From the
Long term administration of 3`-azidothymidine (AZT) for the
treatment of AIDS has led to detrimental clinical side effects in some
patients, the biochemical causes of which are still being delineated.
Base-substituted, azido-nucleotide photoaffinity analogs have routinely
proven to be effective tools for identifying and characterizing
nucleotide-utilizing enzymes. Therefore, we have synthesized
5-azido-3`-azido-2`,3`-dideoxyuridine, which is a potential
photoaffinity analog of two human immunodeficiency virus drugs, AZT and
3`azido-2`,3`-dideoxyuridine. A partially purified herpes simplex virus
type 1 thymidine kinase and [
For the clinical treatment of human immunodeficiency virus and
human herpesvirus infections, nucleoside-based compounds are the major
drugs employed
(1) . Due to the ability of these viruses to
generate drug-resistant strains in humans, the development of new
antiviral drugs and a basic biochemical and pharmacological
understanding of their properties remains a critical priority in
treating these infections
(1, 2, 3) . One way to
achieve this is to understand the structural and catalytic properties
of the target enzymes involved in the metabolism of these drugs. To aid
in accomplishing this for HIV-related enzymes, we report the synthesis
of a photoaffinity analog of 3`-azido-2`,3`-dideoxythymidine
(AZT).
Long term administration of AZT in
patients with AIDS has led to bone marrow suppression and other cytoxic
effects
(7) , as well as the generation of AZT-resistant HIV-1
strains
(2) . It has been frequently observed that AZT
administration results in a large intracellular accumulation of AZT
monophosphate (AZTMP) in lymphocytes, presumbably due to the low
affinity of AZTMP for TMP kinase
(8) . Besides the effects on the
inhibition of HIV reverse transcriptase activity
(8) , this
accumulation of AZTMP has recently been linked to the inhibition of
several viral and cellular enzymes and processes. Addition of AZTMP has
been shown to inhibit UDP-N-acetylglucosamine transport in
Chinese hamster ovary-derived Golgi membranes, which explained an
observed decrease in protein glycosylation in AZT-treated Chinese
hamster ovary cells
(9) . AZTMP has also been reported to inhibit
HIV integrase activity and may be implicated in potential HIV integrase
mutations in AZT-resistant HIV strains
(10) . Additionally, the
effects of AZTMP on associated mitochondrial toxicities and inhibition
of mitochondrial DNA polymerase
As an aid to characterizing the
effects of AZT on the inhibition of HIV replication and its role in
cellular toxicities, we report the synthesis of
5-azido-3`-azido-2`,3`-dideoxyuridine (5N
Solvents, reagents, and AZT were purchased from Aldrich and
Sigma. 3`-Azido-2`,3`-dideoxyuridine (AZddU) was synthesized as
described previously
(13) . The HIV strain I391-4 was obtained
through the AIDS Research and Reference Reagent Program, AIDS Program,
NIAID, NIH; from Dr. Douglas Richman
(14) . Melting points were
obtained on a Thomas-Hoover capillary apparatus and are uncorrected.
Rat liver
microsomes were prepared as described previously
(15) .
Photoaffinity reactions included rat liver microsomal membranes (120
µg), 30 µM
[
It has been previously reported
that the thymidine and TMP sites overlap and are shared in HSV-1 TK
with nucleosides having higher affinity than nucleotide
monophosphates
(18) . This was also observed with studies using
HSV-1 TK and the photoreactive TMP analog,
5N
The possible detrimental clinical effects of long term
administration of AZT in patients infected with HIV-1 are now becoming
clear. However, the underlying biochemical and cellular causes of these
effects are largely uncharacterized. In this report, we have
demonstrated that the photoaffinity analogs 5N
Photolabeling of the rat liver microsomes
with [
One of the original purposes of
this study was to determine whether 5N
The levels of replication of
two HIV-1 strains, IIIB and I391-4, were determined in H9 cells
infected for 12 days in the presence of 1 µM compound
using an HIV-1 p24 enzyme-linked immunosorbent assay. The cytotoxic
effects of the compounds on uninfected H9 cells were determined as
described under ``Materials and Methods'' and expressed as
the inhibitory dose of compound required to kill 50% of H9 cells
(CD
We especially thank Dr. Jackson O. Lay of the National
Center for Toxicological Research, Jefferson, AR for his FAB-MS
analyses and Dr. Alan Toland of University of Arkansas at Little Rock
for NMR analyses.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-
P]ATP were
used to make an AZT monophosphate analog,
[
P]5-azido-3`-azido-2`,3`-dideoxyuridine
monophosphate. The photoaffinity properties of this analog were
initially tested with herpes simplex virus type 1 thymidine kinase.
Photoaffinity labeling of this enzyme was saturable (half-maximal, 30
µM) and could be specifically inhibited by AZT, AZT
monophosphate, thymidine, and thymidine monophosphate. Photolabeling of
rat liver microsomal membranes was also done, and several membrane
proteins that interact with AZT monophosphate were identified. The
antiviral and cytotoxic activities of
5-azido-3`-azido-2`,3`-dideoxyuridine were determined using human
immunodeficiency virus, type 1 strain IIIB and an AZT drug-resistant
strain in human T lymphocyte H9 cells.
(
)
Nucleotide photoaffinity analogs of
2-azidopurines, 8-azidopurines, and 5-azidouridine derivatives have
proven to be very useful in the identification and biochemical
characterization of nucleotide-utilizing enzymes
(4, 5) .
This photoaffinity approach has recently been applied to the study of
wild-type and drug-resistant herpes simplex virus type 1 (HSV-1)
thymidine kinases (TKs) using a TMP analog, 5-azido-deoxyuridine
monophosphate
(6) .
are poorly understood but are
likely to contribute to AZT-associated
myopathies
(11, 12) .
AZddU), a
potential photoaffinity analog of AZT and other structurally related
nucleoside drugs. The effects of 5N
AZddU on HIV-1
replication in a human T cell line are reported, and the photoaffinity
properties of the monophosphate derivative (5N
AZddUMP) are
described using HSV-1 thymidine kinase and rat liver microsomal
membranes.
H NMR spectra were recorded on a General Electric 300-MHz
spectrometer using Me
SO-d
as the
solvent and internal standard. IR spectra were recorded on a
Perkin-Elmer 475 spectrophotometer, and UV spectra were recorded on a
Shimadzu UV-1201 spectrophotometer. FAB-MS analyses were performed on a
Kratos M50 with a fast atom bombardment ionization probe in
thioglycerol by Dr. Jackson O. Lay (National Center for Toxicological
Research, Jefferson, AR). TLC was done on Whatman F254 silica gel
plates, and 70-270 mesh silica gel was used for silica gel
chromatography.
5-Nitro-3`-
A solution of
3`--methanesulfonyl-5`-O-trityl-2`-dideoxyuridine
(2)
-O-methanesulfonyl-5`-O-trityl-2`-dideoxyuridine
(1, 350 mg, 0.64 mmol) prepared as described previously
(13) was dissolved in 10 ml of
N,N-dimethylformamide, and NOBF
(370 mg,
3.2 mmol) was added at room temperature for 10 min. The nitration
reaction was terminated by the addition of 1 ml of H
O. The
reaction solution was poured into ice water with stirring, and the
resulting precipitate was filtered and dried. An analytical sample was
obtained as a crystallization from CH
OH/H
O (325
mg, 85%). UV
(CH
OH) 302 nm (pH 1), 322
nm (pH 12);
H NMR (Me
SO-d
)
2.50-2.70 (m, 2H, 2`-H), 3.22 (s, 3H,
CH
SO
O), 3.55-3.75 (m, 2H, 5`-H), 4.32 (m,
1H, 4`-H), 5.31 (m, 1H, 3`-H), 6.10 (m, 1H, 1`-H), 7.10-7.48 (m,
15H, trityl), 9.45 (s, 1H, 6-H), and 12.10 (s, 1H, 3-NH, D
O
exchangeable); m.p. 114-117 °C.
5-Amino-3`-
Compound 2 (500 mg, 0.84
mmol) was dissolved in 30 ml of CH-O-methanesulfonyl-5`-O-trityl-2`,3`dideoxyuridine
(3)
OH, followed by the
addition of 10 ml of 0.3 M HCl and 4 g of granular zinc. The
reaction solution was stirred until the UV absorbance of the sample
indicated a
of 264 (pH 1) or 290 nm (pH 12). The
product solution was separated from the zinc metal and neutralized with
NH
OH (yields 70-84%). UV
(CH
OH) 264 nm (pH 1), 290 nm (pH 12);
H
NMR (Me
SO-d
) 6.95 (s, 1H, 6-H); m.p.
123-126 °C.
5-Amino-3`-
A mixture of compound 3 (320
mg, 0.57 mmol) and lithium azide (170 mg, 3.4 mmol) in
N,N-dimethylformamide (15 ml) was heated at
85-90 °C for 1.5 h. The solvent was evaporated in vacuo to dryness. The pure product was obtained by silica gel
chromatography using ethyl acetate/benzene (1:1) as an eluent (220 mg,
76%). UV -azido-5`-O-trityl-2`,3`-dideoxyuridine
(4)
(CH
OH) 264 nm (pH 1) and 290
nm (pH 12); m.p. 89-92 °C.
5-Amino-3`-
A suspension of compound 4 (280 mg, 0.55 mmol) in 5 ml of 80% acetic acid was heated at
85-90 °C for 30 min, after which the solution was evaporated
in vacuo to a solid. The product was purified by silica gel
chromatography using chloroform/methanol (4:1) as an eluent (120 mg,
82%). UV (aqueous) -azido-2`,3`-dideoxyuridine
(5)
264 nm (pH 1), 290 nm (pH 12);
IR (KBr) 2090 cm
(azido);
H NMR
(Me
SO-d
) 4.35 (m, 1H, 3`-H) and 6.95
(s, 1H, 6-H); FAB-MS 269 (M+1).
5-Azido-3`-azido-2`,3`-dideoxyuridine
(6)
Compound 5 (100 mg, 0.37
mmol) in 2 ml of 2 N HCl was stirred at 0 °C for 15 min. A
diazonium intermediate was generated by the addition of NaNO (34 mg, 0.49 mmol). After stirring for 2 min at O °C, 4 ml of
4 M NaN
was added with rapid stirring. The
reaction was stirred for 5 min at O °C and for 30 min at room
temperature. The reaction solution was neutralized with
NH
OH and concentrated under reduced pressure. The product
was purified by silica gel chromatography using chloroform/methanol
(4:1) as an eluent (89 mg, 82%). UV (CH
OH)
287 nm (disappears after UV photolysis);
H NMR
(Me
SO-d
) 2.50-2.70 (m, 2H,
2`-H), 3.55-3.75 (m, 2H, 5`-H), 3.82 (m, 1H, 4`-H), 4.38 (m, 1H,
3`-H), 5.40 (s, 1H, 5`-OH), 6.10 (m, 1H, 1`-H), 7.82 (s, 1H, 6-H); IR
(KBr) 2100-2000 cm
(azido); FAB-MS 333
(M+K) m.p. 63-66 °C. Synthesis of
[
P]5-Azido-3`azido-2`,3`-dideoxyuridine
monophosphate-Partially purified HSV-1 thymidine kinase (10
µg), 0.5 µmol 5N
AZddU, 40 mM KCl, 10
mM MgCl
, and 1 mCi of
[
-
P]ATP (3000 mCi/µmol, ICN
Biomedicals) in a total of 0.15 ml was incubated at 37 °C for 20
min
(6) . ATP (2 µmol) was added for 5 min prior to column
purification. The resulting
[
P]5N
AZddUMP was purified by
DEAE-cellulose chromatography as described previously for other
5-azidouridine nucleotides
(5) .
[
P]5N
dUMP was synthesized as
described previously
(6) .
Photoaffinity Labeling of HSV-1 TK and Rat Liver
Microsomes
For photolabeling studies of HSV-1 TK, 10 µg of
DE-52 purified TK
(6) in 20 mM potassium phosphate
buffer, pH 7.6, 5 mM MgCl, 10% glycerol, 1
mM dithiothreitol, and 40 mM KCl was incubated with
the indicated concentration of
[
-
P]5N
AZddUMP for 10 s. The
sample was then irradiated for 90 s with a hand-held UV lamp (254 nm
UVP-11, Ultraviolet Products, Inc.) at a distance of 3 cm. For
competition experiments, varying concentrations of non-radiolabeled
competitors were incubated in the reaction mixture for 20 s prior to
the addition of photoprobe. All reactions were terminated by the
addition of an equal volume of 10% trichloroacetic acid, incubated on
ice for 10 min, and pelleted by centrifugation at 13,000 rpm for 5 min.
The protein was resuspended in a solublization mixture and separated on
10% SDS-polyacrylamide gels
(5) . Dried gels were
autoradiographed for 0.5-3 days. The intensity of the bands
corresponding to TK on the autoradiographs were determined by laser
densitometry (Bio-Rad model GS-670 imaging densitometer).
-
P]5N
AZddUMP, 60 mM
HEPES, pH 6.5, 3 mM MgCl
, and 3 mM
saccharolactone in 50 µl. Photoirradiation and analysis of the
samples were similar to that described above for HSV-1 TK and also have
been previously described (5, 15).
Cytoxicity and Antiviral Assays
The cytotoxic
effects of the compounds on uninfected cells were determined by
incubation of H9 cells with drug concentrations ranging from 50 to 300
µM. The concentration of drug required to kill 50% of the
H9 cells (CD) was determined using a soluble tetrazolium
dye, Alamar blue, as directed by the manufacturer (Alamar, Sacramento,
CA). For anti-HIV assays, H9 cells (3
10
) grown in
RPMI 1640 media supplemented with 20% fetal bovine serum were infected
with HIV-1 strain IIIB or strain I391-4
(14) at a p24
concentration of 5
10
pg/ml
(16) and treated
with different concentrations of drug (0.1-5.0 µM).
The levels of HIV replication were determined by harvesting
supernatants at 4, 8, and 12 days postinfection using HIV-p24
enzyme-linked immunosorbent assay plate assays according to the
instructions of the manufacturer (Incstar, Stillwater, MD).
Synthesis of
5-Azido-3`-azido-2`,3`-dideoxyuridine
The general protocol for
the synthesis of 5-azidouridine nucleotides has been a C-5 nitration
reaction, reduction to the 5-amine, followed by a sodium azide exchange
reaction via a diazonium ion
intermediate
(5, 6, 17) . To obtain the desired
5NAZddU target compound (6), this same general
synthetic strategy was used in combination with protocols commonly used
for the synthesis of anti-HIV 3`-azido-nucleoside
compounds
(13) , as outlined in Fig. 1. The presence of the
3` and 5` azido groups of 5N
AZddU was confirmed by IR
spectra. Each 5-substituted compound also had the characteristic loss
of the H-5 signal and the doublet to singlet conversion at H-6 by
H NMR. The UV spectrum of 5N
AZddU
(
= 287 nm) is characteristic of a
5-azidouridine compound
(5) . As shown in Fig. 2, UV
irradiation of this derivative leads to a loss in absorbance and is
indicative of a photoreactive 5-azidouridine group (5, 17). FAB-MS
analysis was necessary to confirm the final products due to instability
of the 3`- and 5-azides in solid form. Maximal stability of these
compounds was obtained by storing them at -20 °C in methanol.
Figure 1:
Synthesis of
5-azido-3`-azido-2`,3`-dideoxyuridine.
Figure 2:
UV Spectrum of
5-azido-3`-azido-2`,3`-dideoxyuridine. The UV spectrum of
5NAZddU was determined (-UV) (0.06 µmol)
followed by irradiation of the compound for 60 s with a 254 nm UVS-11
lamp and another recording
(+UV).
Photoaffinity Labeling of HSV-1 Thymidine
Kinase
The metabolism of AZT to the pharmacologically active
AZT-triphosphate requires an initial phosphorylation by cytosolic
thymidine kinase, followed by an apparent rate-limiting phosphorylation
step by thymidylate kinase
(8) . To study these enzyme activities
and demonstrate the use of 5NAZddU as a photoaffinity
analog, the multifunctional HSV-1 thymidine kinase was used. This
enzyme has been previously studied with azido-nucleotide
photoprobes
(6) , and it is known to possess distinct thymidine
kinase and thymidylate kinase activities
(18) . Additionally, AZT
has been previously shown to be a substrate of this enzyme
(19) .
Therefore, a [
P]5N
AZddUMP photoprobe
was synthesized following incubation of HSV-1 TK, 5N
AZddU,
and [
-
P]ATP as described under
``Materials and Methods.''
dUMP
(6) . Therefore, the effects that varying
concentrations of thymidine, TMP, AZT, and AZTMP had on
photoincorporation of [
P]5N
AZddUMP
into HSV-1 TK were determined as shown in Fig. 3(A and
B). The concentration of inhibitor required to reduce
photoincorporation by 50% (ICP
) was determined to be 7
µM for AZT and 80 µM for AZTMP
(Fig. 3B). As expected if this photolabeling was active
site-directed
(5, 6) , saturation of photoincorporation
was observed with increasing concentrations of
[
P]5N
AZddUMP (half-maximal, 30
µM) as shown in Fig. 3C. In a previous
study using [
P]5N
dUMP and HSV-1 TK,
half-maximal saturation of photoincorporation of this analog was 6
µM, and the ICP
values for thymidine and TMP
were 1.5 and 12.5 µM, respectively
(6) . Thymidine
and TMP had similar inhibitory effects on
[
P]5N
AZddUMP photoincorporation into
HSV-1 TK (Fig. 3A). For comparison, the inhibitory
effects that AZT and AZTMP had on photoincorporation of
[
P]5N
dUMP are presented in
Fig. 3D. The derived ICP
values of 0.7
µM for AZT and 130 µM for AZTMP (from
Fig. 3D) indicate that AZT is an effective inhibitor of
HSV-1 TK photolabeling with
[
P]5N
dUMP or
[
P]5N
AZddUMP, although AZTMP has
relatively poor affinity for the TMP site of HSV-1 TK.
Figure 3:
Photoaffinity labeling of HSV-1 thymidine
kinase. A and B, competition. HSV-1 TK (10 µg)
was incubated with 10 µM
[-
P]5N
AZddUMP in the presence
of the indicated concentrations of competitor and photolyzed as
described under ``Materials and Methods.'' C,
saturation. Increasing concentrations of
[
-
P]5N
AZddUMP were photolyzed
in the presence of HSV-1 TK (10 µg). A irradiation control sample
(without UV) is also included (-UV). D, the
effect of AZT and AZTMP on photoincorporation of 15 µM
[
-
P]5N
dUMP into HSV-1 TK. For
A-D, photoincorporation was detected by autoradiography
and quantified by laser densitometry.
Photoaffinity Labeling of Rat Liver Microsomal
Membranes
A previous report has indicated that AZTMP can inhibit
UDP-GlcNAc transport in Chinese hamster ovary cells
(9) . Because
we have also been interested in the transport of UDP-glucuronic acid
and UDP-glucose in rat liver microsomal
membranes
(5, 15) ,
[P]5N
AZddUMP was used to photolabel
rat liver microsomes in the presence of different competing nucleotides
and UDP-sugars (Fig. 4). As shown in Fig. 4(lane
1), a protein of 130 kDa was photolabeled with
[
P]5N
AZddUMP and was competively
inhibited by 0.2 mM AZTMP (lane 4). Because one
mechanism for UDP-sugar translocation is UMP antiport
(20) , the
effects of UMP and UDP-sugars on photoincorporation of
[
P]5N
AZddUMP into the 130-kDa
protein were examined. The presence of UMP completely inhibited
photolabeling of the 130-kDa protein, whereas the presence of 0.2
mM UDP-Glc (lane 6) or 0.2 mM UDP-GlcNAc
(lane 8) led to a 3-fold reduction in
[
P]5N
AZddUMP photoincorporation.
Figure 4:
Photoaffinity labeling of rat liver
microsomal membranes.
[-
P]5N
AZddUMP (lanes
1-9) or
[
-
P]5N
UDP-GlcUA (lane
10) (40 µM each) were used to photolabel rat liver
microsomes (50 µg) as described under ``Materials and
Methods.'' Except for lanes 1 and 10, the
following competitors at 200 or 400 µM concentrations were
added in the photolabeling reactions as indicated: lanes 2 and
3, UMP; lanes 4 and 5, AZTMP; lanes 6 and 7, UDP-Glc; lanes 8 and 9,
UDP-GlcNAc.
For comparison, the same rat liver microsomes were photolabeled with
the UDP-glucuronic acid analog,
[P]5N
UDP-GlcUA, as described
previously (5, 15). As shown in Fig. 4(lane 10),
photolabeling with [
P]5N
UDP-GlcUA
resulted in photoincorporation of the 130-kDa protein, and, as
previously reported, the 50-54-kDa UDP-glucuronosyltransferases
were involved in liver detoxification reactions
(5, 15) .
[
P]5N
AZddUMP also photolabeled a
50-kDa protein (lane 1) that was competively inhibited by
AZTMP (lanes 4 and 5). Because AZT is known to be
glucuronidated in human liver microsomes by
UDP-glucuronosyltransferases
(21) , photolabeling of the 50-kDA
protein by [
P]5N
AZddUMP may indicate
that this analog can be used to study the UDP-glucuronosyltransferases
involved in AZT detoxification.
Anti-HIV Testing
The photoaffinity data suggest
that 5NAZddUMP is mimicking AZTMP in isolated enzyme
systems. An important question to answer was whether
5N
AZddU could mimic AZT or its uridine equivalent
AZddU
(22, 23) in HIV-infected cells. Structurally,
5N
AZddU could potentially mimic AZT or AZddU as an anti-HIV
compound. For comparative purposes, the anti-HIV effects of AZT, AZddU,
and 5N
AZddU were determined in the human T
lymphocyte-derived H9 cell line infected with HIV-1 strains IIIB and
I391-4 (a strain shown to have intermediate resistance to
AZT
(14) ). The degree of inhibition was assayed using HIV p24
enzyme-linked immunosorbent assay plates. At the titers tested, only
low levels of HIV replication were detected at days 4 and 8
postinfection, even in the absence of drugs (data not shown).
Therefore, a representative result determined at 1 µM
concentrations of each compound at 12 days postinfection is described
in . Under these conditions, 5N
AZddU was 53-
and 10-fold less effective than AZT at inhibiting HIV-IIIB and
HIV-I391-4 replication, respectively. When compared with AZddU,
5N
AZddU was 10-fold less effective at inhibition of
HIV-IIIB replication, although it was better than AZddU for inhibition
of HIV-I391-4 replication. 5N
AZddU, AZT, and AZddU had few
cytotoxic effects on H9 cells at concentrations up to 300
µM. The anti-HIV-1 properties and cellular metabolism of
5N
AZddU are currently being further characterized, but
these initial studies suggest that 5N
AZddU can mimic the
metabolism of AZT and AZddU in HIV-1-infected cells.
AZddU and
[
P]5N
AZddUMP mimic the biological
activities of AZT and AZTMP and validate a new approach to
characterizing nucleoside/nucleotide drug metabolizing enzymes. As a
model drug-metabolizing enzyme, we have used HSV-1 TK for several
reasons. Because it possesses both thymidine and thymidylate kinase
activities, two reactions that are critical in AZT metabolism,
characterization of [
P]5N
AZddUMP
photolabeling of HSV-1 TK indicates the utility of this approach for
studying the cellular TKs and thymidylate kinases. As shown in
Fig. 3
, AZTMP has a poor affinity for HSV-1 TK, similar to that
reported for AZTMP and cellular thymidylate kinase
(8) .
Determination of the HSV-1 TK amino acids covalently cross-linked with
[
P]5N
AZddUMP may aid in defining the
molecular basis for the weak affinity of thymidylate kinases for AZTMP.
Additionally, HSV-1 TK is being widely used as a toxin gene in
combination with ganciclovir for gene therapy approaches to treating
glioma and other cancers
(24, 25) . It has also been
demonstrated in T cells transfected with HSV-1 TK that the addition of
acyclovir leads to the inhibition of HIV-1 replication
(26) . The
process of HSV-1 TK expression in lymphocytes and its effect on
nucleotide and AZT metabolism is poorly understood, and it is currently
being studied with the aid of 5N
AZddU and related
photoaffinity analogs.
P]5N
AZddUMP led to the
identification of a 130-kDa protein that was specifically labeled.
Because this photolabeling was inhibited by UMP, the proposed antiport
molecule of some UDP-sugar translocators
(20) , this may indicate
a role for this 130-kDa protein in UDP-sugar translocation in the
microsomal membranes. Using
[
P]5N
AZddUMP to photolabel
microsomes prepared from the human T lymphocyte H9 cell line, a similar
protein of 130-kDa mass was identified with similar photolabeling
inhibition properties by competing nucleotides (data not shown). We are
currently characterizing the inhibition properties of AZT and AZTMP on
the inhibition of UDP-glucuronic acid transport and other UDP-sugars in
rat liver and H9 microsomes. Combined with the UDP-sugar photoaffinity
analogs
(5) , these studies suggest that
[
P]5N
AZddUMP could be utilized to
identify putative UDP-sugar transporters and other membrane-associated
enzymes that interact with AZTMP.
AZddU had anti-HIV
activity comparable with AZT or similar thymidine analogs, and, if it
did, could it be used to study cellular and viral enzymes involved in
nucleoside drug metabolism? At least in the human T lymphocyte cell
line used in this study, the cytotoxicity effects of
5N
AZddU were no different than AZT of AZddU. Regarding
inhibition of HIV replication, the results presented in
confirmed our hypothesis in that 5N
AZddU does
effectively inhibit HIV-1 replication at micromolar concentrations,
although at 10-50-fold higher concentrations than clinically
characterized drugs like AZddU and AZT. Because the HIV p24 antigen
assay used did not permit rigorous characterization of
5N
AZddU metabolism as it relates to key features of viral
replication, additional studies using HIV reverse transcriptase assays
and isolation of metabolites in HIV-infected cells are currently in
progress.
Table:
Comparison of cytotoxic and antiviral activities
of AZT, AZddU, and 5NAZddU
).
AZddU,
5-azido-3`-azido-2`,3`-dideoxyuridine; TK, thymidine kinase; HSV,
herpes simplex virus; AZTMP, AZT monophosphate; TMP, thymidine
monophosphate; 5N
AZddUMP, 5N
AZddU
monophosphate; FAB-MS, fast atom bombardment mass spectroscopy;
5N
dUMP, 5-azido-deoxyuridine monophosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.