An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors

Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-γ hypothesis states that this toxicity stems from the analogs'' inhibition of the mitochondrial DNA polymerase (polymerase-γ) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-γ with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication.     

Toxicity of nucleoside analogues used to treat AIDS and the selectivity of the mitochondrial DNA polymerase

Incorporation of nucleoside analogues by the mitochondrial DNA polymerase has been implicated as the primary cause underlying many of the toxic side effects of these drugs in HIV therapy. Recent success in reconstituting recombinant human enzyme has afforded a detailed mechanistic analysis of the reactions governing nucleotide selectivity of the polymerase and the proofreading exonuclease. The toxic side effects of nucleoside analogues are correlated with the kinetics of incorporation by the mitochondrial DNA polymerase, varying over 6 orders of magnitude in the sequence zalcitabine (ddC) > didanosine (ddI metabolized to ddA) > stavudine (d4T) > lamivudine (3TC) > tenofovir (PMPA) > zidovudine (AZT) > abacavir (metabolized to carbovir, CBV). In this review, we summarize our current efforts to examine the mechanistic basis for nucleotide selectivity by the mitochondrial DNA polymerase and its role in mitochondrial toxicity of nucleoside analogues used to treat AIDS and other viral infections. We will also discuss the promise and underlying challenges for the development of new analogues with lower toxicity.     

Structural determinants in human DNA polymerase gamma account for mitochondrial toxicity from nucleoside analogs

Although antiviral nucleoside analog therapy successfully delays progression of HIV infection to AIDS, these drugs cause unwelcome side-effects by inducing mitochondrial toxicity. We and others have demonstrated that the mitochondrial polymerase, DNA polymerase gamma (pol gamma), participates in mitochondrial toxicity by incorporating these chain-terminating antiviral nucleotide analogs into DNA. Here, we explore the role of three highly conserved amino acid residues in the active site of human pol gamma that modulate selection of nucleotide analogs as substrates for incorporation. Sequence alignments, crystal structures and mutagenesis studies of family A DNA polymerases led us to change Tyr951 and Tyr955 in polymerase motif B to Phe and Ala, and Glu895 in polymerase motif A was changed to Ala. The mutant polymerases were tested for their ability to incorporate natural nucleotides and the five antiviral nucleoside analogs currently approved for antiviral therapy: AZT, ddC, D4T, 3TC and carbovir. Steady-state kinetic analysis of the pol gamma derivatives with the normal and antiviral nucleotides demonstrated that Tyr951 is largely responsible for the ability of pol gamma to incorporate dideoxynucleotides and D4T-MP. Mutation of Tyr951 to Phe renders the enzyme resistant to dideoxynucleotides and D4T-TP without compromising the activity of the polymerase. Alteration of Glu895 and Tyr955 to Ala had the largest effect on overall polymerase activity with normal nucleotides, producing dramatic increases in K(m(dNTP)) and large decreases in k(cat). Mutation of Tyr955 in pol gamma causes the degenerative disease progressive external ophthalmoplegia in humans, and we show that this residue partially accounts for the ability of pol gamma to incorporate D4T-MP and carbovir. Alteration of Glu895 to Ala slightly increased discrimination against dideoxynucleotides and D4T-TP. The mechanisms by which pol gamma selects certain nucleotide analogs are discussed.     

5-Fluoro-2'-deoxyuridine has effects on mitochondria in CEM T-lymphoblast cells

Fluoropyrimidines are useful anticancer agents and the compound 5-fluoro-2'-deoxyuridine (FdUrd) plays an important role in chemotherapy of colon cancers. Several nucleoside analogs, such as 3'-azido-2',3'-dideoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC), can be incorporated into and cause depletion of mitochondrial DNA (mtDNA). These drugs are known to cause mitochondrial toxicity after prolonged treatment in patients. In this study we demonstrate that FdUrd reduces the mtDNA content and the expression level of the mtDNA encoded cytochrome c oxidase (COX II) in a CEM T-lymphoblastic cell line.     

The evidence that the DNC (SLC25A19) is not the mitochondrial deoxyribonucleotide carrier

We review the evidence that the function of the SLC25A19 gene product, previously identified as the mitochondrial deoxyribonucleotide carrier (DNC), is actually the transport of thiamine pyrophosphate. This evidence comes from enzyme kinetics, homologous yeast protein alignments, gene knockout studies, and clinical samples from Amish Microcephaly patients. This diverse body of evidence consistently points to the conclusion that SLC25A19 is not the true mitochondrial DNC gene. The identification of the correct mitochondrial DNC is important for research on the genetic diseases of mitochondrial DNA maintenance and the toxicity experienced by many HIV patients undergoing antiretroviral therapy involving nucleoside analogs.     

5‐Fluoro‐2′‐Deoxyuridine Has Effects on Mitochondria in CEM T‐Lymphoblast Cells

Fluoropyrimidines are useful anticancer agents and the compound 5‐fluoro‐2′‐deoxyuridine (FdUrd) plays an important role in chemotherapy of colon cancers. Several nucleoside analogs, such as 3′‐azido‐2′,3′‐dideoxythymidine (AZT) and 2′,3′‐dideoxycytidine (ddC), can be incorporated into and cause depletion of mitochondrial DNA (mtDNA). These drugs are known to cause mitochondrial toxicity after prolonged treatment in patients. In this study we demonstrate that FdUrd reduces the mtDNA content and the expression level of the mtDNA encoded cytochrome c oxidase (COX II) in a CEM T‐lymphoblastic cell line.     

Synthesis and antiviral activity of 3′-azido-3′-deoxythymidine triphosphate distearoylglycerol: a novel phospholipid conjugate of the anti-HIV agent AZT

Phospholipid conjugates of 3′-azido-3′-deoxythymidine (AZT) show activity against the human immunodeficiency virus (HIV) in vitro. In a previous report (K.Y. Hostetler, L.M. Stuhmiller, B.H.M. Lenting, H. van den Bosch and D.D. Richman (1991), J. Biol. Chem. 265, 6112–6117) the syntheses and anti-HIV activities of AZT mono- and diphosphate diglyceride have been described. We now report on the synthesis, characterization and biological activity of 3′-azido-3′-deoxythymidine triphosphate distearoylglycerol (AZTTP-DSG). The compound was prepared by the condensation of AZT diphosphate with distearoylphosphatidic acid morpholidate in anhydrous pyridine at room temperature and purified by means of high-performance liquid chromatography using a silica column. Characterization was performed with ³¹P-NMR and IR analyses and determination of the fatty acid, phosphorus and nucleoside content of the product. AZTTP-DSG inhibited HIV-1 replication in both CEM and HT4-6C cells at a level intermediate in potency between its mono- and diphosphate analogs. The IC₅₀ values of AZTTP-DSG were 0.33 and 0.79 μM in these two cell lines, respectively. In addition, AZTTP-DSG was less toxic to CEM cells in vitro than the other AZT liponucleotides and reduced viable cell numbers in this cell type by 50% at 1000 μM. Initial studies on the metabolism of AZTTP-DSG revealed that both AZT and AZT monophosphate were liberated from the lipid pro-drug by a rat liver mitochondrial enzyme preparation. These phospholipid derivatives of AZT nucleotides represent pro-drugs for the intracellular delivery of phosphorylated antiviral nucleoside analogs.     

Toxicological evaluation of zidovudine and novel chalcogen derivatives in Drosophila melanogaster

Zidovudine (AZT) is the most commonly prescribed antiviral drug for the treatment of human immunodeficiency virus (HIV) infection. However, its chronic administration causes toxic side effects limiting its use. This study aimed to evaluate the toxicity of different concentrations of AZT and novel chalcogen derivatives (7A, 7D, 7G, 7K, 7M) on locomotion, mitochondrial dysfunction, acetylcholinesterase (AChE) activity, and production of reactive oxygen species (ROS) in adult Drosophila melanogaster. Our results show that AZT and its derivative 7K at a concentration of 10 μM impaired flies' locomotor behavior. Furthermore, AZT and the derivatives 7K, 7A, and 7M induced mitochondrial dysfunction observed by a decrease in oxygen flux through mitochondrial complexes I and II. Neither of the compounds tested affected AChE activity or ROS production in flies. According to these data, AZT derivatives presented the following decreasing order of toxicity: 7K > AZT > 7G > 7A > 7M > 7D. Based on the chemical structure, it is possible to infer that the presence of the seleno-phenyl group in 7A and 7G increases their toxicity compared to compounds 7D and 7M. In addition, compounds 7G, 7M, and 7K with three carbon atoms as spacer were more toxic than analogs containing one carbon atom (7A and 7D). Finally, the insertion of a p-methoxyl group enhances toxicity (7K). Based on these results, excepting 7K, all other chalcogen derivatives presented lower toxicity than AZT and are potential drug candidates.     

Identification of pheromones in mouse urine by head-space solid phase microextraction followed by gas chromatography-mass spectrometry

Transmission of HIV from mother to infant can be effectively prevented by zidovudine (3'-azido-3'-deoxythymidine; AZT) alone or in combination with other anti-retroviral drugs; however, significant evidence for genotoxicity, including transplacental carcinogenicity in mice, has been reported for AZT. A method, based upon solid phase extraction (SPE) in the 96-well format, gradient liquid chromatography (LC), and electrospray mass spectrometry (MS), was developed and validated to measure serum concentrations in maternal C57BL/6N and fetal B6C3F1 mice of the nucleoside analogs AZT, lamivudine ((-)2',3'-dideoxy-3'-thiacytidine; 3TC), and several metabolites selected based on importance in detoxification and bioactivation reactions. After intravenous (i.v.) and oral dosing with either 400 mg/kg AZT or 200 mg/kg 3TC, pharmacokinetics were determined for AZT, AZT-5'-glucuronide, 3'-amino-3'-deoxythymidine (AMT), AZT-5'-phosphate, 3TC, and 3TC-5'-phosphate in serum of adult female mice. Pharmacokinetics were also determined in spleen for AZT-5'-phosphate and 3TC-5'-phosphate following i.v. dosing. In addition, a preliminary assessment was made of placental transfer of AZT and 3TC and the presence of metabolites in the fetal compartment. The method described provides a means to evaluate thoroughly metabolism and disposition of anti-retroviral nucleoside analogs in maternal and fetal mice for comprehensive studies of genotoxicity.     

Cross-resistance to anti-HIV nucleoside analogs in multidrug-resistant human cells

Human multidrug-resistant K562/ADM cells showed 12-fold and 31-fold resistance to AZT (3'-azido-2', 3'dideoxythymidine) and DDC (2', 3'-dideoxycytidine), respectively. Other multidrug-resistant human cells CEM/VLB100 and AdrRMCF-7 also showed resistance to these nucleoside analogs. However, verapamil (10 microM) failed to reverse the resistance to the nucleoside analogs. Accumulation of [3H]AZT in human multidrug-resistant K562/ADM, CEM/VLB100 and AdrRMCF-7 cells decreased by 23, 35, and 42% respectively, as compared to their parental cells. These results suggest that anti-HIV nucleoside analogs including AZT, DDC could be transported by outward drug-transport system in the multidrug-resistant cells.     

Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase

To examine the role of the mitochondrial polymerase (Pol gamma) in clinically observed toxicity of nucleoside analogs used to treat AIDS, we examined the kinetics of incorporation catalyzed by Pol gamma for each Food and Drug Administration-approved analog plus 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU), beta-L-(-)-2',3'-dideoxy-3'-thiacytidine (-)3TC, and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). We used recombinant exonuclease-deficient (E200A), reconstituted human Pol gamma holoenzyme in single turnover kinetic studies to measure K(d) (K(m)) and k(pol) (k(cat)) to estimate the specificity constant (k(cat)/K(m)) for each nucleoside analog triphosphate. The specificity constants vary more than 500,000-fold for the series ddC > ddA (ddI) > 2',3'-didehydro-2',3'-dideoxythymidine (d4T) > (+)3TC > (-)3TC > PMPA > azidothymidine (AZT) > Carbovir (CBV). Abacavir (prodrug of CBV) and PMPA are two new drugs that are expected to be least toxic. Notably, the higher toxicities of d4T, ddC, and ddA arose from their 13-36-fold tighter binding relative to the normal dNTP even though their rates of incorporation were comparable with PMPA and AZT. We also examined the rate of exonuclease removal of each analog after incorporation. The rates varied from 0.06 to 0.0004 s(-1) for the series FIAU > (+)3TC approximately equal to (-)3TC > CBV > AZT > PMPA approximately equal to d4T > ddA (ddI) > ddC. Removal of ddC was too slow to measure (<0.00002 s(-1)). The high toxicity of dideoxy compounds, ddC and ddI (metabolized to ddA), may be a combination of high rates of incorporation and ineffective exonuclease removal. Conversely, the more effective excision of (-)3TC, CBV, and AZT may contribute to lower toxicity. FIAU is readily extended by the next correct base pair (0.13 s(-1)) faster than it is removed (0.06 s(-1)) and, therefore, is stably incorporated and highly mutagenic. We define a toxicity index for chain terminators to account for relative rates of incorporation versus removal. These results provide a method to rapidly screen new analogs for potential toxicity.     

Insights into the molecular mechanism of mitochondrial toxicity by AIDS drugs

Several of the nucleoside analogs used in the treatment of AIDS exhibit a delayed clinical toxicity limiting their usefulness. The toxicity of nucleoside analogs may be related to their effects on the human mitochondrial DNA polymerase (Pol gamma), the polymerase responsible for mitochondrial DNA replication. Among the AIDS drugs approved by the FDA for clinical use, two are modified cytosine analogs, Zalcitabine (2',3'-dideoxycytidine (ddC)) and Lamivudine (beta-d-(+)-2',3'-dideoxy-3'-thiacytidine ((-)3TC])). (-)3TC is the only analog containing an unnatural l(-) nucleoside configuration and is well tolerated by patients even after long term administration. In cell culture (-)3TC is less toxic than its d(+) isomer, (+)3TC, containing the natural nucleoside configuration, and both are considerably less toxic than ddC. We have investigated the mechanistic basis for the differential toxicity of these three cytosine analogs by comparing the effects of dideoxy-CTP), (+)3TC-triphosphate (TP), and (-)3TC-TP on the polymerase and exonuclease activities of recombinant human Pol gamma. This analysis reveals that Pol gamma incorporates (-)3TC-triphosphate 16-fold less efficiently than the corresponding (+)isomer and 1140-fold less efficiently than dideoxy-CTP, showing a good correlation between incorporation rate and toxicity. The rates of excision of the incorporated analogs from the chain-terminated 3'-end of the DNA primer by the 3'-5'-exonuclease activity of Pol gamma were similar (0.01 s(-)1) for both 3TC analogs. In marked contrast, the rate of exonuclease removal of a ddC chain-terminated DNA occurs at least 2 orders of magnitude slower, suggesting that the failure of the exonuclease to remove ddC may play a major role in its greater toxicity. This study demonstrates that direct analysis of the mitochondrial DNA polymerase structure/function relationships may provide valuable insights leading to the design of less toxic inhibitors.     

Down-regulation of mitochondrial thymidine kinase 2 and deoxyguanosine kinase by didanosine: Implication for mitochondrial toxicities of anti-HIV nucleoside analogs

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial rate limiting phosphorylation of deoxynucleosides and are essential enzymes for mitochondrial function. Chemotherapy using nucleoside analogs is often associated with mitochondrial toxicities. Here we showed that incubation of U2OS cells with didanosine (ddI, 2′,3′-dideoxyinosine), a purine nucleoside analog used in the highly active antiretroviral therapy (HAART), led to selective degradation of both mitochondrial TK2 and dGK while the cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) were not affected. Addition of guanosine to the ddI-treated cells prevented the degradation of mitochondrial TK2 and dGK. The levels of intracellular reactive oxygen species and protein oxidation in ddI-treated and control cells were also measured. The results suggest that down-regulation of mitochondrial TK2 and dGK may be a mechanism of mitochondrial toxicity caused by antiviral and anticancer nucleoside analogs.     

Synthesis and anti-HIV activity of beta-D-3'-azido-2',3'-unsaturated nucleosides and beta-D-3'-azido-3'-deoxyribofuranosylnucleosides

Since the discovery of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T) as potent and selective inhibitors of the replication of human immunodeficiency virus (HIV), there has been a growing interest for the synthesis of 2',3'-didehydro-2',3'dideoxynucleosides with electron withdrawing groups on the sugar moiety. Here we described an efficient method for the synthesis of such nucleoside analogs bearing structural features of both AZT and d4T The key intermediate, 3-azido-1,2-bis-O-acetyl-5-O-benzoyl-3-deoxy-D-ribofuranose, 5 was synthesized from commercially available D-xylose in five steps, from which a series of pyrimidine and purine nucleosides were synthesized in high yields. The resultant protected nucleosides were converted to target nucleosides using appropriate chemical modifications. The final nucleosides were evaluated as potential anti-HIV agents.     

TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs

Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl–DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs—acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC)—we show that TDP1 is capable of removing the covalently linked corresponding CTNAs from DNA 3′-ends. We also show that Tdp1^−/− cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing CTNA-induced DNA damage. As AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We find that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in Tdp1^−/− cells. Thus, TDP1 seems to be critical for repairing nuclear and mitochondrial DNA damage caused by CTNAs.