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Vol. 52, Issue 6, 1157-1163, 1997
Rega Institute for Medical Research, Katholieke Universtiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium (D.D., J.A.E., M.W., C.P., H.J., E.D.C., A.-M.V.), and Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Italy (S.A., C.-F.P.)
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Summary |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Various analogues of adenosine have been described as inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, and some of these AdoHcy hydrolase inhibitors (e.g., 3-deazaadenosine, 3-deazaaristeromycin, and 3-deazaneplanocin A) have also been reported to inhibit the replication of human immunodeficiency virus type 1 (HIV-1). When evaluated against HIV-1 replication in MT-4 cells, macrophages, or phytohemagglutinin-stimulated peripheral blood lymphocytes infected acutely or chronically with HIV-1IIIB or HIVBaL strains, a wide range of adenosine analogues did not inhibit HIV-1IIIB replication for 50% at subtoxic concentrations. However, they inhibited HIV-1 replication in HeLa CD4+ LTR-LacZ cells at concentrations well below cytotoxicity threshold. A close correlation was found among the inhibitory effect of the compounds on AdoHcy hydrolase activity, their inhibition of HIV-1 replication in Hela CD4+ LTR-LacZ cells, and their inhibition of the HIV-1 Tat-dependent and -independent transactivation of the long terminal repeat, whereas no inhibitory effect was seen on HIV-1 reverse transcription or a Tat-independent cytomegalovirus promoter. Our results suggest that AdoHcy hydrolase and the associated S-adenosylmethionine-dependent methylation mechanism play a role in the process of long terminal repeat transactivation and, hence, HIV replication.
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Introduction |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Various
analogues of adenosine have been described as inhibitors of AdoHcy
hydrolase. AdoHcy hydrolase catalyzes the reversible hydrolysis of
AdoHcy to adenosine and L-Hcy (1). Inhibition of AdoHcy
hydrolase by adenosine analogues results in an accumulation of AdoHcy,
a product inhibitor of methylation reactions that use AdoMet as methyl
donor. Methylations of DNA, RNA, proteins, and phospholipids play a
crucial role in numerous biological processes (for a review, see Ref.
2). Methylation of eukaryotic and viral mRNA (5
-capping) is essential
for the maturation of the mRNA and the translation to proteins and
hence plays an important role in the virus replicative cycle (2).
De Clercq et al. (3-6) described the broad-spectrum
antiviral activity of AdoHcy hydrolase inhibitors; they exhibit a
marked activity against negative stranded (
)-RNA viruses (i.e.,
Paramyxoviridae, Rhabdoviridae, Arenaviridae), double-stranded (±)-RNA
viruses (Reoviridae), and Poxviridae (for a review, see Ref. 3).
Because of the close correlation between the antiviral activity and the affinity for AdoHcy hydrolase (6, 7), it has been postulated that these
inhibitors achieve their antiviral action by targeting the AdoHcy
hydrolase.
Previous reports have described the anti-HIV activity of
3-deazaadenosine, an AdoHcy hydrolase inhibitor (8, 9); when evaluated
in MT-4 cells, no IC50 values could be obtained
due to toxicity to the host cells (4). Mayers et al. (10)
reported the activity of 3-deazaadenosine analogues against
3-azido-3-deoxythymidine-resistant strains of HIV-1 in
vitro. The mechanism of action of these 3-deazaadenosine analogues
as anti-HIV drugs was not explored in detail.
We investigated a series of adenosine analogues that are known to be AdoHcy hydrolase inhibitors for their inhibitory effects on HIV-1 replication, in vitro reverse transcription, and transactivation of the HIV-1 LTR. Replication of HIV requires the function of the virus-encoded Tat protein, which is a strong activator of gene expression directed by the HIV LTR promoter and functions through interaction with the cis-acting TAR (11-17).
We found a close correlation among inhibition of AdoHcy hydrolase, inhibition of HIV-1 transactivation, and inhibition of HIV-1 replication. Our results suggest that AdoHcy hydrolase plays an important role in the activation of the HIV transcription and can be considered as a suitable target for the chemotherapy of HIV infections by AdoHcy hydrolase inhibitors.
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Materials and Methods |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Compounds.
Ro5-3335 (18, 19) was synthesized by Wayne A. Spitze and Frantz Victor at Lilly Research Laboratories (Indianapolis,
IN). TNF-
was purchased from SanverTech (Boechout, Belgium). NPA, 6-(R)-methyl-NPA, 6-(S)-methyl-NPA, NKO-6-16-3
, NOM-14-6-1, 6-(R)- and 6-(S)-ethynyl-NPA,
6-(R)- and 6-(S)-ethenyl-NPA, and
6-(R)- and 6-(S)-ethyl-NPA were obtained from
Dr. S. Shuto, N. Minakawa, and Dr. A. Matsuda (Faculty of
Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan).
C-c3Ado was from J. A. Montgomery
(Kettering-Meyer Laboratory, Southern Research Institute, Birmingham,
AL). Both (R)- and (S)-DHPA were from
A. Holy (Institute of Organic Chemistry and Biochemistry, Czech Academy
of Sciences, Prague, Czech Republic). c3NPA was
from V. E. Marquez (Laboratory of Pharmacology and Experimental therapeutics, National Cancer Institute, Bethesda, MD). DHCaA and
c3DHCaA were from R. T. Borchardt
(Departments of Medical Chemistry and Biochemistry, University of
Kansas, Lawrence, KS). KS605
[(+)-9-(cis-4-methoxycyclopenten-2-yl)-adenine], KS606
[()-9-(trans-2,trans-3-dihydroxy-cis-4-methoxycyclopentyl)adenine], c7DHCaA, and 5-noraristeromycin
[(+)-(1,2
,3,4)-4-(adenin-9-yl)-1,2,3-cyclopentane-triol] was from S. W. Schneller (College of Science and Mathematics, Auburn University, Auburn, AL). The structural formulae of the compounds are presented in Fig. 1.
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Inhibition of HIV-1 transactivation.
Tat-dependent
transactivation was monitored mainly as described previously (19) with
the following modifications. HeLa-tat-III cells [HeLa cells
expressing HIV-1 transactivator; kindly provided by Dr. C. A. Rosen (20)] were transfected with pHIVLacZ [kindly provided by Dr. J. J. Maio (21)] or pCMV (22) plasmid DNA by
electroporation with an Eurogentec Genepulser (260 V, 1050 µF, and
infinite R). pHIVLacZ contains a LacZ gene driven
by the HIV-1 LTR promoter, and pCMV expresses the LacZ
gene under control of the cytomegalovirus promoter. For the
Tat-independent transactivation assay, P4 cells [kindly provided by
Dr. P. Charneau (23)] were transfected with pHIVLacZ or
pCMV plasmid DNA and stimulated with TNF- (50 ng/ml). The
compound Ro5-3335, previously reported as an HIV-1 LTR transactivation
inhibitor (18, 19), was used as a reference compound.
-Gal activity in
20 µl of the cell extracts was quantified by a colorimetric assay as
described by Sambrook et al. (24). Then, 5 µl of cell
extract was used to determine total protein content according to the
Bradford method (BioRad, Hercules, CA). The IC50
value was calculated as being the inhibitor concentration that reduces
-Gal expression by 50%.
Inhibition of AdoHcy hydrolase.
AdoHcy hydrolase was
purified from murine L929 cells using affinity chromatography, and
AdoHcy hydrolase activity was measured in the direction of AdoHcy
synthesis using 8-[14C]Ado (Amersham,
Buckinghamshire, England) and 2 mM DL-Hcy as substrates, as described previously (7). The kinetic properties of the
murine AdoHcy hydrolase enzyme differ only slightly from those of human
AdoHcy hydrolase. For the human enzyme,
Km values of 
1
µM have been reported adenosine, whereas the
Km values for AdoHcy vary from 0.75 to 15 µM (25). The
Km values of purified enzyme from
murine L929 cells are 0.5 µM for adenosine and
4.8 µM for AdoHcy.
Inhibition of HIV-1 replication in MT-4 cells. In MT-4 cells, the anti-HIV-1 activities of the test compounds were determined by measuring virus-induced cytopathogenicity (26). Briefly, MT-4 cells were suspended at 3 × 105 cells/ml and infected with HIV at 100 times the 50% cell culture infective dose/ml. Immediately after infection, 100 µl of the cell suspension was added to each well of flat-bottom 96-well microtiter trays containing various concentrations of the test compound. After 5 days of incubation at 37°, the number of viable cells was determined according to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method as described previously (27).
Inhibition of HIV-1 p24 production in PBLs. The inhibitory effects of the compounds on HIV-1 strain IIIB replication in PBLs were monitored 7 days after infection by quantification of HIV-1 p24 core antigen using an enzyme-linked immunosorbent assay (DuPont, Wilmington, DE). These experiments were performed according to the ACTG protocol.
Inhibition of HIV-1 replication in persistently HIV-1 infected HUT-78/HIV-1IIIB. Persistently infected HUT-78/HIV-1IIIB cells were cultured for 3 days in the presence of various concentrations of test compounds. Anti-HIV-1 activity was determined by monitoring viral p24 antigen expression by enzyme-linked immunosorbent assay (DuPont). The supernatant of the cells incubated in the presence of various concentrations of compound was used to determine virus yield according to the Reed and Muench end point dilution assay (28).
Inhibition of HIV-1 replication in macrophages. Primary macrophages (obtained from blood monocytes) were infected with 300 tissue culture infectious doses of monocytotropic strain HIV-1Ba-L and maintained in culture until day 10, when chronic infection is usually established (29). At this time, macrophages were treated with different concentrations of DHCaA and c3DHCaA. Drugs were maintained in culture throughout the whole experiment. Inhibition of virus replication was assessed in the supernatants of macrophages at different time points by a commercially available immunoassay able to detect the presence of HIV p24 (Abbott Laboratories, Pomezia, Italy). Toxicity was assessed by trypan blue exclusion method on macrophages detached from wells 5-11 days after the beginning of the treatment and compared with the number and viability of macrophages found in control untreated (but infected) cells. Details about the experimental procedure are given elsewhere (29, 30).
Inhibition of HIV-1 replication in P4 cells.
In P4 cells
(HeLa-CD4-LTR-LacZ), the anti-HIV-1 activity of the test
compounds was determined by measuring virus-induced -Gal expression
and p24 antigen production. At day 1, the cells were plated in
microtiter plates at 2 × 104 cells/well and
incubated overnight at 37° with 5% CO2. At day 0, the medium was removed by gentle aspiration, and cells were infected
with an excess of HIV-1IIIB for ~1 hr
(multiplicity of infection >1). After infection, various
concentrations of test compounds were added to the infected cells. Two
days after infection virus replication was monitored by measuring p24
antigen production and by measuring -Gal expression in the cell
extracts. -Gal activity and p24 antigen were measured with the same
assays as described for the transactivation assay (24) and the other
replication assay, respectively. The IC50 value
was calculated as being the inhibitor concentration that reduces
-Gal expression or the p24 production by 50%.
HIV-1 RT assay. The enzymatic activity of recombinant HIV-1 RT was tested using poly(C)/oligo(dG) as template/primer and 3H-labeled dGTP as substrate in an RNA-dependent DNA-polymerase reaction. RT assays were performed in a reaction buffer containing 50 mM Tris·HCl, pH 8.1, 2 mM dithiothreitol, 10 mM MgCl2, 0.01% Triton X-100, 2.5 µM [8-3H]dGTP, 65 µg/ml poly(C)/oligo(dG), 2 nM concentrations of RT enzyme preparations, and appropriate concentrations of the compounds, as described previously (31). The IC50 value was calculated as being the concentration of inhibitor that reduces the RT activity by 50%.
Cellular toxicity. Cellular toxicity of the compounds was measured by on the basis of trypan blue exclusion, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method (27), or total protein content in the cell extracts. The latter was determined according to the Bradford method (BioRad).
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Results |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Inhibition of HIV-1 transactivation.
The -Gal
transactivation assay allowed us to quantify HIV-1 LTR transactivation
by Tat in Hela-tat-III cells after transient transfection
with pHIVLacZ. Tat-independent transactivation of the HIV-1
LTR by TNF- was also measured in pHIVLacZ-transfected P4
cells. After Tat-dependent or -independent transactivation of the HIV-1
LTR, the expression of -Gal activity was increased up to 50-fold;
therefore, these assays provide a sensitive way to study HIV-1 LTR
activity.
-Gal expression (Table
1). The most potent inhibitors were DHCaA
(IC50 = 0.21 µM), NPA
(IC50 = 0.23 µM),
5-noraristeromycin (IC50 = 0.24 µM), c3DHCaA
(IC50 = 0.26 µM), and
c3NPA (IC50 = 0.30 µM). The (R)-isomer of 6-methyl-NPA
(IC50 = 0.32 µM) was >110-fold
more potent than the (S)-isomer (IC50 > 36 µM). The same differential inhibitory activity was
observed for (R)- and (S)-isomers of
6-ethynyl-NPA and 6-ethenyl-NPA. Adenosine analogues that were
inactive as AdoHcy hydrolase inhibitors were also inactive against
transactivation of the HIV-1 LTR. Similar IC50
values were obtained for the inhibition of the HIV-1 LTR activation by
TNF- (Table 1). None of the compounds were able to inhibit the
-Gal expression in cells transiently transfected with pCMV DNA, a
plasmid containing the Tat-independent cytomegalovirus promoter driving
the LacZ gene.
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Inhibition of HIV-1 replication. In the MT-4/3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide HIV-1 replication assay, the anti-HIV-1 activities of c3NPA and C-c3Ado were determined by measuring the inhibition of virus-induced cytopathogenicity (26). At subtoxic concentrations, no inhibition of HIV-induced cytopathogenicity could be found in MT-4 cells (CC50: c3NPA, 0.072 µM; C-c3Ado, 7.1 µM).
Inhibition of replication of HIV-1 IIIB in PBLs was measured by quantifying the p24 antigen production in the presence of varying concentrations of test compound. The IC50 value of DHCaA for HIV-1 replication was 0.052 µM; this was ~2-fold higher than the CC50 value (0.023 µM). When evaluated for their inhibitory effects on HIV-1 replication in persistently infected HUT-78/HIV-1IIIB cells, c3NPA and C-c3Ado at subtoxic concentrations did not reduce HIV-1 progeny yield, as measured by quantification of the p24 core antigen (CC50: c3NPA, 0.38 µM; C-c3Ado, 15 µM). The activity of DHCaA and c3DHCaA was then assessed in chronically infected primary macrophages. We found preliminarily that the CC50 value of either compound was in the range of 4.6 µM. No detectable activity, at least in term of production of p24 antigen assessed in the supernatants at different time points (i.e., day 1, 3, or 5 after treatment with antiviral drugs), was found in three consecutive experiments. Approximately 90% inhibition of virus production was achieved with 5 mM 282-870, a protease inhibitor provided of potent activity both in acutely and chronically infected cells (32). Inhibition of HIV-1 replication in P4 cells was measured by quantifying the transactivation of the HIV-1 LTR by viral Tat using the LacZ reporter gene or quantifying p24 antigen production in the culture medium. P4 cells, HeLa-CD4 cells with stably integrated LTR-LacZ, were infected with HIV-1IIIB and treated with the compounds. Dextran sulfate (IC50 = 0.007 µM), 3-azido-3-deoxythymidine (IC50 = 3.4 × 10
4 µM), and saquinavir
(IC50 = 0.82 µM), known as HIV-1
replication inhibitors, all inhibited the LacZ expression
and p24 production in infected P4 cells in a dose-dependent manner.
Ro5-3335, known as an HIV transactivation inhibitor (18, 19),
inhibited the HIV-1-induced expression of the LacZ gene and
p24 production with an IC50 value of 0.1 µM. The IC50 values obtained for
the adenosine analogues are given in Table
2. All the AdoHcy hydrolase inhibitors tested were able to inhibit the virus induced -Gal expression and
p24 production in P4 cells. The most potent inhibitors were, again,
c3DHCaA (IC50 = 0.29 µM), c3NPA
(IC50 = 0.41 µM), DHCaA
(IC50 = 1.3 µM), and NPA
(IC50 = 1.9 µM). The same
differential activity of the (R)-and (S)-isomers as observed in the transactivation assay was again seen in the replication assay. For example, the (R)-isomer of
6-methyl-NPA (IC50 = 3.5 µM) was
>10-fold more potent than the (S)-isomer
(IC50 = > 36 µM). Those adenosine
analogues that were inactive as AdoHcy hydrolase inhibitors were also
inactive for HIV-1 replication in P4 cells (Tables 1 and 2). With the
exception of NPA (CC50 = 30 µM),
c3NPA (CC50 = 76 µM), DHCaA (CC50 = 55 µM), and c3DHCaA
(CC50 = 37 µM), most of the test
compounds were not toxic to the cells at concentrations tested as
measured by total protein content according to the Bradford method
(Table 2).
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Inhibition of HIV-1 RT. The inhibitory effects of DHCaA and c3DHCaA on the RT were evaluated using poly(C)/oligo(dG) as template/primer. Neither of the test compounds showed any activity against HIV-1 RT.
Correlation among inhibition of AdoHcy hydrolase activity, HIV-1 Tat-transactivation, and HIV-1 replication. The IC50 values of the compounds for AdoHcy hydrolase were compared with their IC50 values on Tat-transactivation and HIV-1 replication in P4 cells. There was a close correlation among the inhibitory effects of the compounds on AdoHcy hydrolase, Tat-transactivation, and HIV-1 replication in P4 cells. When IC50 values of the compounds for AdoHcy hydrolase were plotted on a log-log scale as a function of their IC50 values for Tat-transactivation (Fig. 2), linear regression showed a nice linear correlation (linear r = 0.85 with a slope of 3.8) between the inhibitory effects on AdoHcy hydrolase and Tat-transactivation. The correlation coefficient between the inhibitory effects on virus replication in P4 cells and Tat-transactivation was 0.99 with a slope of 0.15 (Fig. 3). When the IC50 values of the compounds for HIV-1 replication in P4 cells were plotted as a function of their IC50 values for AdoHcy hydrolase (Fig. 4), linear regression again showed a close linear correlation (linear r = 0.91 with a slope of 0.02) between the inhibitory effects on virus replication and AdoHcy hydrolase.
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Discussion |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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The HIV Tat protein stimulates transcriptional initiation and
elongation through interaction with a cis-acting element
located within the HIV LTR, termed the TAR (13-16). The molecular
mechanism by which Tat functions is not yet completely understood, but
several lines of evidence suggest that transactivation of the Tat
protein is mediated by cellular factors: Tat stimulates transactivation in a cell-dependent manner (33), and cellular proteins that bind TAR
in vitro have been described (for reviews, see Refs. 34 and
35). The HIV-1 LTR can also be trans-activated by
lymphocyte-activating factors such as TNF-.
It has been clearly established that adenosine analogues are inhibitory
to AdoHcy hydrolase (3, 36, 37). More recently, Mayers et
al. (10) reported the activity of 3-deazaadenosine analogues
against HIV-1. They postulated that the 5-O-triphosphate of
3-deazaadenosine (38, 39) may act as an inhibitor of viral DNA or RNA
synthesis (10). However, the investigators did not measure the
inhibitory activity of the 5-O-triphosphate derivatives of
3-deazaadenosine analogues against HIV-1 RT in a cell-free system to
confirm this hypothesis. Moreover, we believe that it is rather
unlikely that ribonucleotides are efficiently recognized by DNA
polymerases, such as HIV-1 RT, as a substrate or an inhibitor of
the enzyme reaction. In fact, all compounds we tested contain both 2-
and 3-hydroxyl groups (Fig. 1). Moreover, DHCaA and c3DHCaA cannot be converted in the cell to a
triphosphate form at the 5 position (see Fig. 1) and therefore cannot
inhibit the HIV-1 RT reaction as chain terminator. Furthermore, in
additional experiments, it was determined that DHCaA and
c3DHCaA did not have a direct inhibitory effect
on the HIV-1 RT reaction.
Here, we suggest that these adenosine analogues, described as HIV-1
inhibitors by Mayers et al. (10), most probably act through
their inhibition of AdoHcy hydrolase. We demonstrate that adenosine
analogues that are able to inhibit AdoHcy hydrolase have potent
inhibitory effect on the Tat-dependent and TNF--induced transactivation of a reporter gene driven by the HIV-1 LTR. The most
active agents that emerged were DHCaA, NPA, 5-noraristeromycin, c3DHCaA, and 3-deazaneplanocin A. The fact that
similar IC50 values as obtained for the
Tat-dependent inhibition were obtained for the Tat-independent
inhibition of LTR transactivation means that these adenosine analogues
interfere with the HIV-1 LTR transactivation in general through a
Tat-independent mechanism. The compounds were tested in different host
cells, but no anti-HIV activity could be observed in MT-4 cells or PBLs
at subtoxic concentrations. Moreover, in chronically infected
macrophages, we found no detectable activity. This was not surprising,
however, because previous data, confirmed in this set of experiments,
show that inhibition of virus production in chronically infected
macrophages can be achieved only with protease inhibitors and at
concentrations 
10-50-fold higher than those active in chronically
infected lymphocytes and ~100-1000-fold greater than those active
after de novo infection of lymphocytes. Indeed, Ro5-3335,
known as an HIV transactivation inhibitor, is also completely inactive
in this system. When tested in CD4+ HeLa cells,
all AdoHcy hydrolase inhibitors inhibited
HIV-1IIIB replication at subtoxic concentrations
(Table 2). Of the two diastereoisomers of
6-methyl-NPA, the (R)-isomer proved to be much more active
than the (S)-isomer. This differential inhibitory activity
of the two diastereoisomers was reflected in their inhibitory effect on
HIV-1 LTR transactivation, AdoHcy hydrolase activity, and HIV-1
replication in P4 cells (Tables 1 and 2).
AdoHcy hydrolase is functionally linked to AdoMet-dependent
methyltransferases (4). When serving as the methyl donor for methyltransferase reactions, AdoMet generates AdoHcy, which is not only
a product but also an inhibitor of the methyltransferase reaction. To
avoid this inhibitory effect and allow the methyltransferases to
proceed, AdoHcy must be removed by AdoHcy hydrolase. The fact that
AdoHcy hydrolase inhibitors exert a potent inhibition of Tat-mediated
and TNF--induced transactivation of the LTR points to the
participation of the AdoMet-dependent methyltransferase/AdoHcy hydrolase system in the transactivation process.
We demonstrated a close correlation among the inhibitory effect on
AdoHcy hydrolase, HIV replication, and transactivation activity for a
wide range of adenosine analogues (Figs. 2, 3, 4). A similar correlation
was found earlier between their inhibitory effects on AdoHcy hydrolase
and their activity against a number of viruses (vaccinia virus,
vesicular stomatitis virus, and cytomegalovirus) (6, 7). The strong
correlation between AdoHcy hydrolase inhibition and anti-HIV-1
transactivation suggests that the AdoHcy hydrolase inhibitors may
interfere with the HIV transactivation process via inhibition of AdoHcy
hydrolase (Fig. 2). Furthermore, the close correlation between the
inhibition of virus replication and inhibition of Tat-transactivation
suggests that the anti-HIV-1 activity of the AdoHcy hydrolase
inhibitors is due to inhibition of HIV-1 Tat-transactivation (Fig. 3).
The AdoHcy hydrolase inhibitors may inhibit HIV-1 Tat-transactivation
through interference with the HIV-1 transcription or with the
5-capping of the HIV-1 mRNA. The latter is required for the folding of
the TAR stem structure, which in turn is required for the effective
activation of the LTR. In any case, the mode of action of the AdoHcy
hydrolase inhibitors seems to be specifically related to the HIV-1 LTR
transactivation because the AdoHcy hydrolase inhibitors tested did not
have any effect on the expression of the LacZ gene driven by
a Tat-independent cytomegalovirus promoter.
We suggest that the adenosine analogues exert their anti-HIV-1 activity through inhibition of Tat-transactivation via inhibition of AdoHcy hydrolase. The exact molecular mechanism by which the AdoHcy hydrolase inhibitors interfere with the Tat-transactivation of HIV-1 remains to be elucidated.
Due to the high toxicity in PBLs and T cell lines such as MT-4 and HUT-78 cells, these adenosine analogues might not be immediate candidates for clinical trials. However, they are helpful in improving our understanding of the HIV LTR transactivation mechanism, which opens perspectives for future drug design aimed at interfering with the HIV transactivation.
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Acknowledgments |
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We are grateful to Dr. S. Shuto, Dr. A. Matsuda, and Dr. S. W. Schneller for kindly providing their test compounds. We thank Cindy Heens and Kristien Erven for excellent technical assistance and Jan Balzarini for stimulating discussions.
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Footnotes |
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Received June 6, 1997; Accepted September 10, 1997
This work was supported in part by the Biomedical Research Program of the European Union (Biomed 2 Grant BMH4-LT-951634), the Belgian Geconcerteerde Onderzoeksacties (Project GOA 95/5), and a grant from the "Fonds voor Wetenschappelijk Onderzoek (FWO), Vlaanderen" (Grant G.3304396). D.D. acknowledges a grant from the Flemisch Institute supporting Scientific-Technological Research in Industry (IWT).
Send reprint requests to: Dirk Daelemans, Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail: dirk.daelemans{at}uz.kuleuven.ac.be
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Abbreviations |
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AdoHcy, S-adenosylhomocysteine;
AdoMet, S-adenosylmethionine;
-Gal, -galactosidase;
CC50, 50% cytotoxic concentration;
C-c3Ado, carbocyclic 3-deazaadenosine;
DHCaA, 9-(trans-2,trans-3-dihydroxycyclopentyl)adenine;
c3DHCaA, 9-(trans-2,trans-3-dihydroxycyclopentyl)-3-deazaadenine;
c7DHCaA, 9-(trans-2,trans-3-dihydroxycyclopentyl)-7-deazaadenine;
DHPA, 9-(2,3-dihydroxypropyl)adenine;
Hcy, homocysteine;
HIV-1, human
immunodeficiency virus type 1;
LTR, long terminal repeat;
NKO-6-16-3, 3-deaza-3-fluoro-adenosine, NOM-14-6-1, 3-deaza-3-chloro-adenosine,
NPA, neplanocin A;
c3NPA, 3-deazaneplanocin A;
PBL, peripheral blood lymphocyte;
TAR, transactivation response element;
TNF-, tumor necrosis factor-;
RT, reverse transcription.
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References |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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| 1. |
de la Haba, G. and
G. L Cantoni.
The enzymatic synthesis of S-adenosylhomocysteine from adenosine and homocysteine.
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| 3. | De Clercq, E. S-Adenosylhomocysteine hydrolase inhibitors as broad-spectrum antiviral agents. Biochem. Pharmacol. 36:2567-2575 (1987)[Medline]. |
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R. T. Borchardt,
J. C. Drach,
S. Kitaoka, and
T. Konno.
Broad-spectrum antiviral activities of neplanocin A, 3-deazaneplanocin A, and their 5-nor derivatives.
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