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Selective inhibition of human immunodeficiency virus (HIV) by 3′-Azido-2′,3′-dideoxyguanosine invitro
Vol. 145, No. 3, 1987 June 30, 1987
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 1080-l 086
SELECTIVEINHIBITION OF HUMANIMMDNODEFICIENCY VIRUS (HIV) BY 3'-AZIDO-2',3'-DIDEOXYGUANOSINE --IN VITRO Masanori Baba, Rudi Pauwels, Jan Balzarini. and Erik De Clercq Rega Institute
for Medical Research, Katholieke B-3000 Leuven, Belgium
Piet Herdewijn, Universiteit
Leuven,
Received May 7, 1987
3'-Azido-2',3'-dideoxyguanosine (AzddGuo) is a potent and selective inhibitor of human immunodeficiency virus (HIV) -in vitro. AzddGuo completely inhibits HIV-induced cytopathogeniclty and viral antigen expression in MT-4 cells at a concentration of 5.0 JJM. Its 50 % effective dose for inhibiting HIV-induced cytopathogenicity is 1.4 JJM, as compared to 6.4 JJM for 2',3'-dideoxyadenosine (ddAdo). Thus, AzddGuo Is approximately 4.6-fold more potent as an anti-HIV agent than ddAdo, one of the most promising compounds for the treatment of AIDS. However, AzddGuo is about 4.7 times more cytotoxic than ddAdo, so that its selectivity index, as based on the ratio of the 50 % cytotoxic dose to the 50 % antiviral effective dose, is almost the same as 0 1987 Academic Press, Inc. that of ddAdo (136 and 139, respectively). Acquired immunodeficlency syndrome (AIDS), which was initially
recognized
as a systemic immunosuppressive disorder (l), is an infectious disease caused by human T-cell lymphotroplc virus type IIIllymphadenopathy virus (HTLV-III/ recently termed human immunodeficiency-associated virus (HIV). Since HIV is a retrovirus, viral reverse transcriptase (RT) seems to be a se-
uv)
(2,3),
lective
target
for antiviral
agents. In fact,
been reported to be active
a number of RT inhibitors
against HIV replication in vitro, i.e. -HPA-23 (5), phosphonoformate (6), 3'-azido-2',3'-dideoxythymidine AZT) (7), 2',3'-dideoxynucleosides (8), etc. tial
have
suramin (4), (AzddThd,
In particular AzddThd is promising for the treatment of AIDS, and an iniclinical trial has shown both clinical and immunological improvement upon
a short term (6 week) course of AzddThd in AIDS patients (9). On the other hand, 2'.3'-dideoxyadenosine (ddAdo) is also a promising anti-HIV agent because of its low toxicity for the host cells (8,lO). The selectivity index (SI) of ddAdo, as based on the ratio of 50 X cytotoxic dose (CD50) to 50 % antiviral effective dose (ED50) in ATH8 cells, was more than 8.9-fold higher than that of AzddThd (> 148 versus 16.6) (10). Recently, another purine 2',3'dideoxynucleoside analogue, 2',3'-dideoxy-2,6-diaminopurineriboside (ddDAPR), was found to be slightly (11).
more effective
0006-291X/87$1.50 Copyright All rights
0 I987
4v Arademic Press, in any fbrnr
oJ’ reprodut-rion
Inc. reserved.
1080
than ddAdo against HIV in MT-4 cells
Vol. 145, No. 3, 1987
In this for their
BIOCHEMICAL
study,
we evaluated
anti-retroviral,
cytostatic
found 3'-azido-2',3'-dideoxyguanosine selective
as ddAdo in Inhibiting
AND BIOPHYSICAL
several
RESEARCH COMMUNICATIONS
2',3'-dideoxyguanosine
and antimetabolic
effects
derivatives in vitro,
and
(AzddGuo) to be more potent and equally HIV replication
in vitro. --
MATERIALSANDMETHODS Compounds.ddAdo and ddGuo were purchased from Pharmacia-PL-Biochemicale. The 2',3'-unsaturated derivative of ddGuo, named 2',3'-didgoxyguanosine (ddeGuo), was synthesized by a double elimination reaction of 0 -p-nitrophenylethyl-3'-0-mesyl-2'-deoxyguanosine with sodium methanolate in dimethylformamide. AzddGuo was synthesized by M. Imazawa and F. Eckstein (12) (Fig. 1). Viruses. HIV was obtained from the culture supernatant of a persistently HIV-infected H9 cell line (H9/HTLV-IIIB) (13). Moloney murine sarcoma virus (MSV) was prepared from tumors induced by -in vlvo infection of 3 day-old NMRI mice, as previously described (14). Cells. MT-4 cells (15) and C3H mouse embryo (MO) cells were used for anti-HIV and anti-MSV assays, respectively. The origin and characterization of other cell lines used in this study, i.e. Raji./O, Molt/4F, H9, GEM, and HUT78, have been described elsewhere (16,17). All cell lines were grown and maintained in RPM11640 medium supplemented with 10 X heat-inactivated fetal calf G, and 20 pg/ml gentamycin (culture medium). serum (FCS), 100 IU/ml penicillin Anti-HIV assays. Activity of the compounds against HIV replication was based on the inhibition of virus-induced cvtonathogenic effect (CPE). determined by trypan blue exclusion, as previously described (17).‘Br&fly, MT-4 cells were adjusted at 5 x lo5 cells/ml and tnfected with HIV at 100 CCID / ml. After 90 min incubation at 37"C, 5 x 10 cells/100 ~1 were brought in5?o each well of a flat bottomed 96-well plastic mlcrotiter tray containing various concentrations of test compounds. After 5 days incubation at 37'C, the number of viable cells was determined microscopically in a hematocytometer by trvvan __ blue exclusion. Inhibition of viral antigen expression in HIV-infected MT-4 cells. The inhibitory effects of the compounds on viral antigen expression in infected MT-4 cells were determined at day 4 after infection-by indirect immunofluorescence and laser flow cytofluorography, using a polyclonal antibody as probe, as nreviouslv described (17). Transformation of MG cells by MSV. Confluent MO cell cultures in 24-well multi-dishes were inoculated with 150 foci-forming units of MSV per well. After 90 min of virus adsorption, mediumwas replaced by 1 ml fresh culture medium containing diffferent concentrations of test compounds. After 6 days, the transformation of the cell cultures was examined microscodcallv. Cytostatic and antimetabolic assays. The cytostatic‘effects of the compounds were assessed by measuring inhibition of cell proliferation. The inhi-
ddGuo FIG.
1.
deoxyguanosinene
ddeGuo
AzddGuo -~
Structural formulae of 2',3'-dideoxyguanosine (ddeGuo) and 3'-azido-2',3'-dideoxyguanosine
1081
(ddGuo), 2'.3'-di(AzddGuo).
Vol. 145, No. 3, 1987
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
bitory effects of the compounds on DNA and RNA synthesis of Molt/4F and H9 Sells were detefjmined by measuring inhibition of the incorporation of [methylH]dThd and [5- H]Urd into TCA-insoluble material. The experimental procedures have been described previously (18). RESULTS When ddGuo, ddeGuo and AzddGuo were evaluated for their on the cytopathogenicity
of HIV in MT-4 cells,
in protecting
against destruction
protected
the cells
MT-4 cells
concentration
at a concentration
of 25 UM to achieve full
by the virus.
AzddGuo completely
2). The 2',3'-didehydro less active and more cytotoxic (Fig.
than the parent compound. ddeGuo achieved approximately of 5.0 PM; however, the 50 % protection
tained because of cytotoxicity
25 % protection level
at the higher concentrations
When assayed for cytotoxicity
effect
of 5.0 PM, whereas ddGuo required a At 5.0 JJM, ddGuo afforded protection.
only 20 % protection against cell destruction derivative of ddGuo, ddeGuo, was significantly concentration
inhibitory
AzddGuo proved the most potent
in mock-infected
at a
could not be at(Fig.
MT-4 cells,
2). AzddGuo redu-
ced cell viability to 30 X of control at a concentration of 250 PM, whereas ddGuo effected a 45 % reduction at the highest concentration tested (625 PM) (Fig. 2). The selectivity index (SI), as based on the ratio of CD50 to ED50, was 136 and 64 for AzddGuo and ddGuo, respectively (Table I). The SI of AzddGuo was comparable to that of ddAdo (SI : 139). Next,
the compoundswere evaluated
antigen expression in HIV-infected
MT-4 cells.
z ,\ P 2 " -w "
for their
effect
In these experiments,
on viral AzddGuo
10
5
100
5
0
0 2
10
50
25
125
625
concentration of FIG. 2 ddGuo*o, clusion on day solid columns (0). Data were
inhibitory
0 k-h
0
02
10
50
A25
625
tompound
Inhibition of the cytopathogenicity of HIV and AzddGuo. The cell viability was measured 5 after virus infection. The infected cells Cm) and the mock-infected cells are indicated taken from a representative experiment.
1082
125
for MT-4 cells by by trypan blue exare indicated by by open columns
Vol. 145, No. 3, 1987 TABLE
BIOCHEMICAL Inhibitory effects replication of
I.
Compound
ED50a
ddGuo
ddeGuo
AND BIOPHYSICAL
of ddGuo, ddeGuo, HIV
in
MT-4
AzddGuo cultures
cell
(IJM)
CD5;
ddAdo '50 % antiviral beffect of riIV.
effective
and
and
data represent
ddGuo
achieved
centration were
of
the
5.0
64
1.4 6.4
11 190 890
< 2.2 136 139
mean values
complete
same as those
SIC
486
dose, based on the protection
JJM and
on the
(PM)
against
50 % cytotoxic dose, based on a reduction in the viability host cells. iSelectivity index (ratio of CD50 to ED ). At this concentration, approximately 2 30 % protection was protection could not be attained because of cytotoxicity tions.
All
ddAdo
'a6 >5
AzddGuo
RESEARCH COMMUNICATIONS
for 2 separate
inhibition 25 nM,
required
of
the
cytopathic uninfected
achieved. at higher
The 50 % concentra-
experiments.
viral
antigen
respectively
for
of
the
(Fig.
complete
expression
3).
protection
These
at
a con-
concentrations
against
HIV cytopatho-
genicity. When examined broblast
cells
50 % effective I.IM for
ddGuo
for
their
inhibitory
by MSV, AzddGuo dose
(ED50>
and ddeGuo,
for
effect
was more AzddGuo
respectively
on the
effective
transformation
than
ddGuo
was 13 MM, as compared (data
not
of MO fi-
and ddeGuo. to
160 uM and 36
shown).
50, I ---
40
30 1 20 I
0
0.2
1.0
/
I
I
5.0
25
125
ConcentrationIpMI
FIG. 3. Inhibition of viral antigen expression in HIV-infected MT-4 cells by ddGuo (0). ddeGuo (01, and AzddGuo (A). Antigen-positive cells were measured by indirect immunofluorescence and laser flow cytofluorography on day 4 after virus infection, using a polyclonal antibody as probe. The mean percentage of fluorescent cells in the absence of compound (positive control) was 48.9 % (indicated by broken line), and that of mock-infected cells (negative control) was 1.9 %. 1083
The
BIOCHEMICAL
Vol. 145, No. 3, 1987
TABLEII.
AND BIOPHYSICAL
Inhibitory effects of ddGuo, ddeGuo,AzddGuo,and ddAdoon the replication of humantumor cell lines
Compound
ID50a (PM) Raji
ddGuo
229
ddeGuo
46
AzddGuo ddAdo
185 > 1000
a50 % inhibitory All
RESEARCH COMMUNICATIONS
Molt/4F
MT-4
H9
CEM
HUT-78
> 690
288 33 2 275 > 1000
288 41 > 150 2 1000
90 31 1 150 564
297 50 2 275 768
152 > 275 720
dose required to inhibit
data represent
mean values
for
cell proliferation
3 separate
by 50 %.
experiments.
When the cytostatic effect of the compounds against several human cell lines, i.e. Rajl, Molt/4F, MT-4, H9, CEM, and HUT-78, was evaluated, ddeGuo was of all test compounds the most cytostatic against all cell lines (Table XI).
The order of cytostatic
this
order completely
potency was ddeGuo > AzddGuo 2 ddGuo > ddAdo, and
corresponded with
the order of cytotoxicity
noted for
these compoundsin mock-infected MT-4 cells (Table I). The ID5C of AzddGuo and ddGuo for cellular DNAand RNA synthesis in H9 and Molt/4F cells, as monitored by the incorporation
maof [methyl-3 H]dThd and [5-3H1Urd Into acid-insoluble was higher than the highest concentrations tested for
terial, respectively, the compounds (> 139 JJMfor AzddGuo and > 688 JJMfor ddGuo) (data not shown). DISCUSSION Recently,
ddAdo has been recognized as a prime candidate for the chemo-
therapy of AIDS because of its mann -et al.
mentioned that
(19). The results derable potential
low toxicity
and high selectivity
AzddGuo may also be an effective
(8,lO). anti-HIV
Hartagent
presented here indicate that, indeed, AzddGuo offers consifor the treatment of AIDS. In comparison with ddAdo, the cy-
4.7-fold higher and the activity totoxicity of AzddGuo was approximately against HIV was about 4.6-fold higher, which means that the selectivity of AzddGuo was almost Identical to that of ddAdo (Table I). In addition, since AzddGuo proved to be a potent inhibitor of MO transformation by MSV -in vitro : 13 JJM), the investigation on the inhibitory effect of this compoundon (ID50 tumor induction by MSV in newborn NMRI mice can be considered. The latter seemsto be a representative model for retrovlrus infection -in vivo (14). A selective inhibition of HIV replication in MT-4 cells could also be achieved with ddGuo. However, its 2',3'-didehydro derivative, ddeGuo, was quite toxic and not effective at non-toxic concentrations (Fig. 2). We have derivatives of 2',3'-dideoxythymipreviously shown that the 2' ,3'-didehydro dine (ddThd) and 2',3'-dideoxycytidine (ddCyd), namely 2',3'-dldeoxythymidi1084
Vol. 145, No. 3, 1987
BIOCHEMICAL
AND BIOPHYSICAL
nene (ddeThd) and 2',3'-dideoxycytidinene and selective vative
inhibitors
tion
(ddeCyd) respectively,
of HIV (10,20,21),
of ddAdo (ddeAdo) is not effective
dro derivative
RESEARCH COMMUNICATIONS
are potent
whereas the 2',3'-didehydro (10,Zl).
Moreover,
of ddDAPR (ddeDAPR) also proved non-inhibitory
deri-
the 2',3'-didehyto HIV replica-
at nontoxic
concentrations (11). Taken together, these results suggest of purines, in conas a rule, 2',3'-didehydro-2',3'-dideoxyribosides do not appear promising as anti-HIV with their pyrimidine counterparts,
that,
trast agents.
The mechanism by which AzddGuo inhibits subject
of further
study.
the replication
Like AzddThd and other
2',3'-dideoxynucleosides,
AzddGuo may be assumed to act as a chain terminator phosphorylated
by cellular
kinases.
Furthermore,
of HIV remains
for RT after the recent
it
has been
observations
of
Dalal -et al. (24) demonstrate that ddAdo is rapidly deaminated in Molt/4F cells to 2',3'-dldeoxyinosine and subsequently metabolized to the normal adenosine nucleotides. cells.
It
is not known how AzddGuo is metabolized
Should AzddGuo prove resistant
to degradative
within
enzymes (purine
the
nucleo-
side phosphorylases) and apt to a straightforward conversion to Its 5'-triphosphate, the compoundmay be advantageous over ddAdo from a chemotherapeutic viewpoint. The results luated
for
its
presented here indicate efficacy
in the treatment
that AzddGuo should be further of retrovirus
infections
eva-
including
AIDS. ACKNOWLEDGEMENTS HIV (HTLV-IIIB) was kindly provided by Dr. R.C. Gallo (National Cancer Institute, Bethesda, MD, USA). MT-4 cells were a gift from Dr. L. Montagnier (Pasteur Institute, Paris, France) and 3' -azido-2',3'-dideoxyguanosine was kindly supplied by Dr. F. Eckstein (Max-Planck-Institut fiir Experlmentelle Medizin, Gbttingen, W.-Germany). Dr. M. Baba is a recipient of a grant from the J.S.P.S. (Japan Society for the Promotion of Science, 2-438). Dr. P. Herdewljn is a Research Associate of the Belgian N.F.W.O. (Nationaal Fonda voor Wetenschappelijk Onderzoek). These investigations were supported in part by the AIDS Basic Research Programme of the European Community, by grants from the Belgian F.G.W.O. (Fond8 voor Geneeskundig Wetenschappelijk Onderzoek: Project no. 3.0040.83), and by the Belgian G.O.A. (Geconcerteerde OnderzoeksacLizette Van Berckelaer, ties: Project no. 85190-79). We thank Ann Absillis, and Francis Vanlangendonck for excellent technical assistance and Christiane Callebaut for fine editorial help. REFERENCES 1. 2. 3.
Gottlieb, M.S., Schroff, R., Schanker, H.M., Weisman, J-D-S Fan, P-T-, Wolf, R.A., and Saxon, A. (1981) New Engl. J. Med. 305, 1425-1431. Gallo, R.C., Sarin, P.S., Gelmann, E.P., Robert-Guroff. M., Richardson, V.S., Mann, D.L., Sidhu, G., Stahl, R-E., Eolla-Pazner, E., Kalyanarman, S., Leibowitch, J., and Popovic, M. (1983) Science 220, 865-867. BarrG-Sinoussi, F., Chermann, J.C., Rey, R., Nugeyre, M-T., Chamaret, Se, Gr-uest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W., and Montagnier, L. (1983) Science 220, 868-871. 1085
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6. 7. 8. 0_.
10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20.
21. 22.
23. 24.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Mftsuya, H., Guo, H.-G., Cossman, .J., Megson, M., Reitz, M.S. JR., and Broder, S. (1584) Science 225, 1484-1486. Dormont, D., Spire, B., Barr&Sinoussi, F., Nontagnier, I.., and Chermann, J.C. (1985j Ann. Viral. (Inst. Pasteur) 136E, 75-83. Sandstrom, E.G., Kaplan, J.C., Byington, R.E., and Hirsch, M.S. (1985) Lancet i, 148C-1482. Mitsuya, H., Weinhold, K.J., Furman, P.A., St. Clair, M.H., Nusinoff T.ehrman, S., Gallo, R.C., Bolognesi, D., Barry, D.W., and Broder, S. (1985) Proc. Natl. Acad. Sci. USA 82, 7096-7100. Mitsuya, ., and Broder, S. (1986) Proc. Natl. Acad. Sci. USA 83, 19111915.
Sarchoan a HR., Klecker, R.W.. Weinhold, K.J., Markham, P.D., Lyerly, H.E., Durack, D.T., Gelmann, E., Nusinoff Lehrmann, S., Blum, R.M., Barry, D.W., Shearer, G.M., Fischl, M.A., Mitsuya, H., Gallo, R.C., Collins, J.M., Bolognesi, D.P., Myers, C.E., and Broder, S. (1986) Lancet i, 575580. Balzarini, J., Kang, C.-J., Dalal, M., Herdewijn, P., De Clercq, E., Broder, S., and Johns, D.G. (1987) Molec. Pharmacol., in press. Eialzarini, J., Fauwels, R., Baba, M., Robins, M.J., Zou, R., Herdewijn, P and De Clercq, E. (1987) Biochem. Biophys. Res. Commun., in press. Jiizawa, M., and Eckstein, F. (1978) J. Org. Chem. 43, 3044-3048. Popovic, N., Sarngadharan, M.G., Read, E., and Callo, R.C. (1984) Science 224, 497-500. De Clercq, E., and Irerigan, T.C. (1971) Yroc. Sot. Exp. Biol. Med. 137, 590-593. Harada, S., Koyanagi, Y., and Pamamoto, N. (1985) Science 229, 563-566. Balzarini, J., Mitsuya, H., De Clercq, E., and Broder, S. (1986) Biochem. Biophys. Res. Commun.136, 64-71. Pauwels, R., De Clercq, E., Desmyter, J., Balzarini, J., Goubau, P., Herdewijn, P., Vanderhaeghe, H., and Vandeputte, M. (1987) J. Viral. heth., in press. De Clercq, E., Balzarini, J., Torrence, P.F., Mertes, M.P., Schmidt, C.L., Shugar, D., Barr, P.J., Jones, A.S., Verhelst, G., and Walker, ROT. (1981) Mol. Pharmacol. 19, 321-330. Hartmann, H., Hunsmann, G., and Eckstein, F. (1987) LanCet is 40-41. Balzarini, J., Pauwels, R., Herdewijn, P., De Clercq, E-9 Cooney, D.A.9 Kang, G.-J., Dalal, M., Johns, D.G., and Broder, S. (1986) Biochem. Biophys. Res. Commun.140, 735-742. Baba, M., Pauwels, R., Herdewijn, P., De ClerCq, E., Desmyter, J-9 and Vandeputte, M. (1987) Biochem. Biophys. Res. Commun.142, 128-134. Furman, P.A., Fyfe, J.A., St. Clair, M.H., Weinhold, K., Rideout, J-L*, Freeman, G.A., Nusinoff Lehrmann, S., Bolognesi, D.P., Broder, S., Mitouya, H., and Barry, D.W. (1986) Proc. Natl. AC&~. Sci. USA 83, 8333~ 8337. Cooney, D.A., Dalal, M., Mitsuya, H., McKahon, J.B., Nadkarni, M.9 Balzarini, J., Broder, S., and Johns, D.G. (1986) Biochem. Pharmacol. 35, 2065-2068. Dalal, M., Cooney, D.A., Mitsuya, H., Balzarini, J., Hao, Z.. Nadkarni, r'., Broder, S., and Johns, D.G. (1987) Proc. Am. Assoc. Cancer Res., in press.
1086
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 1080-l 086
SELECTIVEINHIBITION OF HUMANIMMDNODEFICIENCY VIRUS (HIV) BY 3'-AZIDO-2',3'-DIDEOXYGUANOSINE --IN VITRO Masanori Baba, Rudi Pauwels, Jan Balzarini. and Erik De Clercq Rega Institute
for Medical Research, Katholieke B-3000 Leuven, Belgium
Piet Herdewijn, Universiteit
Leuven,
Received May 7, 1987
3'-Azido-2',3'-dideoxyguanosine (AzddGuo) is a potent and selective inhibitor of human immunodeficiency virus (HIV) -in vitro. AzddGuo completely inhibits HIV-induced cytopathogeniclty and viral antigen expression in MT-4 cells at a concentration of 5.0 JJM. Its 50 % effective dose for inhibiting HIV-induced cytopathogenicity is 1.4 JJM, as compared to 6.4 JJM for 2',3'-dideoxyadenosine (ddAdo). Thus, AzddGuo Is approximately 4.6-fold more potent as an anti-HIV agent than ddAdo, one of the most promising compounds for the treatment of AIDS. However, AzddGuo is about 4.7 times more cytotoxic than ddAdo, so that its selectivity index, as based on the ratio of the 50 % cytotoxic dose to the 50 % antiviral effective dose, is almost the same as 0 1987 Academic Press, Inc. that of ddAdo (136 and 139, respectively). Acquired immunodeficlency syndrome (AIDS), which was initially
recognized
as a systemic immunosuppressive disorder (l), is an infectious disease caused by human T-cell lymphotroplc virus type IIIllymphadenopathy virus (HTLV-III/ recently termed human immunodeficiency-associated virus (HIV). Since HIV is a retrovirus, viral reverse transcriptase (RT) seems to be a se-
uv)
(2,3),
lective
target
for antiviral
agents. In fact,
been reported to be active
a number of RT inhibitors
against HIV replication in vitro, i.e. -HPA-23 (5), phosphonoformate (6), 3'-azido-2',3'-dideoxythymidine AZT) (7), 2',3'-dideoxynucleosides (8), etc. tial
have
suramin (4), (AzddThd,
In particular AzddThd is promising for the treatment of AIDS, and an iniclinical trial has shown both clinical and immunological improvement upon
a short term (6 week) course of AzddThd in AIDS patients (9). On the other hand, 2'.3'-dideoxyadenosine (ddAdo) is also a promising anti-HIV agent because of its low toxicity for the host cells (8,lO). The selectivity index (SI) of ddAdo, as based on the ratio of 50 X cytotoxic dose (CD50) to 50 % antiviral effective dose (ED50) in ATH8 cells, was more than 8.9-fold higher than that of AzddThd (> 148 versus 16.6) (10). Recently, another purine 2',3'dideoxynucleoside analogue, 2',3'-dideoxy-2,6-diaminopurineriboside (ddDAPR), was found to be slightly (11).
more effective
0006-291X/87$1.50 Copyright All rights
0 I987
4v Arademic Press, in any fbrnr
oJ’ reprodut-rion
Inc. reserved.
1080
than ddAdo against HIV in MT-4 cells
Vol. 145, No. 3, 1987
In this for their
BIOCHEMICAL
study,
we evaluated
anti-retroviral,
cytostatic
found 3'-azido-2',3'-dideoxyguanosine selective
as ddAdo in Inhibiting
AND BIOPHYSICAL
several
RESEARCH COMMUNICATIONS
2',3'-dideoxyguanosine
and antimetabolic
effects
derivatives in vitro,
and
(AzddGuo) to be more potent and equally HIV replication
in vitro. --
MATERIALSANDMETHODS Compounds.ddAdo and ddGuo were purchased from Pharmacia-PL-Biochemicale. The 2',3'-unsaturated derivative of ddGuo, named 2',3'-didgoxyguanosine (ddeGuo), was synthesized by a double elimination reaction of 0 -p-nitrophenylethyl-3'-0-mesyl-2'-deoxyguanosine with sodium methanolate in dimethylformamide. AzddGuo was synthesized by M. Imazawa and F. Eckstein (12) (Fig. 1). Viruses. HIV was obtained from the culture supernatant of a persistently HIV-infected H9 cell line (H9/HTLV-IIIB) (13). Moloney murine sarcoma virus (MSV) was prepared from tumors induced by -in vlvo infection of 3 day-old NMRI mice, as previously described (14). Cells. MT-4 cells (15) and C3H mouse embryo (MO) cells were used for anti-HIV and anti-MSV assays, respectively. The origin and characterization of other cell lines used in this study, i.e. Raji./O, Molt/4F, H9, GEM, and HUT78, have been described elsewhere (16,17). All cell lines were grown and maintained in RPM11640 medium supplemented with 10 X heat-inactivated fetal calf G, and 20 pg/ml gentamycin (culture medium). serum (FCS), 100 IU/ml penicillin Anti-HIV assays. Activity of the compounds against HIV replication was based on the inhibition of virus-induced cvtonathogenic effect (CPE). determined by trypan blue exclusion, as previously described (17).‘Br&fly, MT-4 cells were adjusted at 5 x lo5 cells/ml and tnfected with HIV at 100 CCID / ml. After 90 min incubation at 37"C, 5 x 10 cells/100 ~1 were brought in5?o each well of a flat bottomed 96-well plastic mlcrotiter tray containing various concentrations of test compounds. After 5 days incubation at 37'C, the number of viable cells was determined microscopically in a hematocytometer by trvvan __ blue exclusion. Inhibition of viral antigen expression in HIV-infected MT-4 cells. The inhibitory effects of the compounds on viral antigen expression in infected MT-4 cells were determined at day 4 after infection-by indirect immunofluorescence and laser flow cytofluorography, using a polyclonal antibody as probe, as nreviouslv described (17). Transformation of MG cells by MSV. Confluent MO cell cultures in 24-well multi-dishes were inoculated with 150 foci-forming units of MSV per well. After 90 min of virus adsorption, mediumwas replaced by 1 ml fresh culture medium containing diffferent concentrations of test compounds. After 6 days, the transformation of the cell cultures was examined microscodcallv. Cytostatic and antimetabolic assays. The cytostatic‘effects of the compounds were assessed by measuring inhibition of cell proliferation. The inhi-
ddGuo FIG.
1.
deoxyguanosinene
ddeGuo
AzddGuo -~
Structural formulae of 2',3'-dideoxyguanosine (ddeGuo) and 3'-azido-2',3'-dideoxyguanosine
1081
(ddGuo), 2'.3'-di(AzddGuo).
Vol. 145, No. 3, 1987
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
bitory effects of the compounds on DNA and RNA synthesis of Molt/4F and H9 Sells were detefjmined by measuring inhibition of the incorporation of [methylH]dThd and [5- H]Urd into TCA-insoluble material. The experimental procedures have been described previously (18). RESULTS When ddGuo, ddeGuo and AzddGuo were evaluated for their on the cytopathogenicity
of HIV in MT-4 cells,
in protecting
against destruction
protected
the cells
MT-4 cells
concentration
at a concentration
of 25 UM to achieve full
by the virus.
AzddGuo completely
2). The 2',3'-didehydro less active and more cytotoxic (Fig.
than the parent compound. ddeGuo achieved approximately of 5.0 PM; however, the 50 % protection
tained because of cytotoxicity
25 % protection level
at the higher concentrations
When assayed for cytotoxicity
effect
of 5.0 PM, whereas ddGuo required a At 5.0 JJM, ddGuo afforded protection.
only 20 % protection against cell destruction derivative of ddGuo, ddeGuo, was significantly concentration
inhibitory
AzddGuo proved the most potent
in mock-infected
at a
could not be at(Fig.
MT-4 cells,
2). AzddGuo redu-
ced cell viability to 30 X of control at a concentration of 250 PM, whereas ddGuo effected a 45 % reduction at the highest concentration tested (625 PM) (Fig. 2). The selectivity index (SI), as based on the ratio of CD50 to ED50, was 136 and 64 for AzddGuo and ddGuo, respectively (Table I). The SI of AzddGuo was comparable to that of ddAdo (SI : 139). Next,
the compoundswere evaluated
antigen expression in HIV-infected
MT-4 cells.
z ,\ P 2 " -w "
for their
effect
In these experiments,
on viral AzddGuo
10
5
100
5
0
0 2
10
50
25
125
625
concentration of FIG. 2 ddGuo*o, clusion on day solid columns (0). Data were
inhibitory
0 k-h
0
02
10
50
A25
625
tompound
Inhibition of the cytopathogenicity of HIV and AzddGuo. The cell viability was measured 5 after virus infection. The infected cells Cm) and the mock-infected cells are indicated taken from a representative experiment.
1082
125
for MT-4 cells by by trypan blue exare indicated by by open columns
Vol. 145, No. 3, 1987 TABLE
BIOCHEMICAL Inhibitory effects replication of
I.
Compound
ED50a
ddGuo
ddeGuo
AND BIOPHYSICAL
of ddGuo, ddeGuo, HIV
in
MT-4
AzddGuo cultures
cell
(IJM)
CD5;
ddAdo '50 % antiviral beffect of riIV.
effective
and
and
data represent
ddGuo
achieved
centration were
of
the
5.0
64
1.4 6.4
11 190 890
< 2.2 136 139
mean values
complete
same as those
SIC
486
dose, based on the protection
JJM and
on the
(PM)
against
50 % cytotoxic dose, based on a reduction in the viability host cells. iSelectivity index (ratio of CD50 to ED ). At this concentration, approximately 2 30 % protection was protection could not be attained because of cytotoxicity tions.
All
ddAdo
'a6 >5
AzddGuo
RESEARCH COMMUNICATIONS
for 2 separate
inhibition 25 nM,
required
of
the
cytopathic uninfected
achieved. at higher
The 50 % concentra-
experiments.
viral
antigen
respectively
for
of
the
(Fig.
complete
expression
3).
protection
These
at
a con-
concentrations
against
HIV cytopatho-
genicity. When examined broblast
cells
50 % effective I.IM for
ddGuo
for
their
inhibitory
by MSV, AzddGuo dose
(ED50>
and ddeGuo,
for
effect
was more AzddGuo
respectively
on the
effective
transformation
than
ddGuo
was 13 MM, as compared (data
not
of MO fi-
and ddeGuo. to
160 uM and 36
shown).
50, I ---
40
30 1 20 I
0
0.2
1.0
/
I
I
5.0
25
125
ConcentrationIpMI
FIG. 3. Inhibition of viral antigen expression in HIV-infected MT-4 cells by ddGuo (0). ddeGuo (01, and AzddGuo (A). Antigen-positive cells were measured by indirect immunofluorescence and laser flow cytofluorography on day 4 after virus infection, using a polyclonal antibody as probe. The mean percentage of fluorescent cells in the absence of compound (positive control) was 48.9 % (indicated by broken line), and that of mock-infected cells (negative control) was 1.9 %. 1083
The
BIOCHEMICAL
Vol. 145, No. 3, 1987
TABLEII.
AND BIOPHYSICAL
Inhibitory effects of ddGuo, ddeGuo,AzddGuo,and ddAdoon the replication of humantumor cell lines
Compound
ID50a (PM) Raji
ddGuo
229
ddeGuo
46
AzddGuo ddAdo
185 > 1000
a50 % inhibitory All
RESEARCH COMMUNICATIONS
Molt/4F
MT-4
H9
CEM
HUT-78
> 690
288 33 2 275 > 1000
288 41 > 150 2 1000
90 31 1 150 564
297 50 2 275 768
152 > 275 720
dose required to inhibit
data represent
mean values
for
cell proliferation
3 separate
by 50 %.
experiments.
When the cytostatic effect of the compounds against several human cell lines, i.e. Rajl, Molt/4F, MT-4, H9, CEM, and HUT-78, was evaluated, ddeGuo was of all test compounds the most cytostatic against all cell lines (Table XI).
The order of cytostatic
this
order completely
potency was ddeGuo > AzddGuo 2 ddGuo > ddAdo, and
corresponded with
the order of cytotoxicity
noted for
these compoundsin mock-infected MT-4 cells (Table I). The ID5C of AzddGuo and ddGuo for cellular DNAand RNA synthesis in H9 and Molt/4F cells, as monitored by the incorporation
maof [methyl-3 H]dThd and [5-3H1Urd Into acid-insoluble was higher than the highest concentrations tested for
terial, respectively, the compounds (> 139 JJMfor AzddGuo and > 688 JJMfor ddGuo) (data not shown). DISCUSSION Recently,
ddAdo has been recognized as a prime candidate for the chemo-
therapy of AIDS because of its mann -et al.
mentioned that
(19). The results derable potential
low toxicity
and high selectivity
AzddGuo may also be an effective
(8,lO). anti-HIV
Hartagent
presented here indicate that, indeed, AzddGuo offers consifor the treatment of AIDS. In comparison with ddAdo, the cy-
4.7-fold higher and the activity totoxicity of AzddGuo was approximately against HIV was about 4.6-fold higher, which means that the selectivity of AzddGuo was almost Identical to that of ddAdo (Table I). In addition, since AzddGuo proved to be a potent inhibitor of MO transformation by MSV -in vitro : 13 JJM), the investigation on the inhibitory effect of this compoundon (ID50 tumor induction by MSV in newborn NMRI mice can be considered. The latter seemsto be a representative model for retrovlrus infection -in vivo (14). A selective inhibition of HIV replication in MT-4 cells could also be achieved with ddGuo. However, its 2',3'-didehydro derivative, ddeGuo, was quite toxic and not effective at non-toxic concentrations (Fig. 2). We have derivatives of 2',3'-dideoxythymipreviously shown that the 2' ,3'-didehydro dine (ddThd) and 2',3'-dideoxycytidine (ddCyd), namely 2',3'-dldeoxythymidi1084
Vol. 145, No. 3, 1987
BIOCHEMICAL
AND BIOPHYSICAL
nene (ddeThd) and 2',3'-dideoxycytidinene and selective vative
inhibitors
tion
(ddeCyd) respectively,
of HIV (10,20,21),
of ddAdo (ddeAdo) is not effective
dro derivative
RESEARCH COMMUNICATIONS
are potent
whereas the 2',3'-didehydro (10,Zl).
Moreover,
of ddDAPR (ddeDAPR) also proved non-inhibitory
deri-
the 2',3'-didehyto HIV replica-
at nontoxic
concentrations (11). Taken together, these results suggest of purines, in conas a rule, 2',3'-didehydro-2',3'-dideoxyribosides do not appear promising as anti-HIV with their pyrimidine counterparts,
that,
trast agents.
The mechanism by which AzddGuo inhibits subject
of further
study.
the replication
Like AzddThd and other
2',3'-dideoxynucleosides,
AzddGuo may be assumed to act as a chain terminator phosphorylated
by cellular
kinases.
Furthermore,
of HIV remains
for RT after the recent
it
has been
observations
of
Dalal -et al. (24) demonstrate that ddAdo is rapidly deaminated in Molt/4F cells to 2',3'-dldeoxyinosine and subsequently metabolized to the normal adenosine nucleotides. cells.
It
is not known how AzddGuo is metabolized
Should AzddGuo prove resistant
to degradative
within
enzymes (purine
the
nucleo-
side phosphorylases) and apt to a straightforward conversion to Its 5'-triphosphate, the compoundmay be advantageous over ddAdo from a chemotherapeutic viewpoint. The results luated
for
its
presented here indicate efficacy
in the treatment
that AzddGuo should be further of retrovirus
infections
eva-
including
AIDS. ACKNOWLEDGEMENTS HIV (HTLV-IIIB) was kindly provided by Dr. R.C. Gallo (National Cancer Institute, Bethesda, MD, USA). MT-4 cells were a gift from Dr. L. Montagnier (Pasteur Institute, Paris, France) and 3' -azido-2',3'-dideoxyguanosine was kindly supplied by Dr. F. Eckstein (Max-Planck-Institut fiir Experlmentelle Medizin, Gbttingen, W.-Germany). Dr. M. Baba is a recipient of a grant from the J.S.P.S. (Japan Society for the Promotion of Science, 2-438). Dr. P. Herdewljn is a Research Associate of the Belgian N.F.W.O. (Nationaal Fonda voor Wetenschappelijk Onderzoek). These investigations were supported in part by the AIDS Basic Research Programme of the European Community, by grants from the Belgian F.G.W.O. (Fond8 voor Geneeskundig Wetenschappelijk Onderzoek: Project no. 3.0040.83), and by the Belgian G.O.A. (Geconcerteerde OnderzoeksacLizette Van Berckelaer, ties: Project no. 85190-79). We thank Ann Absillis, and Francis Vanlangendonck for excellent technical assistance and Christiane Callebaut for fine editorial help. REFERENCES 1. 2. 3.
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