Azidothymidine Triphosphate Research Articles

Evidence and Possible Consequences of the Phosphorylation of Nucleoside Reverse Transcriptase Inhibitors in Human Red Blood Cells

The intracellular metabolism of nucleoside reverse transcriptase inhibitors (NRTI) in mononuclear cells has been thoroughly studied, but that in red blood cells (RBC) has been disregarded. However, the phosphorylation of other analogous nucleosides (in particular, ribavirin) has been described previously. In this study, we investigated for the first time the phosphorylation of NRTI in human RBC. The presence of intracellular zidovudine (AZT) monophosphate, AZT triphosphate, lamivudine (3TC) triphosphate, and tenofovir (TFV) diphosphate, as well as endogenous dATP, dGTP, and dTTP, in RBC collected from human immunodeficiency virus-infected patients was examined. We observed evidence of a selective phosphorylation of 3TC, TFV, and endogenous purine deoxynucleosides to generate their triphosphate moieties. Conversely, no trace of AZT phosphate metabolites was found, and only faint dTTP signals were visible. A comparison of intracellular TFV diphosphate and 3TC triphosphate levels in RBC and peripheral blood mononuclear cells (PBMC) further highlighted the specificity of NRTI metabolism in each cell type. These findings raise the issue of RBC involvement in drug-drug interaction, drug pharmacokinetics, and drug-induced toxicity. Moreover, the typical preparation of PBMC samples by gradient density centrifugation does not prevent their contamination with RBC. We demonstrated that the presence of RBC within PBMC hampers an accurate determination of intracellular TFV diphosphate and dATP levels in clinical PBMC samples. Thus, we recommend removing RBC during PBMC preparation by using an ammonium chloride solution to enhance both the accuracy and the precision of intracellular drug monitoring.

Zidovudine Inhibits Thymidine Phosphorylation in the Isolated Perfused Rat Heart

Zidovudine (AZT; 3'-azido-3'-deoxythymidine), a thymidine analog, has been a staple of highly active antiretroviral therapy. It is phosphorylated in the host to the triphosphate and functions by inhibiting the viral reverse transcriptase. However, long-term use of AZT is linked to various tissue toxicities, including cardiomyopathy. These toxicities are associated with mitochondrial DNA depletion, which is hypothesized to be caused by AZT triphosphate inhibition of mitochondrial DNA polymerase gamma. In previous work with isolated heart mitochondria, we demonstrated that AZT phosphorylation beyond the monophosphate was not detected and that AZT itself was a potent inhibitor of thymidine phosphorylation. This suggests an alternative hypothesis in which depletion of the TTP pool may limit mitochondrial DNA replication. The present work extends these studies to the whole cell by investigating the metabolism of thymidine and AZT in the intact isolated perfused rat heart. [3H]thymidine is converted to [3H]TTP in a time- and concentration-dependent manner. The level of [3H]TMP is low, suggesting that the reaction catalyzed by thymidine kinase is the rate-limiting step in phosphorylation. [3H]AZT is converted in a time- and concentration-dependent manner to AZT monophosphate, the only phosphorylated product detected after 3 h of perfusion. Both compounds display negative cooperativity, similar to the observations with cloned and purified mitochondrial thymidine kinase 2. The presence of AZT in the perfusate inhibits the phosphorylation of [3H]thymidine with a 50% inhibitory concentration of 24+/-4 microM. These data support the hypothesis that AZT-induced mitochondrial cardiotoxicity may be caused by a limiting pool of TTP that lowers mitochondrial DNA replication.

3‘-Azido-3‘-deoxythymidine-(5‘)-tetraphospho-(5‘)-adenosine, the Product of ATP-Mediated Excision of Chain-Terminating AZTMP, Is a Potent Chain-Terminating Substrate for HIV-1 Reverse Transcriptase

The resistance of HIV-1 to 3'-azido-3'-deoxythymidine (AZT) involves phosphorolytic excision of chain-terminating AZT-5'-monophosphate (AZTMP). Both pyrophosphate (PPi) and ATP act as excision substrates in vitro, but the intracellular substrate used during replication of AZT-resistant HIV is still unknown. PPi-mediated excision produces AZT-5'-triphosphate (AZTTP), which could be immediately re-used as a substrate for viral DNA chain termination. In contrast, ATP-mediated excision produces the novel compound AZT-(5')-tetraphospho-(5')-adenosine (AZTp4A). Since little is known of the interaction of AZTp4A with HIV-1 RT, we carried out kinetic and molecular modeling studies to probe this. AZTp4A was found to be a potent inhibitor of HIV-1 RT-catalyzed DNA synthesis and of both ATP- and PPi-mediated AZTMP excision. AZTp4A is in fact an excellent chain-terminating substrate for AZT-resistant RT-catalyzed DNA synthesis, better than AZTTP (k(pol)/Kd = 6.2 and 11.9 for AZTTP and AZTp4A, respectively). The affinity of AZT-resistant HIV-1 RT for AZTp4A is at least 30,000-fold greater than that for the excision substrate ATP and approximately 10-fold greater than that for AZTTP. Dissociation of newly formed AZTp4A from RT may therefore provide a significant rate-limiting step for continued HIV-1 DNA synthesis. Our studies show that the products of PPi- and ATP-mediated excision of chain-terminating AZTMP (AZTTP and AZTp4A, respectively) are both potent chain-terminating substrates for HIV-1 RT, suggesting that there is no obvious benefit to HIV using ATP instead of PPi as the excision substrate.

Inhibition of DNA polymerase η by oxetanocin derivatives

DNA polymerase eta is recently found as a responsible gene product of xeroderma pigmentosum variant. Differently from other eukaryotic DNA polymerases, such as alpha, beta or gamma, eta polymerase is categorized in Y family. Specific inhibitors for DNA polymerases are useful tools to study the exact role of enzyme in the living cells, however, the inhibitor for DNA polymerase eta has not been developed. We examined the inhibitory effects of several sugar-modified nucleotide analogs on DNA polymerase eta. The arabinonucleotides (araCTP), dideoxynucleotides (ddTTP) and 3'-modified nucleotides (3'-dCTP and 3'-azidothymidine tri-phosphate) did not show any inhibitory effect on DNA polymerase eta. On the other hand, the oxetanocin derivatives those have the oxetane ring as a sugar moiety, OXT-GTP and OXT-ATP, strongly inhibited this polymerase. These results suggest that DNA polymerase eta has a unique recognition mode against the sugar moiety of nucleotide compared with other eukaryotic nucleotide polymerases.

Encapsulation of mono- and oligo-nucleotides into aqueous-core nanocapsules in presence of various water-soluble polymers

In a previous study, we have shown that cidofovir (CDV) and azidothymidine-triphosphate (AZT-TP) were poorly encapsulated in poly( iso-butylcyanoacrylate) (PIBCA) aqueous-core nanocapsules. This was attributed to the rapid leakage of these small and hydrophilic molecules through the thin polymer wall of the nanocapsules. In the present study, we have selected various water-soluble polymers as increasing Mw adjuvants and investigated their influence on the entrapment of mononucleotides (CDV, AZT-TP) as well as of oligonucleotides (ODN) into these PIBCA aqueous-core nanocapsules. We show here that the presence of cationic polymers (i.e. poly(ethyleneimine) (PEI) or chitosan) in the nanocapsule aqueous compartment allowed successful encapsulation of AZT-TP and ODN.

Hybrid polymer nanocapsules enhance in vitro delivery of azidothymidine-triphosphate to macrophages

One of the main limitations in the use of nucleoside reverse transcriptase inhibitors (NRTIs) such as azidothymidine (AZT) lies in their poor intracellular activation by cellular kinases into their active tri-phosphorylated form. Thus, the direct administration of triphosphate NRTIs like azidothymidine-triphosphate (AZT-TP), has been considered for bypassing this metabolic bottleneck, but these molecules do not diffuse intracellularly, due to their too hydrophilic character. Therefore, poly( iso-butylcyanoacrylate) (PIBCA) aqueous-cored nanocapsules have been tested as carriers to overcome the cellular delivery of AZT-TP. However, encapsulation of AZT-TP remained challenging because this molecule, due to its relatively low molecular weight, rapidly leaked out of the nanocapsules. In this study, we show that association of AZT-TP to a cationic polymer such as poly(ethyleneimine) (PEI) allowed to reach high entrapment efficiency of AZT-TP in PIBCA nanocapsules (up to 90%) as well as gradual in vitro release. The resulting hybrid PIBCA/PEI nanocapsules efficiently delivered AZT-TP in vitro to macrophages: the cellular uptake was increased by 30-fold compared to the free molecule, reaching relevant cellular concentrations for therapeutic purposes.

Differential Susceptibility of HIV-1 Reverse Transcriptase to Inhibition by RNA Aptamers in Enzymatic Reactions Monitoring Specific Steps during Genome Replication

Nucleic acid aptamers to HIV-1 reverse transcriptase (RT) are potent inhibitors of DNA polymerase function in vitro, and they have been shown to inhibit viral replication when expressed in cultured T-lymphoid lines. We monitored RT inhibition by five RNA pseudoknot RNA aptamers in a series of biochemical assays designed to mimic discrete steps of viral reverse transcription. Our results demonstrate potent aptamer inhibition (IC50 values in the low nanomolar range) of all RT functions assayed, including RNA- and DNA-primed DNA polymerization, strand displacement synthesis, and polymerase-independent RNase H activity. Additionally, we observe differences in the time dependence of aptamer inhibition. Polymerase-independent RNase H activity is the most resistant to long term aptamer suppression, and RNA-dependent DNA polymerization is the most susceptible. Finally, when DNA polymerization was monitored in the presence of an RNA aptamer in combination with each of four different small molecule inhibitors, significant synergy was observed between the aptamer and the two nucleoside analog RT inhibitors (azidothymidine triphosphate or ddCTP), whereas two non-nucleoside analog RT inhibitors showed either weak synergy (efavirenz) or antagonism (nevirapine). Together, these results support a model wherein aptamers suppress viral replication by cumulative inhibition of RT at every stage of genome replication.

Inhibition of endogenous reverse transcription of human and nonhuman primate lentiviruses: Potential for development of lentivirucides

In the current study, we extended our previous works on natural endogenous reverse transcription (NERT) and further examined its potential as a virucide molecular target in sexual transmission of primate lentiviruses. HIV-1 and SIV virions were pretreated with select nucleoside (NRTIs) and nonnucleoside RT inhibitors (NNRTIs), either alone or in combination with NERT-stimulating substances. The effects of these antiretrovirals on virion inactivation were analyzed in human T cell lines and primary cell cultures. Pretreatment of HIV-1 virions with physiologic NERT-stimulants and 3′-azido-3′-deoxythymidine 5′-triphosphate (AZT-TP) or nevirapine potently inactivated cell-free HIV-1 virions and resulted in strong inhibition of the viral infectivity. Pretreatment of chimeric SHIV-RT virions with NERT-stimulating cocktail and select antiretrovirals also resulted in virion inactivation and inhibition of viral infectivity in T cell lines. Our findings demonstrate the potential clinical utility of approaches based on inhibiting NERT in sexual transmission of HIV-1, through the development of effective anti-HIV-1 microbicides, such as NRTIs and NNRTIs.

Encapsulation of antiviral nucleotide analogues azidothymidine-triphosphate and cidofovir in poly( iso-butylcyanoacrylate) nanocapsules

Nucleoside analogues are widely used in the treatment of various viral infections. However, the poor in vivo conversion of the nucleoside analogues like azidothymidine (AZT) into their active triphosphate nucleotide counterpart limits their pharmacological efficacy. This could be overcome by the direct administration of azidothymidine triphosphate (AZT-TP), but it requires an appropriate drug delivery approach. Besides nucleoside analogues, nucleotide analogues like cidofovir (CDV) are also used in the treatment of viral infections. CDV has raised recent interest because of its promising activity against smallpox, but its use is limited by its poor bioavailability and nephrotoxicity. Here again, a proper drug delivery system should address these issues. In this study, we investigated the encapsulation of the nucleotide analogues AZT-TP and CDV into poly( iso-butylcyanoacrylate) aqueous core nanocapsules, known to efficiently entrap oligonucleotides. We show here that the encapsulation of these mono-nucleotides is less efficient than with oligonucleotides and that a rapid release of AZT-TP from the nanocapsules occurred in vitro. This highlights the importance of the molecular weight of the entrapped molecules which, if they are too small, are diffusing through the thin polymer membrane of the nanocapsules. On the other hand, a good protection of the encapsulated AZT-TP was observed.

3′-Azido-3′-deoxythymidine (AZT) is a competitive inhibitor of thymidine phosphorylation in isolated rat heart and liver mitochondria

Long-term use of 3′-azido-3′-deoxythymidine (AZT) is associated with various tissue toxicities, including hepatotoxicity and cardiomyopathy, and with mitochondrial DNA depletion. AZT-5′-triphosphate (AZTTP) is a known inhibitor of the mitochondrial DNA polymerase γ and has been targeted as the source of the mitochondrial DNA depletion. However, in previous work from this laboratory with isolated rat heart and liver mitochondria, AZT itself was shown to be a more potent inhibitor of thymidine phosphorylation (IC 50 of 7.0 ± 1.0 μM AZT in heart mitochondria and of 14.4 ± 2.6 μM AZT in liver mitochondria) than AZTTP is of polymerase γ (IC 50 of >100 μM AZTTP), suggesting that depletion of mitochondrial stores of TTP may limit replication and could be the cause of the mitochondrial DNA depletion observed in tissues affected by AZT toxicity. The purpose of this work is to characterize the nature of AZT inhibition of thymidine phosphorylation in isolated rat heart and rat liver mitochondria. In both of these tissues, AZT was found to be a competitive inhibitor of the phosphorylation of thymidine to TMP, catalyzed by thymidine kinase 2. The inhibition constant ( K i) for heart mitochondria is 10.6 ± 4.5 μM AZT, and for liver mitochondria K i is 14.0 ± 2.5 μM AZT. Since AZT is functioning as a competitive inhibitor, increasing thymidine concentrations may be one mechanism to overcome the inhibition and decrease AZT-related toxicity in these tissues.

3′-Azido-3′-deoxythymidine (AZT) inhibits thymidine phosphorylation in isolated rat liver mitochondria: A possible mechanism of AZT hepatotoxicity

3′-Azido-3′-deoxythymidine (AZT) is a staple of highly active antiretroviral therapy (HAART). Prior to HAART, long-term use of high-dosage AZT caused myopathy, cardiomyopathy, and hepatotoxicity, associated with mitochondrial DNA depletion. As a component of HARRT, AZT causes cytopenias and lipodystrophy. AZT-5′-triphosphate (AZTTP) is a known inhibitor of the mitochondrial polymerase γ and has been targeted as the source of the mitochondrial DNA depletion. However, in previous work from this laboratory with isolated rat heart mitochondria, AZT phosphorylation beyond AZT-5′-monophosphate (AZTMP) was not detected. Rather, AZT was shown to be a more potent inhibitor of thymidine phosphorylation (50% inhibitory concentration (IC 50) of 7.0 ± 1.0 μM) than AZTTP is of polymerase γ (IC 50 of >100 μM), suggesting that depletion of mitochondrial stores of TTP may limit replication. This work has investigated whether an identical mechanism might account for the hepatotoxicity seen with long-term use of AZT. Isolated rat liver mitochondria were incubated with labeled thymidine or AZT, and the rate and extent of phosphorylation were determined by HPLC analysis of acid-soluble extracts of the incubated mitochondria. The results showed that in the phosphorylation of thymidine to TMP, liver mitochondria exhibit a higher V max and K m than heart mitochondria, but otherwise heart and liver mitochondria display similar kinetics. AZT is phosphorylated to AZTMP, but no further phosphorylated forms were detected. In addition, AZT inhibited the production of TTP, with an IC 50 of 14.4 ± 2.6 μM AZT. This is higher, but comparable to, the results seen in isolated rat heart mitochondria.

A Novel Suicide Gene Therapy Approach for Reduction of GvHD Using Lentiviral Delivery of a Modified Human Thymidylate Monophosphate Kinase.

The infusion of donor lymphocytes in allogenic BMT recipients provides potent antitumor activity to treat recurrent malignancies. One complication, however, is severe GvHD, which is mediated by T cells in the graft. One approach to control GvHD is to employ ‘suicide' gene therapy. In the customary adaptation of this approach, the herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene, combined with the antiviral prodrug ganciclovir (GCV), is used to control GvHD after introduction of this suicide gene into donor T lymphocytes. However, the efficiency of HSV1-tk is suboptimal and the issue of host immunogenicity against this heterologous effector gene product can hamper outcomes. In addition, prophylactic GCV is often used to control cytomegalovirus infection after BMT. This confounds the broad clinical implementation of this approach. Our novel suicide gene therapy strategy combines the use of human thymidylate monophosphate kinase (tmpk) and the prodrug Zidovudine (AZT) in a lentiviral vector (LV) format. Since tmpk is endogenously expressed in human cells, immunogenic responses will be limited. We constructed a LV expression system carrying wild-type or one of two modified forms of tmpk. These engineered tmpk mutants (F105Y and BL) show substantially increased catalytic conversion of AZT compared to wild-type tmpk. Our vector also includes a truncated form of human CD19 (hCD19Δ), not normally expressed on the T cell lineage, that can be used to enrich and track transduced cells. Highly efficient (95%) transduction of Jurkat cells (human T cell leukemia line) was attained by a single infection with our LVs (MOI of 10). Transduced cells were efficiently and selectively killed in a dose-dependent manner by AZT (IC50 of 2 μM), while wild-type tmpk transduced cells were unaffected by AZT up to 100 μM. In response to AZT treatment, the apoptotic cell indices of cells transduced with wild-type tmpk, F105Y, or BL were 6.2 ± 0.3 %, 40.7 ± 1.7 %, and 46.1 ± 4.6 %, respectively (n=3). We next established by HPLC that cells transduced with a LV encoding a mutant form of tmpk effectively convert AZT into its active anti-metabolite form, AZT-triphosphate (AZT-TP). Intracellular ratio of AZT-TP to AZT-monophosphate (MP) is 11.3 in cells transduced with a LV encoding the BL mutant of tmpk, compared to 0.02 in non-transduced cells and 0.10 in wild-type tmpk transduced cells. Our findings also revealed that following incubation with indirubin-3′-oxime, which inhibits cellular proliferation, and AZT treatment, transduced cells were successfully killed. Thus the cytotoxic mechanism differs from HSV1-tk mediated cell killing and is independent of cell proliferation. We also succeeded in the infection of primary mouse and human T cells to over 40% and 70% transduction efficiency, respectively. Lastly, we have shown that in vivo growth of tumor cells transduced with these mutant tmpk LVs was totally inhibited by treatment with AZT. These results demonstrate that our novel suicide gene therapy system has significant potential for many clinical applications.

Effect of Cell Cycle Arrest on the Activity of Nucleoside Analogues against Human Immunodeficiency Virus Type 1

Human immunodeficiency virus (HIV) reverse transcription can be notably affected by cellular activation, differentiation, and division. We hypothesized that changes in the cell cycle could also affect HIV susceptibility to nucleoside analogues, which compete with natural nucleotides for incorporation into viral DNA and inhibit viral replication through premature termination of reverse transcription. Proliferating HeLa-derived indicator cells were arrested in the S/G2 phase with etoposide, a topoisomerase II inhibitor, or in the G1/S phase with aphidicolin, a polymerase alpha inhibitor. Cell cycle arrest by both agents induced a remarkable decrease in HIV susceptibility to zidovudine (AZT). This decrease was seen both with a single-cycle infectivity assay and with a viral DNA quantitation assay, indicating that the effect of cell cycle arrest was exerted at the reverse transcription stage. The increase in the 50% inhibitory concentration (IC50) seen with arrested cells was strongest for AZT (23-fold) and stavudine (21-fold) but more modest for other drugs (lamivudine, 11-fold; dideoxyinosine, 7-fold; and nevirapine, 3-fold). In drug-resistant reverse transcriptase mutants, the increase in AZT IC50 (relative to that in dividing cells) was most prominent with a Q151M mutant and was comparable to the wild type in other drug-resistant mutants. Quantitation of intracellular pools of dTTP and AZT 5'-triphosphate (AZTTP) showed that etoposide treatment induced a significant increase in intracellular dTTP and consequently a decrease in AZTTP/dTTP ratios, suggesting that the decrease in viral susceptibility to AZT was caused by reduced incorporation of the analogue into nascent viral DNA. These results emphasize the importance of cellular proliferation and deoxynucleoside triphosphate metabolism in HIV susceptibility to nucleoside analogues and underscore the need to study the activities of drugs of this class with natural target cells under physiological conditions of activation and proliferation.

Cyclic Induction of Senescence with Intermittent AZT Treatment Accelerates both Apoptosis and Telomere Loss

3'-azido-2',3'-dideoxythymidine (AZT) is phosphorylated intracellularly to 3'-azido-3'-deoxythymidine-5'-triphosphate (AZT-TP), which is incorporated into telomeric DNA, thereby blocking chain elongation. AZT is also known to inhibit reverse transcriptase, as well as other cellular enzymes including DNA polymerase gamma, thymidine kinase, and telomerase. We induced cancer cell senescence by treating MCF-7 cells with AZT in dosages of IC10 and IC20 for an extended period (about 120 population doublings (PD)). We then investigated the sequential changes in cellular growth, expression of telomerase subunits and transcription factors (c-Myc, Mad1), telomerase activity and telomere length. Senescence, apoptosis, growth delay, inhibition of telomerase activity and shortening of telomere length were all observed in a dose- and time-dependent manner. After the onset of senescence, the apoptosis rate increased slowly during early PDs. In contrast to senescence, the apoptotic rate showed little change after AZT removal, while it increased suddenly and significantly in a dose-dependent manner upon the second introduction of AZT. Continuous shortening of the telomeric length was observed with AZT, and, upon re-exposure to AZT, shortening of the telomere occurred more rapidly than with first exposure. Of the telomerase subunits, telomerase reverse transcriptase (hTERT) and c-Myc were the first to show a reduction in activity after AZT treatment, followed by changes in hTER , Mad1 and hTEP-1. Cyclic treatment with AZT initially suppressed hTERT and c-Myc, followed by suppression of hTER, Mad1 and hTEP-1. Furthermore, the treatment accelerated both telomere loss and apoptosis, even when administered at a senescence-inducing dosage level.

Nucleotide specificity of HIV-1 reverse transcriptases with amino acid substitutions affecting Ala-114

Ala-114, together with Asp-113, Tyr-115 and Gln-151, form the pocket that accommodates the 3'-OH of the incoming dNTP in the HIV-1 RT (reverse transcriptase). Four mutant RTs having serine, glycine, threonine or valine instead of Ala-114 were obtained by site-directed mutagenesis. While mutants A114S and A114G retained significant DNA polymerase activity, A114T and A114V showed very low catalytic efficiency in nucleotide incorporation assays, due to their high apparent K(m) values for dNTP. Discrimination between AZTTP (3'-azido-3'-deoxythymidine triphosphate) and dTTP was not significantly affected by mutations A114S and A114G in assays carried out with heteropolymeric template/primers. However, both mutants showed decreased susceptibility to AZTTP when poly(rA)/(dT)16 was used as substrate. Steady-state kinetic analysis of the incorporation of ddNTPs compared with dNTPs showed that substituting glycine for Ala-114 produced a 5-6-fold increase in the RT's ability to discriminate against ddNTPs (including the physiologically relevant metabolites of zalcitabine and didanosine), a result that was confirmed in primer-extension assays. In contrast, A114S and A114V showed wild-type ddNTP/dNTP discrimination efficiencies. Discrimination against ribonucleotides was not affected by mutations at position 114. Misinsertion and mispair extension fidelity assays as well as determinations of G-->A mutation frequencies using a lacZ complementation assay showed that, unlike Tyr-115 or Gln-151 mutants, the fidelity of HIV-1 RT was not largely affected by substitutions of Ala-114. The role of the side-chain of Ala-114 in ddNTP/dNTP discrimination appears to be determined by its participation in van der Waals interactions with the ribose moiety of the incoming nucleotide.

Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor Combinations on HIV-1 Reverse-Transcriptase Activities

Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H) activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. The nucleoside analogue 3'-azido-3'-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid (DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.

Synthesis of AZT 5‘-Triphosphate Mimics and Their Inhibitory Effects on HIV-1 Reverse Transcriptase

In search of active nucleoside 5'-triphosphate mimics, we have synthesized a series of AZT triphosphate mimics (AZT P3Ms) and evaluated their inhibitory effects on HIV-1 reverse transcriptase as well as their stability in fetal calf serum and in CEM cell extracts. Reaction of AZT with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, followed by treatment of the phosphite intermediate 2 with pyrophosphate analogues, yielded the cyclic triphosphate intermediates 4b-4f, which were subjected to boronation and subsequent hydrolysis to give AZT 5'-alpha-borano-beta,gamma-bridge-modified triphosphates 6b-6f in moderate to good yields. Reaction of the cyclic intermediate 4d with iodine, followed by treatment with a series of nucleophiles, afforded the AZT 5'-beta,gamma-difluoromethylene-gamma-substituted triphosphates (7b-7i). Several different types of AZT P3Ms containing alpha-P-thio (or dithio) and beta,gamma-difluoromethylene (13,14), alpha,beta-difluoromethylene and gamma-P-methyl(or phenyl) (15,16), and alpha-borano-beta,gamma-difluoromethylene and gamma-O-methyl/phenyl (11,12) were also synthesized. The effectiveness of the compounds as inhibitors of HIV-1 reverse transcriptase was determined using a fluorometric assay and a poly(A) homopolymer as a template. A number of AZT P3Ms exhibited very potent inhibition of HIV-1 reverse transcriptase. Modifications at the beta,gamma-bridge of triphosphate rendered the AZT P3Ms 6b-6f with varied activities (K(i) from 9.5 to >>500 nM) while modification at the alpha,beta-bridge of triphosphate led to weak AZT P3M inhibitors. The results imply that the AZT P3Ms were substrate inhibitors, as is AZT triphosphate. The most active compound, AZT 5'-alpha-R(p)()-borano-beta,gamma-(difluoromethylene)triphosphate (AZT 5'-alphaB-betagammaCF(2)TP) (6d-I), is as potent as AZT triphosphate with a K(i)() value of 9.5 nM and at least 20-fold more stable than AZT triphosphate in the serum and cell extracts. Therefore, for the first time, a highly active and stable nucleoside triphosphate mimic has been identified, which is potentially useful as a new type of antiviral drug. The promising triphosphate mimic, 5'-alpha-borano-beta,gamma-(difluoromethylene)triphosphate, is expected to be valuable to the discovery of nucleotide mimic antiviral drugs.

Azidothymidine promotes free radical generation by activated macrophages and hydrogen peroxide-iron-mediated oxidation in a cell-free system.

Azidothymidine (AZT) and AZT monophosphate (AZT-MP) in concentrations as low as 10 and 50 microM, respectively, promote oxidation of chemically deacetylated 2',7'-dichlorodihydrofluorescein (DCDHF) to 2',7'-dichlorofluorescein (DCF) by rat peritoneal macrophages activated with latex. Cells were incubated with AZT and AZT-MP for 18 h, washed out from residual AZT or AZT-MP and activated with latex for 30 or 60 min in the presence of DCDHF. Latex-activated cells oxidize DCDHF extracellularly due to release of hydrogen peroxide and low-molecular iron complexes, which is verified using catalase, desferal and the peroxidase inhibitor sodium azide. AZT and AZT-MP increase DCDHF oxidation due to additional release of hydrogen peroxide as demonstrated by catalase inhibition of DCDHF oxidation and direct H(2)O(2) measurement. Thymidine and thymidine phosphates did not show any effect on macrophage activation. In separate experiments we evaluated the in vitro prooxidant activity of AZT, AZT-MP, AZT triphosphate (AZT-TP), AZT glucuronide (GAZT) and 3'-amino-3'-deoxythymidine (AMT) in a cell-free system using the hydrogen peroxide-iron-mediated oxidation of DCDHF. Under these conditions, AZT and AZT phosphates exhibit a prooxidant effect in concentrations as low as 100 microM. Furthermore, GAZT is a less effective prooxidant and AMT acts like an antioxidant. Thymidine did not show any effect.

Phosphorylation of Thymidine and AZT in Heart Mitochondria: Elucidation of a Novel Mechanism of AZT Cardiotoxicity

Antiretroviral nucleoside analogs used in highly active antiretroviral therapy (HAART) are associated with cardiovascular and other tissue toxicity associated with mitochondrial DNA depletion, suggesting a block in mitochondrial (mt)-DNA replication. Because the triphosphate forms of these analogs variably inhibit mt-DNA polymerase, this enzyme has been promoted as the major target of toxicity associated with HAART. We have used isolated mitochondria from rat heart to study the mitochondrial transport and phosphorylation of thymidine and AZT (azidothymidine, or zidovudine), a component used in HAART. We demonstrate that isolated mitochondria readily transport thymidine and phosphorylate it to thymidine 5'-triphosphate (TTP) within the matrix. Under identical conditions, AZT is phosphorylated only to AZT-5'-monophosphate (AZT-MP). The kinetics of thymidine and AZT suggest negative cooperativity of substrate interaction with the enzyme, consistent with work by others on mitochondrial thymidine kinase 2. Results show that TMP and AZT-MP are not transported across the inner membrane, suggesting that AZT-MP may accumulate with time in the matrix. Given the lack of AZT-5'-triphosphate (AZT-TP), it seems unlikely that the toxicity of AZT in the heart is mediated by AZT-TP inhibition of DNA polymerase gamma. Rather, our work shows that AZT is a potent inhibitor of thymidine phosphorylation in heart mitochondria, having an inhibitory concentration (IC)(50) of 7.0 +/- 0.9 microM. Thus, the toxicity of AZT in some tissues may be mediated by disrupting the substrate supply of TTP for mt-DNA replication.

Telomerase Inhibition Using Azidothymidine in the HT-29 Colon Cancer Cell Line

We investigated the effects of telomerase inhibition by using the reverse transcriptase inhibitor azidothymidine (AZT) in the human colorectal cancer cell line HT-29 in the presence and absence of 5-fluorouracil (5-FU). HT-29 cells were cultured in the presence of AZT. Telomerase activity was measured by using the telomerase repeat amplification protocol. Telomere length was determined by Southern analysis. The colorimetric microtiter assay was performed to determine the cytotoxic effects of AZT, alone and in combination with 5-FU. The presence of 3'-azido-3'-deoxythymidine triphosphate (AZT-TP) effectively inhibited telomerase extracted from HT-29 cells. HT-29 cells cultured with 125 microM of AZT underwent fewer total population doublings over 91 days. Southern analysis revealed that telomere attrition occurred within this period. Exposure to 125 microM of AZT resulted in slightly reduced viability (10%) of HT-29 cells. However, the presence of AZT increased 5-FU cytotoxicity, suggesting that the effects of these two drugs are synergistic. The data are consistent with telomerase inhibition having growth-inhibitory effects in addition to those predicted to accompany loss of telomere function. Further studies using specific small-molecule inhibitors will confirm whether the growth-inhibitory and 5-FU-sensitivity effects seen here are a direct result of telomerase inhibition.