. Natl. Acad. Sci. USA
92:2760-2764[Abstract/Free Full Text].
|
| 14.
|
Gu, Z.,
Y. Quan,
Z. Li, and M. A. Wainberg.
1995.
Effects of non-nucleoside inhibitors of human immunodeficiency virus type 1 in cell-free recombinant reverse transcriptase.
J. Biol. Chem.
270:31046-31051[Abstract/Free Full Text].
|
| 15.
|
Hart, G. J.,
D. C. Orr,
C. R. Penn,
H. T. Figueiredo,
N. M. Gray,
R. E. Boehme, and J. M. Cameron.
1992.
Effects of ( )-2'-deoxy-3'-thiacytidine (3TC) 5'-triphosphate on human immunodeficiency virus reverse transcriptase and mammalian DNA polymerases alpha, beta, and gamma.
Antimicrob. Agents Chemother.
36:1688-1694[Abstract/Free Full Text].
|
| 16.
|
Ho, D.,
A. U. Neumann,
A. S. Perelson,
W. Chen,
J. M. Leonard, and M. Markowitz.
1995.
Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection.
Science
273:123-126.
|
| 17.
|
Kim, H. O.,
R. F. Schinazi,
S. Nampalli,
K. Shanmuganathan,
D. L. Cannon,
A. J. Alves,
L. S. Jeong,
J. W. Beach, and C. K. Chu.
1993.
1,3-Dioxolanylpurine nucleosides (2R, 4R) and (2R, 4S) with selective anti-HIV-1 activity in human lymphocytes.
J. Med. Chem.
36:30-37[Medline].
|
| 18.
|
Kolstaedt, L. A.,
J. Wang,
J. M. Friedman,
P. A. Rice, and T. A. Steitz.
1993.
The structure of HIV-1 reverse transcriptase, p. 223-249.
In
A. M. Skalka, and S. P. Goff (ed.), Reverse transcriptase. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 19.
|
Mellors, J. W.,
H. Bazmi,
C. K. Chu, and R. F. Schinazi.
1996.
K65R mutation in HIV-1 reverse transcriptase causes resistance to ( )- -D-dioxolane-guanosine and reverses AZT resistance, abstr. 7.
In
Proceedings of the 5th International Workshop on HIV Drug Resistance, Whistler, Canada, 3 to 6 July 1996.
|
| 20.
|
Mitsuya, H., and S. Broder.
1986.
Inhibition of the in vitro infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) by 2',3'-dideoxynucleoside.
Proc. Natl. Acad. Sci. USA
83:1911-1915[Abstract/Free Full Text].
|
| 21.
|
Norbeck, D. W.,
S. Spanton,
S. Broder, and H. Mitsuya.
1989.
(±)-Dioxolane-T ((±)-1-[(2 ,4)-2-(hydroxymethyl)-4-dioxolanyl]thymine): a new 2',3'-dideoxynucleoside prototype with in vitro activity against HIV.
Tetrahedron Lett.
30:6263-6266.
|
| 22.
|
Perelson, A. S.,
P. Essunger,
Y. Cao,
M. Vesanen,
A. Hurley,
K. Saksela,
M. Markowitz, and D. Ho.
1997.
Decay characteristics of HIV-1 infected compartments during combination therapy.
Nature
387:188-191[Medline].
|
| 23.
|
Rajagopalan, P.,
F. D. Boudinot,
C. K. Chu,
H. M. McClure, and R. F. Schinazi.
1994.
Pharmacokinetics of ( )- -D-2,6-diaminopurine dioxolane and its metabolite guanosine in rhesus monkeys.
Pharm. Res.
11(Suppl.):381-386.
|
| 24.
|
Rajagopalan, P.,
F. D. Boudinot,
C. K. Chu,
B. C. Tennant,
B. H. Baldwin, and R. F. Schinazi.
1996.
Pharmacokinetics of ( )- -D-2,6-diaminopurine dioxolane and its metabolite, dioxolane guanosine, in woodchucks (Marmota monax).
Antivir. Chem. Chemother.
7:65-70.
|
| 25.
|
Rando, R.,
J. Ojwang,
A. Elbaggari,
G. R. Reyes,
R. Tinder,
M. S. McGrath, and M. E. Hogan.
1995.
Suppression of human immunodeficiency virus type 1 activity in vivo by oligonucleotide which form intramolecular tetrads.
J. Biol. Chem.
270:1754-1760[Abstract/Free Full Text].
|
| 26.
|
Rittinger, K.,
G. Divita, and R. S. Goody.
1995.
Human immunodeficiency virus reverse transcriptase substrate-induced conformational changes and the mechanism of inhibition by nonnucleoside inhibitors.
Proc. Natl. Acad. Sci. USA
92:8046-8049[Abstract/Free Full Text].
|
| 27.
|
Rooke, R.,
M. Tremblay, and M. A. Wainberg.
1990.
AZT (zidovudine) may act postintegrationally to inhibit generation of HIV-1 progeny virus in chronically infected cells.
Virology
176:205-215[Medline].
|
| 28.
|
Salomon, H.,
A. Belmonte,
K. Nguyen,
Z. Gu,
M. Gelfand, and M. A. Wainberg.
1994.
Comparison of cord blood and peripheral blood mononuclear cells as targets for viral isolation and drug sensitivity studies involving human immunodeficiency virus type 1.
J. Clin. Microbiol.
32:2000-2002[Abstract/Free Full Text].
|
| 29.
|
Schinazi, R. F.,
B. A. Larder, and J. W. Mellors.
1997.
Mutations in retroviral genes associated with drug resistance.
Int. Antivir. News
4:95-107.
|
| 30.
|
Schinazi, R. F.,
H. M. McClure,
F. D. Boudinot,
Y. Jiang, and C. K. Chu.
1994.
Development of ( )- -D-2,6-diaminopurine dioxolane as a potential antiviral agent.
Antivir. Res.
23:81. (Abstract.)
|
| 31.
|
Shirasaka, T.,
M. F. Kavlick,
T. Ueno,
W.-Y. Gao,
E. Kojima,
M. L. Alcaide,
S. Chokekijchai,
B. M. Roy,
E. Arnold,
R. Yarchoan, and H. Mitsuya.
1995.
Emergence of human immunodeficiency virus type 1 variants with resistance to multiple dideoxynucleosides in patients receiving therapy with dideoxynucleosides.
Proc. Natl. Acad. Sci. USA
92:2398-2402[Abstract/Free Full Text].
|
| 32.
|
Siddiqui, M. A.,
W. L. Brown,
N. Nguyen-Ba,
D. M. Dixit, and T. S. Mansour.
1993.
Antiviral optically pure dioxolane purine nucleoside analogs.
Bioorg. Med. Chem. Lett.
3:1543-1546.
|
| 33.
|
Spence, J. C.,
W. M. Kati,
K. S. Anderson, and K. A. Johnson.
1995.
Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside inhibitors.
Science
276:988-992.
|
| 34.
|
St. Clair, M. H.,
C. A. Richards,
T. Spector,
K. J. Weinhold,
W. H. Miller,
A. J. Langlois, and P. A. Furman.
1987.
3'-Azido-3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase.
Antimicrob. Agents Chemother.
31:1972-1977[Abstract/Free Full Text].
|
| 35.
|
St. Clair, M. H.,
J. L. Martin,
G. Tudor-Williams,
M. C. Bach,
C. L. Vavro,
D. M. King,
P. Kellam,
S. D. Kemp, and B. A. Laeder.
1991.
Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase.
Science
153:1557-1559.
|
| 36.
|
Tisdale, M.,
S. D. Kemp,
N. R. Parry, and B. A. Larder.
1993.
Rapid in vitro selection of human immunodeficiency virus type 1 resistance to 3'-thiacytidine inhibitors due to a mutation in the YMDD region of reverse transcriptase.
Proc. Natl. Acad. Sci. USA
90:5653-5656[Abstract/Free Full Text].
|
| 37.
|
Wei, X.,
S. K. Ghosh,
M. E. Taylor,
V. A. Johnson,
E. A. Emini,
P. Deutsch,
J. D. Lifson,
S. Bonhoeffer,
M. A. Nowak,
B. H. Hahn,
M. S. Saag, and G. M. Shaw.
1995.
Viral dynamics in human immunodeficiency virus type 1 infection.
Science
273:117-122.
|
| 38.
|
Wu, J. C.,
T. C. Warren,
J. Adams,
J. Proudfoot,
J. Skiles,
P. Raghavan,
C. Perry,
I. Potocki,
P. R. Farina, and P. M. Grob.
1991.
A novel dipyridodiazepinone inhibitor of HIV-1 reverse transcriptase acts through a nonsubstrate binding site.
Biochemistry
30:2022-2026[Medline].
|
| 39.
|
Yarchoan, R.,
H. Mitsuya,
C. E. Myers, and S. Broder.
1989.
Clinical pharmacology of 3'-azido-2',3'-dideoxythymidine (zidovudine) and related dideoxynucleosides.
N. Engl. J. Med.
321:726-738[Medline].
|