Structural basis for processivity and antiviral drug toxicity in human mitochondrial DNA replicase.

The human DNA polymerase gamma (Pol γ) is responsible for DNA replication in mitochondria. Pol γ is particularly susceptible to inhibition by dideoxynucleoside-based inhibitors designed to fight viral infection. Here, we report crystal structures of the replicating Pol γ-DNA complex bound to either substrate or zalcitabine, an inhibitor used for HIV reverse transcriptase. The structures reveal that zalcitabine binds to the Pol γ active site almost identically to the substrate dCTP, providing a structural basis for Pol γ-mediated drug toxicity. When compared to the apo form, Pol γ undergoes intra- and inter-subunit conformational changes upon formation of the ternary complex with primer/template DNA and substrate. We also find that the accessory subunit Pol γB, which lacks intrinsic enzymatic activity and does not contact the primer/template DNA directly, serves as an allosteric regulator of holoenzyme activities. The structures presented here suggest a mechanism for processivity of the holoenzyme and provide a model for understanding the deleterious effects of Pol γ mutations in human disease. Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity.

Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML.

Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2'3'-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML.

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.

Generation of Mammalian Cells Devoid of Mitochondrial DNA (ρ0 cells).

To cope with DNA damage, mitochondria have developed a pathway whereby severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules can be discarded and degraded, after which new molecules are synthesized using intact templates. In this unit, we describe a method that harnesses this pathway to eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1) in mitochondria. We also provide alternate protocols for mtDNA elimination using either combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC) or clustered regulatory interspersed short palindromic repeat (CRISPR)-Cas9-mediated knockout of TFAM or other genes essential for mtDNA replication. Support protocols detail approaches for several processes: (1) genotyping ρ0 cells of human, mouse, and rat origin by polymerase chain reaction (PCR); (2) quantification of mtDNA by quantitative PCR (qPCR); (3) preparation of calibrator plasmids for mtDNA quantification; and (4) quantification of mtDNA by direct droplet digital PCR (dddPCR). © 2023 Wiley Periodicals LLC. Basic Protocol: Inducing mtDNA loss with mUNG1 Alternate Protocol 1: Generation of ρ0 cells by mtDNA depletion with EtBr and ddC Alternate Protocol 2: Generation of ρ0 cells by knocking out genes critical for mtDNA replication Support Protocol 1: Genotyping ρ0 cells by DirectPCR Support Protocol 2: Determination of mtDNA copy number by qPCR Support Protocol 3: Preparation of calibrator plasmid for qPCR Support Protocol 4: Determination of mtCN by direct droplet digital PCR (dddPCR).

Transgenic cardiac-targeted overexpression of human thymidylate kinase.

Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes phosphorylation of thymidine monophosphate to thymidine diphosphate. TMPK also mediates phosphorylation of monophosphates of thymidine nucleoside analog (NA) prodrugs on the pathway to their active triphosphate antiviral or antitumor moieties. Novel transgenic mice (TG) expressing human (h) TMPK were genetically engineered using the alpha-myosin heavy chain promoter to drive its cardiac-targeted overexpression. In '2 by 2' protocols, TMPK TGs and wild-type (WT) littermates were treated with the NA zidovudine (a deoxythymidine analog, 3'-azido-3'deoxythymidine (AZT)) or vehicle for 35 days. Alternatively, TGs and WTs were treated with a deoxycytidine NA (racivir, RCV) or vehicle. Changes in mitochondrial DNA (mtDNA) abundance and mitochondrial ultrastructure were defined quantitatively by real-time PCR and transmission electron microscopy, respectively. Cardiac performance was determined echocardiographically. Results showed TMPK TGs treated with either AZT or RCV exhibited decreased cardiac mtDNA abundance. Cardiac ultrastructural changes were seen only with AZT. AZT-treated TGs exhibited increased left ventricle (LV) mass. In contrast, LV mass in RCV-treated TGs and WTs remained unchanged. In all cohorts, LV end-diastolic dimension remained unchanged. This novel cardiac-targeted overexpression of hTMPK helps define the role of TMPK in mitochondrial toxicity of antiretrovirals.

Cellular pharmacology of the anti-hepatitis B virus agent beta-L-2',3'-didehydro-2',3'-dideoxy-N4-hydroxycytidine: relevance for activation in HepG2 cells.

Beta-l-2',3'-didehydro-2',3'-dideoxy-N(4)-hydroxycytidine (l-Hyd4C) was demonstrated to be an effective and highly selective inhibitor of hepatitis B virus (HBV) replication in HepG2.2.15 cells (50% effective dose [ED(50)] = 0.03 microM; 50% cytotoxic dose [CD(50)] = 2,500 microM). In the present study, we investigated the intracellular pharmacology of tritiated l-Hyd4C in HepG2 cells. l-[(3)H]Hyd4C was shown to be phosphorylated extensively and rapidly to the 5'-mono-, 5'-di-, and 5'-triphosphate derivatives. Other metabolites deriving from a reduction or removal of the NHOH group of l-Hyd4C could not be detected, although both reactions were described as the primary catabolic pathways of the stereoisomer ss-d-N(4)-hydroxycytidine in HepG2 cells. Also, the formation of liponucleotide metabolites, such as the 5'-diphosphocholine derivative of l-Hyd4C, as described for some l-deoxycytidine analogues, seems to be unlikely. After incubation of HepG2 cells with 10 microM l-[(3)H]Hyd4C for 24 h, the 5'-triphosphate accumulated to 19.4 +/- 2.7 pmol/10(6) cells. The predominant peak belonged to 5-diphosphate, with 43.5 +/- 4.3 pmol/10(6) cells. The intracellular half-life of the 5'-triphosphate was estimated to be 29.7 h. This extended half-life probably reflects a generally low affinity of 5'-phosphorylated l-deoxycytidine derivatives for phosphate-degrading enzymes but may additionally be caused by an efficient rephosphorylation of the 5'-diphosphate during a drug-free incubation. The high 5'-triphosphate level and its extended half-life in HepG2 cells are consistent with the potent antiviral activity of l-Hyd4C.

Structural studies of nucleoside analog and feedback inhibitor binding to Drosophila melanogaster multisubstrate deoxyribonucleoside kinase.

The Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (dNK; EC 2.7.1.145) has a high turnover rate and a wide substrate range that makes it a very good candidate for gene therapy. This concept is based on introducing a suicide gene into malignant cells in order to activate a prodrug that eventually may kill the cell. To be able to optimize the function of dNK, it is vital to have structural information of dNK complexes. In this study we present crystal structures of dNK complexed with four different nucleoside analogs (floxuridine, brivudine, zidovudine and zalcitabine) and relate them to the binding of substrate and feedback inhibitors. dCTP and dGTP bind with the base in the substrate site, similarly to the binding of the feedback inhibitor dTTP. All nucleoside analogs investigated bound in a manner similar to that of the pyrimidine substrates, with many interactions in common. In contrast, the base of dGTP adopted a syn-conformation to adapt to the available space of the active site.

Elimination of Mitochondrial DNA from Mammalian Cells.

To cope with DNA damage, mitochondria developed a pathway by which severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules are abandoned and degraded, and new molecules are resynthesized using intact templates, if available. In this unit, we describe a method that harnesses this pathway to completely eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1). We also provide an alternate protocol for mtDNA depletion using combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC). Support protocols detail approaches for (1) genotyping ρ° cells of human, mouse, and rat origin by PCR; (2) quantitation of mtDNA by quantitative PCR (qPCR); and (3) preparation of calibrator plasmids for mtDNA quantitation. © 2018 by John Wiley & Sons, Inc.

Insights on resistance to reverse transcriptase: the different patterns of interaction of the nucleoside reverse transcriptase inhibitors in the deoxyribonucleotide triphosphate binding site relative to the normal substrate.

It is presently known that the long-term failure in the treatment of AIDS with the currently available nucleotide reverse transcriptase inhibitors (NRTIs) is related to the development of resistance by reverse transcriptase (RT) at the binding or incorporation level or both, or subsequent to the nucleotide incorporation (excision). To achieve greater insight on the differential interactions of two NRTIs that are mainly discriminated by different mechanisms, 2',3'-didehydro-2',3'-dideoxythymidine-5'-triphosphate (d4TTP, that is, phosphorylated stavudine) and 2',3'-dideoxycytidine-5'-triphosphate (ddCTP, that is, phosphorylated zalcitabine), with the primer/template (p/t) and with the N binding site of reverse transcriptase (RT) in relation to the normal substrate (dNTP), we have conducted a series of molecular dynamics (MD) simulations. We propose that the different resistance profiles arise from the different conformations adopted by the inhibitors at the N site. d4TTP adopts an ideal conformation for catalysis because it forms an ion-dipole intramolecular interaction with the beta-phosphate oxygen of the triphosphate, as does the normal substrate. In ddCTP, the lack of this essential interaction results in a different, noncatalytic conformation.

Identification of residues involved in the specificity and regulation of the highly efficient multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster.

In contrast to all known deoxyribonucleoside kinases, a single highly efficient deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) is able to phosphorylate all precursor nucleosides for DNA synthesis. Dm-dNK was mutated in vitro by high-frequency random mutagenesis, expressed in the thymidine kinase-deficient Escherichia coli strain KY895 and clones were selected for sensitivity to the nucleoside analogs 1-beta-d-arabinofuranosylcytosine (AraC, Cytarabine), 3'-azido-2', 3'-dideoxythymidine (AZT, Zidovudine, Retrovir, 2', 3'-dideoxyadenosine (ddA) and 2',3'-dideoxycytidine (ddC, Zalcitabine, Hivid. Thirteen mutants with increased sensitivity compared to the wild-type Dm-dNK were isolated from a relatively small pool of less than 10,000 clones. Eight mutant Dm-dNKs increased the sensitivity of KY895 to more than one analog, and two of these mutants even to all four nucleoside analogs. Surprisingly, the mutations did not map to the five regions which are highly conserved among deoxyribonucleoside kinases. The molecular background of improved sensitivity was characterized for the double-mutant MuD (N45D, N64D), where the LD(100) value of transformed KY895 decreased 316-fold for AZT and more than 11-fold for ddC when compared to wild-type Dm-dNK. Purified recombinant MuD displayed higher K(m) values for the native substrates than wild-type Dm-dNK and the V(max) values were substantially lower. On the other hand, the K(m) and V(max) values for AZT and the K(m) value for ddC were nearly unchanged between MuD and wild-type Dm-dNK. Additionally, a decrease in feedback inhibition of MuD by thymidine triphosphate (TTP) was found. This study demonstrates how high-frequency mutagenesis combined with a parallel selection for desired properties provides an insight into the structure-function relationships of the multisubstrate kinase from D. melanogaster. At the same time these mutant enzymes exhibit properties useful in biotechnological and medical applications.

Multidrug-resistant HIV-1 reverse transcriptase: involvement of ribonucleotide-dependent phosphorolysis in cross-resistance to nucleoside analogue inhibitors.

Human immunodeficiency virus type 1 (HIV-1) strains having a dipeptide insertion between codons 69 and 70 of the viral reverse transcriptase (RT) have been observed in isolates from patients treated with 3'-azido-3'-deoxythymidine (AZT) and other nucleoside analogues. These viruses contain additional mutations related to drug resistance and display reduced susceptibility to most nucleoside analogue inhibitors, including AZT. The mechanism of AZT resistance implies an increased ability of the multidrug-resistant (SS) RT to remove AZT-monophosphate (AZTMP) from blocked primers through a nucleotide-dependent reaction. We show that its higher ATP-dependent phosphorolytic activity is also detectable with primers terminated with 2',3'-didehydro-3'-deoxythymidine-5'-monophosphate (d4TMP) or 2',3'-dideoxythymidine-5'-monophosphate (ddTMP), but is significantly reduced when the dipeptide insertion is deleted. Removal of AZTMP, d4TMP and ddTMP can be inhibited by the next complementary deoxynucleoside triphosphate (dNTP). AZTMP removal reactions catalysed by SS RT were highly resistant to dNTP inhibition (IC(50)>0.25mM), while unblocking of d4TMP- and ddTMP-terminated primers was around tenfold more sensitive to inhibition by the next complementary dNTP. Both SS and mutant 2S0S RTs were able to unblock and extend primers terminated with 2',3'-dideoxycytidine-5'-monophosphate (ddCMP) in the presence of ATP, albeit very poorly. Under these conditions, none of the RTs was able to remove 2',3'-dideoxy-3'-thiacytidine-5'-monophosphate (3TCMP) from a terminated DNA primer. Resistance mediated by ATP-dependent phosphorolysis depends on the intracellular levels of dNTP. High levels as found in transformed cell lines (i.e. H-9, CEM lymphoblasts, SupT1 cells, etc.) may prevent repair of primers terminated with d4TMP. However, ATP-dependent phosphorolysis could be relevant for d4T resistance in cells having low levels of dNTPs. This proposal could explain why insertion-containing HIV-1 variants have been detected in the absence of AZT, during d4T treatment.

In vitro screening of nucleoside analog combinations for potential use in anti-HIV therapy.

With the results from the Delta and ACTG 175 clinical trials clearly showing an increased benefit of two drugs over monotherapy, combination nucleoside analog therapy looks set to play a major role in the battle against HIV. It is therefore essential that suitable combinations of drugs are used in clinical trials. We investigated the intracellular activation of zidovudine (ZDV), zalcitabine (ddC), and lamivudine (3TC) in MOLT-4 cells in two- and three-drug combinations at clinically achieved concentrations. The phosphorylation of ZDV and 3TC to their active triphosphate anabolites was not affected by the presence of the other drugs studied. However, the phosphorylation of ddC was significantly inhibited when incubated with 3TC, resulting in levels of ddC triphosphate (ddC-TP) less than 50% of control values. This can be explained by the requirement of both nucleoside analogs for the enzyme deoxycytidine kinase to carry out the initial step in their phosphorylation pathways, and by the comparatively low plasma concentrations of ddC achieved in vivo. These results suggest that regimens containing nucleoside analogs should be designed taking into account potential interactions affecting phosphorylation.

Effect of stereoisomerism on the cellular pharmacology of beta-enantiomers of cytidine analogs in Hep-G2 cells.

The beta-L enantiomers of 2',3'-dideoxycytidine (beta-L-ddC) and its 5-fuoro derivative, 2',3'-dideoxy-5-fluorocytidine (beta-L-FddC), were demonstrated to be active against human immunodeficiency virus (HIV) and hepatitis B virus (HBV) replication in vitro. In the present study, we investigated the cellular pharmacology of beta-L-ddC and beta-L-FddC and compared it with that of beta-D-2',3'-dideoxy-5-fluorocytidine (beta-D-FddC). Beta-L-FddC (10 microM) was found to be phosphorylated rapidly in Hep-G2 cells to its 5'-mono-, di-, and triphosphate derivatives with intracellular triphosphate levels achieving 26.6 +/- 10.9 pmol/10(6) cells after 72 hr. In contrast, the active 5'-phosphorylated derivative of beta-D-FddC achieved lower levels with triphosphate levels of only 2.3 +/- 0.5 pmol/ (10(6) cells under the same conditions. Beta-L-ddC was also phosphorylated rapidly. A 5'-diphosphocholine (18 +/- 5.8 pmol/10(6) cells) and a 5'-diphosphoethanolamine (13.6 +/- 0.9 pmol/10(6) cells) derivative were detected in beta-D-FddC-treated cells after 72 hr, whereas in beta-L-FddC- and beta-L-ddC-treated cells, only the 5'-diphosphocholine derivative (10.9 +/- 2.8 and 60.4 +/- 5.7 pmol/10(6) cells, respectively) was detected. Beta-L-FddC-5'-triphosphate (beta-L-FddCTP), beta-D-FddC-5'-triphosphate (beta-D-FddCTP), and beta-L-ddC-5'-triphosphate (beta-L-ddCTP) followed a single phase elimination process with an intracellular half-life (T1/2) of 10.5, 5.7, and 12.3 hr, respectively. Furth ermore, beta-L-FddCTP, beta-D-FddCTP, and beta-L-ddCTP levels of 6.7 +/- 2.3, 0.3 +/- 0.1, and 12.0 pmol/10(6) cells, respectively, were still detectable 24 hr following drug removal. The higher intracellular 5'-triphosphate levels of beta-L-FddC and the extended T1/2 of its 5'-triphosphate are consistent with the more potent in vitro antiviral activity of beta-L-FddC in Hep-G2 cells when compared with its beta-D enantiomer, beta-D-FddC.

Substrate specificity of Escherichia coli thymidine phosphorylase for pyrimidine nucleosides with anti-human immunodeficiency virus activity.

Various nucleoside antiviral agents and their metabolites were examined for their ability to be cleaved across the glycosidic bond by Escherichia coli thymidine phosphorylase. The increasing order of susceptibility to cleavage was U greater than T much greater than C derivatives. Nucleosides that were unsaturated in the sugar moiety were more susceptible than saturated ones. 3'-Deoxy-2',3'-didehydrothymidine was a substrate, whereas 3'-azido-, 3'-fluoro-, 3'-oxo- and 3'-thiapyrimidine nucleosides were resistant to this enzyme.

Intracellular metabolism of 2',3'-dideoxynucleosides in duck hepatocyte primary cultures.

The intracellular fate of the potent duck hepatitis B virus (DHBV) inhibitor 2,6-diaminopurine 2',3'-dideoxyriboside (ddDAPR), its deamination product 2',3'-dideoxyguanosine (ddG), and the less effective DHBV-inhibitor 2',3'-dideoxycytidine (ddC) was investigated in duck hepatocyte primary cultures. After a 1-min exposure of [3H]ddDAPR to duck blood, 95% of the compound was converted to ddG. Similarly, [3H]ddDAPR was converted rapidly to ddG in duck hepatocyte primary cultures, with ddG exhibiting resistance to further catabolism. The major pathway of ddG utilization in these cells was phosphorylation, yielding a concentration of 2.1 and 1.9 microM total ddG nucleotides after 5 and 26 hr, respectively, of exposure to 4 microM ddG. Removal of exogenous ddG led to a rapid (T1/2 = 1.6 hr) decrease in the total intracellular ddG nucleotide pools. Duck hepatocytes treated with 4 microM ddC exhibited a time-dependent accumulation of ddC nucleotides, culminating in a maximum intracellular total ddC nucleotide concentration of 1.4 microM after 24-26 hr. The intracellular total ddC nucleotide level decreased with a T1/2 of 4.4 hr following the removal of exogenous ddC. The formation of ddC nucleotides was reduced in the presence of excess 2'-dideoxycytidine implicating deoxycytidine kinase in the initial step of ddC phosphorylation. A 25-fold excess of 2'-deoxycytidine had no effect on ddG phosphorylation in duck hepatocytes. However, a 92% inhibition of ddG nucleotide formation occurred in duck hepatocytes treated for 5 hr with 4 microM [3H]dG + 100 microM adenosine in the presence of the adenosine deaminase inhibitor 2'-deoxycoformycin, suggesting that, in these cells, adenosine kinase is involved in the ddG phosphorylation process.

Removal of anti-human immunodeficiency virus 2',3'-dideoxynucleoside monophosphates from DNA by a novel human cytosolic 3'-->5' exonuclease.

A 3'-->5' exonuclease has been highly purified from the cytosol of human acute lymphoblastic leukemia H9 cells. The apparent molecular weight of this enzyme was approximately 50,000, as indicated by its sedimentation in glycerol gradients. The exonuclease did not copurify with DNA polymerase activity, required MgCl2 for its exonucleolytic activity, and was inhibited by KCl above 60 mM. The enzyme was active on single-stranded DNA, DNA duplexes and DNA/RNA duplexes, and it was efficient at removing 3'-terminal mispairs from DNA. The products of the exonucleolytic reaction were deoxynucleoside 5'-monophosphates. The behavior of the exonuclease was examined on DNA terminated at the 3' end with a variety of dideoxynucleosides that are potent against human immunodeficiency virus type 1. The exonuclease has a broad substrate specificity; however, the rate of the enzymatic reaction varied among the D dideoxynucleosides tested (ddAMP = ddCMP > d4TMP > AZTMP). Similarly, the enzyme was examined for its reactivity with DNA terminated by either the D or L enantiomers of ddC, SddC or FddC. The removal of analogs with the native D configuration was at least 6-fold more rapid than that of the L-compounds, and the type of structural modification had an impact on the rate at which the D enantiomers were removed (SddCMP > ddCMP > FddCMP). The monophosphate forms of AZT, D4T, L-FddC and L-ddC were potent inhibitors of the exonuclease at micromolar concentrations, while D-ddCMP partially inhibited the enzyme at millimolar concentrations. Based on its physical and enzymatic properties, this exonuclease represents a novel enzyme that may have an important role in determining the relative potencies of dideoxynucleosides against human immunodeficiency virus type 1.

The intracellular phosphorylation of (-)-2'-deoxy-3'-thiacytidine (3TC) and the incorporation of 3TC 5'-monophosphate into DNA by HIV-1 reverse transcriptase and human DNA polymerase gamma.

(-)-2'-deoxy-3'-thiacytidine (3TC) has been shown to be a potent, selective inhibitor of HIV replication in vitro, which requires phosphorylation to its 5'-triphosphate for antiviral activity. The intracellular concentration of 3TC 5'-triphosphate in phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBL) shows a linear dependence on the extracellular concentration of 3TC up to an extracellular 3TC concentration of 10 microM. At this extracellular concentration of 3TC, the resulting intracellular concentration of 3TC 5'-triphosphate is 5 microM. This value is similar to the inhibition constant (Ki) values for the competitive inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and human DNA polymerases (10-16 microM) by 3TC 5'-triphosphate. Since the concentration of 3TC producing 90% inhibition (IC90) of HIV replication in PBLs has been reported to be 76 nM, the antiviral activity of 3TC requires intracellular concentrations of 3TC 5'-triphosphate, which would result in very little inhibition of reverse transcriptase if its sole mode of action was competitive inhibition. This apparent discrepency may be explained by the ability of 3TC 5'-triphosphate to act as a substrate for reverse transcriptase. Primer extension assays have shown that 3TC 5'-triphosphate is a substrate for HIV-1 reverse transcriptase and DNA polymerase gamma, resulting in the incorporation of 3TC 5'-monophosphate into DNA. In the case of DNA polymerase gamma, the product of this reaction (i.e. double-stranded DNA with 3TC 5'-monophosphate incorporated at the 3'-terminus of the primer strand) is also a substrate for the 3'-5' exonuclease activity of this enzyme. This may explain the low levels of mitochondrial toxicity observed with 3TC.

Cellular metabolism of (-) enantiomeric 2'-deoxy-3'-thiacytidine.

The metabolism of (-) enantiomeric 2'-deoxy-3'-thiacytidine (3TC) was examined in human immunodeficiency virus type 1 (HIV-1)-infected and mock-infected human cells. 3TC 5'-triphosphate levels accumulated comparably in HIV-1-infected and mock-infected phytohaemagglutinin-stimulated peripheral blood lymphocytes (PBL) and reached 40% or more of total intracellular 3TC metabolites after 4 hr. The rate of decay of 3TC triphosphate in HIV-1-infected and mock-infected PBL measured as a half-life (T1/2) ranged from 10.5 to 15.5 hr. 3TC did not significantly affect metabolism of deoxynucleotides in the U937 cell line, and was shown to be resistant to the action of human platelet pyrimidine nucleoside phosphorylase.

Catabolic disposition of 3'-azido-2',3'-dideoxyuridine in hepatocytes with evidence of azido reduction being a general catabolic pathway of 3'-azido-2',3'-dideoxynucleosides.

3'-Azido-2',3'-dideoxyuridine (AzddU, CS-87) is a potent inhibitor of human immunodeficiency virus replication in vitro with low bone marrow toxicity. Although AzddU is currently being evaluated in clinical trials, its catabolic disposition is unknown. Pharmacokinetic studies in rhesus monkeys have demonstrated that a 5'-O-glucuronide is excreted in urine. The present study examined the catabolic disposition of AzddU is isolated rat hepatocytes, a model for the study at the cellular level of biosynthetic, catabolic and transport phenomena in the liver. Following exposure of cells to 10 microM [3H]AzddU, low intracellular levels of two catabolites, identified as 3'-azido-2',3'-dideoxy-5'-beta-D-glucopyranosyluridine (GAzddU) and 3'-amino-2',3'-dideoxyuridine (AMddU), were detected. Studies using rat microsomes demonstrated that GAzddU formation was only detected in the presence of uridine 5'-diphosphoglucuronic acid, and that the rate of AMddU formation increased significantly in the presence of NADPH. Under similar conditions, reduction of the 3'-azido function was also demonstrated herein with 3'-azido-2',3'-dideoxycytidine (AzddC), 3'-azido-2',3'-dideoxy-5-methylcytidine (AzddMeC) and 3'-azido-2',3'-dideoxyguanine (AzddG), suggesting that enzymatic reduction to a 3'-amino derivative is a general catabolic pathway of 3'-azido-2',3'-dideoxynucleosides at the hepatic site.

Cross-resistance of dideoxycytidine-resistant cell lines to azidothymidine.

2',3'-Dideoxycytidine (ddC) and azidothymidine (AZT) inhibit HIV-1 replication and currently are used in AIDS therapy. Long-term use of the drugs is associated with the selection of drug-resistant HIV strains, thus limiting their effectiveness. Another mechanism, associated with their altered metabolism in host cells, also can cause "cellular" drug resistance. Human lymphocytic H9 cell lines (H9-ddC0.5w and H9-ddC5.0w) selected for ddC resistance by exposure to 0.5 and 5.0 microM ddC were found to be cross-resistant to AZT. Compared with controls, the thymidine kinase (TK) activities in H9-ddC0.5w and H9-ddC5.0w cells were 56.7 and 51.4% (with thymidine as a substrate) and 50.3 and 42% (with AZT as a substrate). Consequently the cellular incorporation of AZT and thymidine (24-hr incubation) also was reduced to 51.3 and 70.0% in H9-ddC0.5w cells and to 12.1 and 17.3% in H9-ddC5.0w cells. A 3-hr incubation with 25 microM AZT and ddC decreased their cellular incorporation to 50.5 and 76.15% in H9-ddC0.5w cells and to 12.95 and 47.8% in H9-ddC5.0w cells compared with H9 cells. Thus, the change in AZT accumulation did not correlate exactly with the decrease in TK activity and far exceeded the effect on ddC accumulation. Evidence is presented that ddC, in addition to deoxycytidine kinase, affected TK1 activity. The involvement of multidrug resistance proteins in the mechanism of the resistance was ruled out by the failure of trifluoperazine and verapamil to alter cellular accumulations of AZT, ddC, daunorubicin, and rhodamine-123. Development of cellular ddC and AZT cross-resistance may affect the therapeutic efficacy of these antiviral agents.