MRP4: A previously unidentified factor in resistance to nucleoside-based antiviral drugs.

Dideoxynucleosides, which are potent inhibitors of HIV reverse transcriptase and other viral DNA polymerases, are a common component of highly active anti-retroviral therapy (HAART) (ref. 1). Six reverse transcriptase inhibitors have been approved for human use: azidothymidine; 2'3'-dideoxycytidine; 2'3'-dideoxyinosine; 2', 3'-didehydro-3'deoxythymidine; 2',3'-dideoxy-3'-thiacytidine; and 4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-++ +metha nol. Although drug-resistant HIV strains resulting from genetic mutation have emerged in patients treated with HAART (ref. 1), some patients show signs of drug resistance in the absence of drug-resistant viruses. In our study of alternative or additional mechanisms of resistance operating during antiviral therapy, overexpression and amplification of the MRP4 gene correlated with ATP-dependent efflux of PMEA (9-(2-phosphonylmethoxyethyl)adenine) and azidothymidine monophosphate from cells and, thus, with resistance to these drugs. Overexpression of MRP4 mRNA and MRP4 protein severely impaired the antiviral efficacy of PMEA, azidothymidine and other nucleoside analogs. Increased resistance to PMEA and amplification of the MRP4 gene correlated with enhanced drug efflux; transfer of chromosome 13 containing the amplified MRP4 gene conferred resistance to PMEA. MRP4 is the first transporter, to our knowledge, directly linked to the efflux of nucleoside monophosphate analogs from mammalian cells.

Tiazofurin metabolism in human lymphoblastoid cells: evidence for phosphorylation by adenosine kinase and 5'-nucleotidase.

The exact route of metabolism of tiazofurin, a novel nucleoside with antitumor activity, is controversial. Using human cell lines severely deficient in salvage nucleotide enzymes, we were able to identify the route of activation in tiazofurin metabolism. With loss of adenosine kinase activity by mutation in two lymphoblastoid cell lines, CCRF-CEM and WI-L2, the growth sensitivity to tiazofurin decreased by 6- and 3-fold, respectively. In contrast, the mutant lines were about 3000- to 1500- and 16- to 4-fold more resistant to the structurally similar tiazofurin analogues pyrazofurin and ribavirin, respectively. Other mutants with defective deoxycytidine or uridine kinase activity showed normal sensitivity to all three analogues. Both cell lines with defective adenosine kinase activity accumulated about 50% wild-type levels of tiazofurin-5'-monophosphate and thiazole-4-carboxamide adenine dinucleotide analogue of tiazofurin at cytotoxic concentrations of the drug. Extracts of wild-type lymphoblasts catalyzed the phosphorylation of tiazofurin in the presence of adenosine 5'-triphosphate and Mg2+. Loss of adenosine kinase activity in the mutant extract eliminated this phosphorylating activity for tiazofurin consistent with the notion that adenosine kinase catalyzes phosphorylation of tiazofurin. However, an enzyme activity that catalyzed the phosphorylation of tiazofurin in the presence of inosine-5'-monophosphate as donor and Mg2+ was detected in the extracts of both wild-type cells and adenosine kinase-deficient mutants. The monophosphate donor specificity, divalent metal, high salt requirement, and nucleoside acceptor specificity of this enzyme activity paralleled that of a 5'-nucleotidase (EC 3.1.3.5) which catalyzes inosine phosphorylation. In addition, tiazofurin phosphorylation was competitively inhibited by inosine and the apparent Ki value was similar to the apparent Km value for inosine phosphorylation. These results indicate that two enzymes, adenosine kinase and a cytoplasmic 5'-nucleotidase, are functionally important anabolizing enzymes for tiazofurin in human cells.

Detection of membrane and intracellular antigens by flow cytometry following ORTHO PermeaFix fixation.

In the present study, we explored the suitability of a new cell fixative (ORTHO PermeaFix, OPF) for the detection by flow cytometry of intracellular molecules while preserving the cell surface immunoreactivity, scatter features and morphology. The effect of OPF was investigated on whole blood of ten normal donors, and on separated blasts of 17 leukemic patients. OPF fixation for 45 min to 24 h maintained the morphology of lymphoid cells with minimal cellular distortion and scatter changes, and only slightly modified cell surface immunoreactivity. For at least 1 week following fixation, the cells were still suitable for immunostaining with monoclonal antibodies that recognize the main lymphoid populations. These included CD3, CD4 and CD8 for T-cell subsets, CD19 and CD16 for B lymphocytes and NK cells, and CD45 for leukocyte common antigen (LCA). The OPF fixation of leukemic cells allowed the simultaneous detection of nuclear TdT in conjunction with membrane CD19, and with membrane and/or cytoplasmic CD22 in common-ALL, as well as with cytoplasmic CD3 in T-ALL cases. Our findings suggest that with the introduction of this new fixative into the routine laboratory service, a number of convenient and practical arrangements can be made which increase the efficiency of immunodiagnosis. Small laboratories with no inhouse flow-cytometric facilities can now accumulate OPF-treated whole blood samples for at least 3-4 days and send these to reference laboratories. In addition, the immunodiagnosis of acute leukemia is greatly facilitated by combination staining for membrane and intracellular antigens both at diagnosis and when the analysis of minority populations is warranted for detecting minimal disease.

Cellular factors for resistance against antiretroviral agents.

Substantial advancements have been made in our understanding of the complex replication cycle of, and immunopathology associated with HIV infection as well as the drugs used to treat the disease. The nucleoside reverse transcriptase inhibitors remain the cornerstones of current antiviral treatment modalities. Unfortunately, their longterm use often leads to adverse reactions and the emergence of virus mutants with decreased susceptibility to therapeutic agents. In addition to viral resistance, prolonged antiviral treatment may affect metabolic changes in the host cells that can diminish the efficacy of the treatment. Thus, both viral and cellular resistance mechanisms must be considered in the context of failing antiviral chemotherapy. This review article concerns the intracellular pharmacology of antiviral nucleoside analogues in human lymphoid cells and the possible impact of a newly identified nucleotide transporter on drug resistance.

Increased host cell-Trypanosoma cruzi interaction following phospholipase D treatment of the parasite surface.

We examined the effect of phospholipase D (PLD) treatment on the ability of Trypanosoma cruzi to interact with phagocytic and nonphagocytic host cells. The presence of PLD during the incubation of parasites with mouse peritoneal macrophages caused significant increases in both the number of parasites per 100 macrophages and the percentage of macrophages associated with parasites. Parasites pretreated with PLD, washed, and then incubated with untreated macrophages showed a marked increase in parasite-host cell association. In contrast, when only the macrophages were pretreated with PLD, there was no significant change in the association. Parasites required 45 min of PLD treatment before a significant enhancement in parasite-host cell association was observed. The action of PLD could be blocked by the presence of a competitive substrate, phosphatidylethanolamine, during enzyme treatment. The enhancing effect of PLD treatment of the parasites was relatively long lasting since it was still seen 3 h after the enzyme had been removed. The enhancing effect of PLD probably reflected an increased capacity of T. cruzi to associate with host cells rather than increased phagocytosis of PLD-altered parasites by macrophages since similar results were obtained when rat heart myoblasts, which are not phagocytic, were used as host cells. Neither the presence of phospholipids or PLD phospholipid cleavage products during the incubation of T. cruzi with macrophages had any effect on parasite-host cell association. These results show that PLD-mediated alterations to parasite phospholipids increase parasite-host cell association, and suggest that these phospholipids play a role in the initial stages of host cell infection by T. cruzi.

Mechanism of uptake of the phosphonate analog (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) in Vero cells.

The cellular uptake of phosphonylmethoxypropyl cytosine (HPMPC) was characterized to gain insight into the molecular properties that allow this anticytomegalovirus drug to permeate cell membranes. The time course of uptake of HPMPC into Vero cells was linear between 10 and 75 min and proportional to the concentration in the medium from 10(-6) to 10(-2) M. HPMPC uptake was temperature sensitive and the rate of uptake was considerably lower at 27 degrees than at 37 degrees and almost totally inhibited at 4 degrees. In competition studies with naturally occurring nucleosides, nucleotides or the phosphonylmethoxyethyl derivatives, none affected the uptake of HPMPC at concentrations up to 2000-fold molar excess. The uptake of [3H]HPMPC into Vero cells was compared with that of [14C]sucrose, a probe for fluid-phase endocytosis. Kinetics for both compounds were very similar, as were the effects of the microtubule antagonist colchicine and the tumor promoting agent phorbol myristate acetate. Colchicine and the phorbol ester are known to, respectively, inhibit and stimulate endocytosis. It is concluded from these data that HPMPC enters Vero cells by fluid-phase endocytosis and that once internalized it may accumulate in the lysosome. Protonation of the negative charge on the phosphonyl group in HPMPC may allow its diffusion across the lysosome membrane and eventual activation to its putative active diphosphorylated form in the cell cytoplasm.

Chemical and immunochemical studies on lipopolysaccharides from pyocin 103-sensitive and -resistant Neisseria gonorrhoeae.

The chemical and immunochemical properties of lipopolysaccharides (LPS) isolated from pyocin 103-sensitive and -resistant Neisseria gonorrheae were investigated. Marked differences were found in immunochemical behavior of LPS from pyocin-sensitive gonococcal strain JW31 and its isogenic pyocin-resistant variant JW31R. JW31 LPS readily precipitated wheat-germ agglutinin, soybean lectin, and rabbit anti-Streptococcus faecalis or horse anti-type 14 pneumococcal antibody. In contrast, JW31R LPS precipitated only soybean lectin. The combining-site specificity of anti-S. faecalis cross-precipitated by JW31 LPS, or type 14 pneumococcal capsular polysaccharide, was examined by hapten inhibition, and lactose found to be the most potent inhibitor. Horse anti-pneumococcal type 14 antibodies, cross-precipitated by JW31 LPS and streptococcal lactose polymer, exhibited heterogeneity with respect to combining site specificity. Gel filtration of LPS-derived core oligosaccharide showed both strain JW31 and JW31 R to possess R-type lipopolysaccharide with cores having a Mr approximately 1800. JW31R LPS contains more galactose but less hexosamine than JW31 LPS. Both JW31 and JW31R core oligosaccharides possess D-glucosamine and D-galactosamine, probably N-acetylated, as the only nonreducing end-groups, and (1 leads to 4)-linked D-glucose residues. Chemical data support immunochemical findings which indicate that lactose units occur as a structural feature of JW31 gonococcal LPS.

Effects of alpha- and beta-adrenergic agonists on Trypanosoma cruzi interaction with host cells.

We studied the effects of adrenergic agonists on the capacity of blood trypomastigote forms of Trypanosoma cruzi to associate with (i.e., bind and/or penetrate) host cells in vitro. The extent of T. cruzi association with mouse macrophages in the presence of the beta-adrenergic agonist L-isoproterenol was significantly decreased with respect to mock-treated controls. Similar results were obtained when the parasite was pretreated with L-isoproterenol and was then allowed to interact with untreated macrophages. In contrast, pretreatment of trypomastigotes with either L-phenylephrine or methoxamine-alpha-adrenergic agonists--enhanced their reactivity with macrophages. Interaction with a nonphagocytic host cell was also decreased and increased by parasite pretreatment with beta- and alpha-adrenergic agonists, respectively. The L-isoproterenol and L-phenylephrine effects were no longer detectable 2 and 3 hr after their removal, respectively, and were therefore reversible. Atenolol, a specific beta 1 adrenoreceptor blocker inhibited the L-isoproterenol effect, whereas butoxamine, a specific beta 2 blocker, did not. Thus, beta 1-like but not beta 2-like binding sites appeared to be expressed on T. cruzi. Both prazosin and yohimbine, preferential alpha 1- and alpha 2-receptor blockers, respectively, abolished the L-phenylephrine effect. The opposite effects of alpha- and beta-adrenergic agonists suggested that the infectivity of T. cruzi may be regulated by activation of surface components comparable to the adreno-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of MagniSep chromium dioxide particles in an immunoassay system.

The use of chromium dioxide particles as a solid support for very sensitive and rapid immunoassays, is the result of the combination of large surface area (40 m2) and high protein uptake capacity (40 mg/g) allowing rapid capture kinetics and high binding capacity. Magnetic and physical properties of these particles give a rapid separation, a complete resuspension and a rapid high-efficiency washing, highly desirable characteristics for efficient automation of immunoassays. Good precision and accuracy, exemplified by excellent recovery, parallelism and correlation were demonstrated. Test results prove that the technology is highly flexible and applicable to a variety of assay formats.

Antiplasmodial activity and cytotoxicity of 10ß-aminoquinolinylethylethers of artemisinin.

Each year roughly 800 000 people die of malaria, with 95% being African children. The shortcomings of the current drugs and the emergence of P. falciparum resistance to the artemisinin class of compounds warrant the search for new classes or derivatives. In search for such compounds, a series of 10ß-amino-quinolinylethylethers of artemisinin, previously synthesized from this laboratory were screened for antimalarial activity against both the chloroquine-susceptible 3D7 and -resistant K1 strains of P. falciparum. Their cytotoxicity was also assessed against HEK 293 and HepG2 cell lines.The parasitic and mammalian cells were incubated with compounds at various concentrations for 72 h. The antimalarial activity was determined using SYBR Green I-based fluorescence. For cytotoxicity determination, cells were grown to confluence and CellTiter-Glo luminescent cell viability assay was used.All derivatives proved to be active against both strains with good selectivity towards the parasitic cells. The derivative 11 featuring 2 artemisinin moieties and an aminoethylpiperazine linker was the most active of all. It possessed 17- and 166-fold more potency than artemether against 3D7 (EC50: 9.5 vs. 166 nM) and K1 (10.9 vs. 1723.3 nM), respectively, while was found to be as potent as artesunate against both strains.Derivative 11 stands as a good candidate to be further investigated primarily in vitro in comparison with an equimolar combination of chloroquine (CQ) and artemisinin to ascertain its advantages, if any, over the combination.

Modulation of macrophage interaction with Trypanosoma cruzi by phospholipase A2-sensitive components of the parasite membrane.

The presence of phospholipase A2 (PLA2) significantly increased the association between Trypanosoma cruzi and macrophages. This effect reflected alterations to the parasite membrane since it was reproduced only when the parasite but not the macrophage was pretreated with PLA2. That PLA2 activity was responsible for the noted enhancement was indicated by the ability of the specific substrate phosphatidylcholine to block it. The presence of the PLA2 inhibitors quinacrine, 4-bromophenacyl bromide or phentermine markedly inhibited parasite-macrophage association. Quinacrine also inhibited association of the parasite with a non-phagocytic host cell. These results suggested a role for endogenous PLA2 in the initial stages of cell infection by T. cruzi.

Antigenic specificity and heterogeneity of lipopolysaccharides from pyocin-sensitive and -resistant strains of Neisseria gonorrhoeae.

Homologous antisera were raised against lipopolysaccharides (LPSs) isolated from pyocin 103-sensitive JW31 strain Neisseria gonorrhoeae and its isogenic, pyocin-resistant variant, JW31R. Changes in immunochemical reactivity of LPS antigen associated with pyocin-resistance were examined by enzyme-linked immunosorbent assay, employing homologous and heterologous anti-LPS immune sera. The acquisition of pyocin 103 resistance is accompanied by a loss in LPS antigen reactivity with homologous anti-LPS. The variant LPS of pyocin 103-resistant mutants is immunogenic and displays a new, distinct antigenic specificity shared with other pyocin 103-resistant variant gonococcal strains. The acquisition of pyocin 103 resistance by JW31 strain gonococci is also accompanied by a striking loss of LPS cross-reactivity with antistreptococcal polysaccharide reagents having an antibody combining site specificity directed against the chemically defined lactose polymer from Streptococcus faecalis cell wall and pneumococcal type 14 capsular polysaccharide. When examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, JW31 and JW31R LPSs show banding patterns characteristic of microheterogeneous, rough-type LPS devoid of O-side chains. Immunoblot transfer analysis of gel-separated gonococcal LPS antigens shows a difference in the pattern of antibody binding by homologous versus cross-reactive anti-LPS, which suggests a heterogeneity in the distribution of cross-reactive determinants among LPS molecules.

Rapid single-step method for flow cytometric detection of surface and intracellular antigens using whole blood.

Fixation/permeabilization methods used for the detection of intracellular antigens by flow cytometry often result in the destruction of cellular morphology and surface immunoreactivity, properties useful in flow cytometry for the characterization of cells in heterogeneous populations. In addition, a majority of these methods are incompatible with whole blood and require that peripheral blood leukocytes (PBLs) be purified prior to fixation. This article describes a new technique for the rapid detection of both intracellular and cell surface antigens, while preserving cell morphology, through the use of a single-step fixation/permeabilization reagent, ORTHO PermeaFix (OPF). OPF is compatible with whole blood, allowing for the direct preparation of PBLs without prior cell separation. An additional red blood cell lysing reagent was not required because RBC lysis occurred upon resuspension of OPF-treated whole blood samples in isotonic solution. Discrimination of leukocyte populations by light scatter after OPF treatment was comparable to matched unfixed live cells. In addition, absolute lymphocyte and white blood cell (WBC) counts were not significantly affected when OPF-treated cells were compared with unfixed cells. Treatment of whole blood from 7 normal donors showed no significant difference in percentage of cells positive for CD2, CD3, CD4, CD8, CD16, or CD19 between fixed and unfixed samples when cells were stained before fixation, and no difference in CD3, CD4, CD8, CD16, or CD19 percentages when cells were stained following fixation. Monoclonal antibodies specific for intracellular antigens located at various sites within the cell were tested on fixed samples. OPF-treated peripheral blood lymphocytes showed greater than 95% reactivity for the inner mitochondrial membrane protein bcl-2, and the cytoskeletal cytoplasmic protein vimentin. TIA-1, a cytolytic granule-associated protein, showed differential reactivity within lymphocyte subsets, from a low of 8 +/- 2% in CD4+ cells to 89 +/- 6% in CD16+ cells, when whole blood from five normal donors was fixed and stained. Reh cells treated with OPF showed greater than 95% reactivity for the internuclear protein TdT. A comparison of OPF with two other fixation/permeabilization procedures, 1% paraformaldehyde followed by 45% ethanol and 0.25% paraformaldehyde followed by 0.2% Tween 20, showed that only OPF could be used both prior to or following cell surface staining with no effect on antigen detection while allowing optimal detection of all of the intracellular antigens tested.

Relationship of deoxynucleotide changes to inhibition of DNA synthesis induced by the antiretroviral agent 3'-azido-3'-deoxythymidine and release of its monophosphate by human lymphoid cells (CCRF-CEM).

The metabolism and cytostatic effects of 3'-azido-3'-deoxythymidine (AZT), one of the most effective agents being used in the treatment of acquired immunodeficiency syndrome, were investigated in the CCRF-CEM line of human T lymphoid cells. The concentration of drug required to inhibit cell growth by 50% (CD50) was significantly lower when the cells were exposed to AZT for 24 hr (CD50 = 50 microM), as compared with 48 or 96 hr (CD50 = 225 and greater than 300 microM, respectively). AZT at 25 microM blocked the progression of cells in S phase for about 12 hr, but this effect was reversed by 24 hr, despite the continued presence of drug in the medium. At this drug concentration, the level of dTTP decreased to about 75% of the control level by 4 hr but rebounded to 30% above normal by 8 hr of drug exposure. dGTP and dATP pool sizes were unchanged, whereas the dCTP pool increased 5-fold. The time course of these biochemical changes indicated that the onset of S phase arrest was not directly related to the decrease in deoxynucleoside triphosphate pools. CCRF-CEM cells incubated with 25 microM AZT accumulated about 0.9 mM 5'-monophosphate (AZTMP) after 4 hr whereas levels of the 5'-di- and 5'-triphosphates (AZTDP and AZTTP) plateaued at about 2 and 5 microM, respectively. After this period, there was a rapid decrease in AZTMP accumulation, to one third its initial level by 24 hr, whereas AZTDP and AZTTP pools decreased to only about 70%. The loss in AZT nucleotide formation with time of drug exposure was associated with a concomitant accumulation of AZTMP in the medium. Cellular excretion of AZTMP was not associated with any detectable cell lysis or leakage of other cellular metabolites. The ability of CCRF-CEM cells to excrete AZTMP may be an important factor limiting the biochemical and biological effects of the drug.

Interaction of lectins with Neisseria gonorrhoeae.

Fifty-four strains of Neisseria gonorrhoeae were examined for their ability to be agglutinated by various lectins. Wheat germ agglutinin, ricin, soybean lectin, and peanut agglutinin agglutinated all strains tested. Dolichos biflorus, Sophora japonica, Maclura pomifera, Ulex Europaeus, Lens culinaris, Canavalia ensiformis, Phaseolus lunatus, and Bandeiraea simplicifolia BS I and BS II lectins either failed to agglutinate or agglutinated some strains but not others. Agglutination of gonococci by wheat germ agglutinin, ricin, and soybean lectin is sugar specific and most effectively inhibited by ligands known to interact with lectin combining sites. Lectin reactive groupings appear to be independent of antigenic determinants conferring Gc serogroup specificity. Interactions with wheat germ agglutinin and ricin suggest the occurrence of multiple beta-linked N-acetyl-D-glucosamine (D-GlcNAc) and beta-D-galactosyl (beta-D-Gal) units as common structural features of gonococcal cell envelope polysaccharide. Interactions with soybean agglutinin, peanut agglutinin, and Dolichos biflorus lectins suggest that alpha-N-acetyl-D-galactosamine (alpha-D-GalNAc) units and beta-D-Gal linked to GalNAc and (or) GlcNAc may also occur as structural features of the polysaccharide components of the cell envelope of some gonococci.

Intracellular metabolism and action of acyclic nucleoside phosphonates on DNA replication.

9-(2-phosphonylmethoxyethyl)guanine (PMEG) is an acyclic nucleoside phosphonate derivative that has demonstrated significant anticancer activity in a number of in vitro and in vivo animal model systems. In this study, we compared the cellular metabolism of PMEG and 9-(2-phosphonylmethoxyethyl)adenine (PMEA), a clinically active anti-HIV and antihepatitis agent, and the inhibitory activities of their putative active diphosphate derivatives, PMEGpp and PMEApp, respectively, toward human cellular DNA polymerases. PMEG was significantly more cytotoxic than PMEA against a panel of human leukemic cells. The diphosphate derivatives were the major metabolites formed in cells on both these agents, with PMEGpp reaching cellular concentration approximately 4-fold higher than that achieved for PMEApp. These differences in cellular accumulation of the diphosphate derivatives were not, however, sufficient to account for the 30-fold difference in cytotoxicity between the two analogs. PMEGpp was also at least a 7-fold more effective inhibitor of in vitro simian vacuolating virus 40 DNA replication system than that of PMEApp (IC50 = 4.6 microM). Studies with a defined primed DNA template showed that PMEGpp was a potent inhibitor of both human polymerases alpha and delta, two key enzymes involved in cellular DNA replication, whereas PMEApp inhibited these enzymes relatively poorly. From these studies, we can conclude that the factors that contribute to the enhanced antileukemic activity of PMEG derives both from its increased anabolic phosphorylation and the increased potency of the diphosphate derivative to target the cellular replicative DNA polymerases.

Metabolic pathways for activation of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine in human lymphoid cells.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA), the acyclic phosphonate analog of adenine monophosphate, is a promising antiviral drug with activity against herpesviruses, Epstein-Barr virus, and retroviruses, including the human immunodeficiency virus. In order to be active, it must be converted to the diphosphate derivative, the putative inhibitor of viral DNA polymerases. The metabolic pathway responsible for activation of PMEA is unclear. The metabolism of PMEA was investigated in human T-lymphoid cells (CEMss) and a PMEA-resistant subline (CEMss(r-1)) with a partial deficiency in adenylate kinase activity. Experiments with [3H]PMEA showed that extracts of CEMss phosphorylated PMEA to its mono- and diphosphate in the presence of ATP as the phosphate donor. No other nucleotides or 5-phosphoribosyl pyrophosphate displayed appreciable activity as a phosphate donor. Subcellular fractionation experiments showed that CEMss cells contained two nucleotide kinase activities, one in mitochondria and one in the cytosol, which phosphorylated PMEA. The PMEA-resistant CEMss mutant proved to have a deficiency in the mitochondrial adenylate kinase activity, indicating that this enzyme was important in the phosphorylation of PMEA. Other effective antiviral purine phosphonate derivatives of PMEA showed a profile of phosphorylating activity similar to that of PMEA. By comparison, phosphorylation of the pyrimidine analog (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine proceeded by an enzyme present in the cytosol. We conclude from these studies that adenylate kinase which has been localized in the intermembrane space of mitochondria is the major route for PMEA phosphorylation in CEMss cells but that another hitherto unidentified enzyme(s) present in the cytosol may contribute to the anabolism of the phosphonates.

Metabolic diversity and antiviral activities of acyclic nucleoside phosphonates.

The acyclic nucleoside phosphonates (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) inhibited herpes simplex virus-1 replication in Vero cells, and the IC50 values ranged from 4 microM (for HPMPC and HPMPA) to 40 microM (for PMEA). Pretreatment of cells with HPMPC for 12-24 hr induced an effective antiviral state, and the cells maintained this antiviral state for > 7 days. In contrast, much larger amounts (approximately 2.5-5 x IC50 doses) of PMEA or HPMPA were required to establish an antiviral state, which lasted for only approximately 24 or 72 hr, respectively. A 12-hr treatment of the cells with the phosphonates was required for the establishment of optimal antiviral activity; surprisingly, longer durations of exposure to PMEA (but not HPMPA or HPMPC) resulted in diminished antiviral effect. We investigated the metabolism of PMEA and HPMPC to determine the cellular basis for these differences. The cellular uptake of HPMPC was approximately 8-fold greater than that of PMEA. The levels of the PMEA metabolites PMEA monophosphate and PMEA diphosphate increased for approximately 12 hr and plateaued thereafter. PMEA and its metabolites were cleared from the cells with a half-life of 4.9 hr. In contrast, the HPMPC metabolites HPMPC monophosphate (HPMPCp) and HPMPC diphosphate (HPMPCpp) accumulated throughout the 24-hr study period and, at equimolar drug concentrations (25 microM), reached intracellular levels approximately 2-3-fold greater than those of the PMEA metabolites. HPMPC also differed from PMEA in its capacity to generate a phosphodiester metabolite (HMPCp-choline), which was a predominant metabolite in HPMPC-treated cells. In addition, the rates of disappearance of intracellular metabolites of the two drugs were significantly different. Thus, the decay of HPMPCpp was quite slow and biphasic (t1/2 = 24 and 65 hr) and that of HMPCp-choline was monophasic (t1/2 = 87 hr). Together, these factors can explain the differing antiviral potencies seen with PMEA and HPMPC. The possible role of the choline adduct in the expression of antiviral activity of the drug remains to be elucidated, but the adduct may serve as an intracellular store for the long term maintenance of active HPMPCpp in cells. The results also highlight the extent of diversity in the cellular pharmacology and antiviral activities of the acyclic nucleoside phosphonates.

A human T lymphoid cell variant resistant to the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine shows a unique combination of a phosphorylation defect and increased efflux of the agent.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a new antiviral agent with activity against herpes viruses and retroviruses, including human immunodeficiency virus, but its metabolism and mechanism of action remain unclear. We have isolated a human T lymphoid cell line (CEMr-1) that is resistant to the antiproliferative effects of PMEA. The antiviral effects of PMEA against human immunodeficiency virus-1 infection were also greatly reduced in CEMr-1 cells, compared with the parental cells. This mutant showed cross-resistance to the related acyclic nucleoside phosphonates 9-(2-phosphonylmethoxyethyl)diaminopurine and 9-(2-phosphonylmethoxyethyl)guanine and the lipophilic prodrug bis(pivaloyloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine-( bispome-PMEA), as well as partial resistance to the purine nucleosides 2-chlorodeoxyadenosine, 2-fluro-9-beta-D-arabinosylfuranosyladenine, and adenosine, but did not show resistance to 2'-deoxyadenosine or 9-beta-D-arabinosylfuranosyladenine. We compared the uptake and metabolism of [3H]PMEA and [3H]-bispom-PMEA in the mutant and parental cells. The analysis of radioactive products by high pressure liquid chromatography revealed marked alterations in the ability of the mutant cell line to accumulate PMEA and its anabolites, compared with the parental cells. Accumulation of PMEA, PMEA monophosphate, and PMEA bisphosphate (major metabolites formed with either PMEA or bispom-PMEA) decreased by 50, 95, and 97%, respectively. Compared with the parental cells, the variant cells showed a approximately 7-fold increase in the rate of efflux of PMEA and a 2-fold decrease in the activity of adenylate kinase. In contrast, other enzymes of nucleotide metabolism, such as adenosine kinase, deoxycytidine kinase, and 5-phosphoribosyl-1-pyrophosphate synthetase, showed no significant change in the two cell lines. Overall, these results suggest that the mutation in this resistant cell line is of a novel type, involving an alteration in the cellular efflux of PMEA as the major basis for the resistant phenotype.

Metabolic pathways for the activation of the antiretroviral agent 2',3'-dideoxyadenosine in human lymphoid cells.

The pathways of 2',3'-dideoxyadenosine (ddAdo) metabolism, a selective inhibitor of the replication of human immunodeficiency virus, were investigated with use of the human T-lymphoid cell line CCRF-CEM which is deficient in either deoxycytidine kinase or adenosine kinase activity, or both. At an extracellular concentration of 10 microM, which blocks the cytopathic effect of human immunodeficiency virus in vitro, ddAdo was found to be metabolized to its mono-, di-, and triphosphates and to dideoxyinosine monophosphate (ddIMP). The metabolism of ddAdo in the kinase-deficient mutants was found to be unchanged by comparison with that in parental cells; however, the inhibition of ddAdo deamination to 2',3'-dideoxyinosine (ddIno) by the adenosine deaminase inhibitor, 2'-deoxycoformycin, reduced ddAdo nucleotide formation in deoxycytidine kinase-deficient, adenosine kinase-deficient, and doubly kinase-deficient mutants by 42, 54, and 80%, respectively. Incubation of the CCRF-CEM cells with 20 microM L-alanosine, an amino acid antagonist that inhibits purine biosynthesis at the level of adenylosuccinate/lyase synthetase, resulted in 80% inhibition in the accumulation of ddAdo nucleotides in both wild-type and kinase-deficient mutants and also increased ddIMP accumulation 2- to 3-fold. These findings indicate that ddAdo activation in human T-lymphoblasts can occur by three metabolic pathways: directly, by phosphorylation to ddAMP by the action of either deoxycytidine kinase or adenosine kinase and, indirectly, through deamination to ddIno with consequent phosphorylation of ddIno to ddIMP, and reamination to ddAMP in a reaction catalyzed by adenylosuccinate synthetase/lyase. However, in the absence of 2'-deoxycoformycin, the activation of ddAdo to ddATP in T-lymphoid cells is primarily a function of the indirect route.