©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Synthesis of a Photoaffinity Analog of 3`-Azidothymidine, 5-Azido-3`-azido-2`,3`-dideoxyuridine
INTERACTIONS WITH HERPESVIRUS THYMIDINE KINASE AND CELLULAR ENZYMES (*)

Feng Mao (1), Tammy M. Rechtin (1), Robyn Jones (1), Alejandro A. Cantu (3), L. Sheri Anderson (3), Anna Radominska (2), Mary Pat Moyer (3), Richard R. Drake (1)(§)

From the (1) Department of Biochemistry and Molecular Biology and the (2) Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 and the (3) Department of Surgery, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 [-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.


INTRODUCTION

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).() 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) .

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 are poorly understood but are likely to contribute to AZT-associated myopathies (11, 12) .

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 (5NAZddU), a potential photoaffinity analog of AZT and other structurally related nucleoside drugs. The effects of 5NAZddU on HIV-1 replication in a human T cell line are reported, and the photoaffinity properties of the monophosphate derivative (5NAZddUMP) are described using HSV-1 thymidine kinase and rat liver microsomal membranes.


MATERIALS AND METHODS

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. H NMR spectra were recorded on a General Electric 300-MHz spectrometer using MeSO-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`--methanesulfonyl-5`-O-trityl-2`-dideoxyuridine (2)

A solution of 3`--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 HO. 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 CHOH/HO (325 mg, 85%). UV (CHOH) 302 nm (pH 1), 322 nm (pH 12); H NMR (MeSO-d) 2.50-2.70 (m, 2H, 2`-H), 3.22 (s, 3H, CHSOO), 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, DO exchangeable); m.p. 114-117 °C.

5-Amino-3`--O-methanesulfonyl-5`-O-trityl-2`,3`dideoxyuridine (3)

Compound 2 (500 mg, 0.84 mmol) was dissolved in 30 ml of CHOH, 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 NHOH (yields 70-84%). UV (CHOH) 264 nm (pH 1), 290 nm (pH 12); H NMR (MeSO-d) 6.95 (s, 1H, 6-H); m.p. 123-126 °C.

5-Amino-3`--azido-5`-O-trityl-2`,3`-dideoxyuridine (4)

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 (CHOH) 264 nm (pH 1) and 290 nm (pH 12); m.p. 89-92 °C.

5-Amino-3`--azido-2`,3`-dideoxyuridine (5)

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) 264 nm (pH 1), 290 nm (pH 12); IR (KBr) 2090 cm (azido); H NMR (MeSO-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 NHOH 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 (CHOH) 287 nm (disappears after UV photolysis); H NMR (MeSO-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 5NAZddU, 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]5NAZddUMP was purified by DEAE-cellulose chromatography as described previously for other 5-azidouridine nucleotides (5) . [P]5NdUMP 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]5NAZddUMP 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).

Rat liver microsomes were prepared as described previously (15) . Photoaffinity reactions included rat liver microsomal membranes (120 µg), 30 µM [-P]5NAZddUMP, 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).


RESULTS

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 5NAZddU 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 5NAZddU ( = 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]5NAZddUMP photoprobe was synthesized following incubation of HSV-1 TK, 5NAZddU, and [-P]ATP as described under ``Materials and Methods.''

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, 5NdUMP (6) . Therefore, the effects that varying concentrations of thymidine, TMP, AZT, and AZTMP had on photoincorporation of [P]5NAZddUMP 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]5NAZddUMP (half-maximal, 30 µM) as shown in Fig. 3C. In a previous study using [P]5NdUMP 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]5NAZddUMP photoincorporation into HSV-1 TK (Fig. 3A). For comparison, the inhibitory effects that AZT and AZTMP had on photoincorporation of [P]5NdUMP 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]5NdUMP or [P]5NAZddUMP, 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]5NAZddUMP in the presence of the indicated concentrations of competitor and photolyzed as described under ``Materials and Methods.'' C, saturation. Increasing concentrations of [-P]5NAZddUMP 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]5NdUMP 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]5NAZddUMP 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]5NAZddUMP 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]5NAZddUMP 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]5NAZddUMP photoincorporation.


Figure 4: Photoaffinity labeling of rat liver microsomal membranes. [-P]5NAZddUMP (lanes 1-9) or [-P]5NUDP-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]5NUDP-GlcUA, as described previously (5, 15). As shown in Fig. 4(lane 10), photolabeling with [P]5NUDP-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]5NAZddUMP 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]5NAZddUMP 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 5NAZddU could mimic AZT or its uridine equivalent AZddU (22, 23) in HIV-infected cells. Structurally, 5NAZddU could potentially mimic AZT or AZddU as an anti-HIV compound. For comparative purposes, the anti-HIV effects of AZT, AZddU, and 5NAZddU 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, 5NAZddU was 53- and 10-fold less effective than AZT at inhibiting HIV-IIIB and HIV-I391-4 replication, respectively. When compared with AZddU, 5NAZddU was 10-fold less effective at inhibition of HIV-IIIB replication, although it was better than AZddU for inhibition of HIV-I391-4 replication. 5NAZddU, 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 5NAZddU are currently being further characterized, but these initial studies suggest that 5NAZddU can mimic the metabolism of AZT and AZddU in HIV-1-infected cells.


DISCUSSION

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 5NAZddU and [P]5NAZddUMP 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]5NAZddUMP 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]5NAZddUMP 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 5NAZddU and related photoaffinity analogs.

Photolabeling of the rat liver microsomes with [P]5NAZddUMP 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]5NAZddUMP 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]5NAZddUMP could be utilized to identify putative UDP-sugar transporters and other membrane-associated enzymes that interact with AZTMP.

One of the original purposes of this study was to determine whether 5NAZddU 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 5NAZddU were no different than AZT of AZddU. Regarding inhibition of HIV replication, the results presented in confirmed our hypothesis in that 5NAZddU 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 5NAZddU 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

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).



FOOTNOTES

*
This research was supported in part by a grant from the Arkansas Science and Technology Authority (to R. R. D.) and by Grant HL48497 from the National Institutes of Health (to M. P. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Slot 516, University of Arkansas for Medical Sciences, 4301 W. Markham, Little Rock, AR 72205. Tel.: 501-686-5419; Fax: 501-686-8169.

The abbreviations used are: AZT, 3`-azido-2`,3`-dideoxythymidine; HIV, human immunodeficiency virus; AZddU, 3`-azido-2`,3`-dideoxyuridine; 5NAZddU, 5-azido-3`-azido-2`,3`-dideoxyuridine; TK, thymidine kinase; HSV, herpes simplex virus; AZTMP, AZT monophosphate; TMP, thymidine monophosphate; 5NAZddUMP, 5NAZddU monophosphate; FAB-MS, fast atom bombardment mass spectroscopy; 5NdUMP, 5-azido-deoxyuridine monophosphate.


ACKNOWLEDGEMENTS

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.


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