Purine nucleoside metabolism in the erythrocytes of patients with adenosine deaminase deficiency and severe combined immunodeficiency.

Deficiency of erythrocytic and lymphocytic adenosine deaminase (ADA) occurs in some patients with severe combined immunodeficiency disease (SCID). SCID with ADA deficiency is inherited as an autosomal recessive trait. ADA is markedly reduced or undetectable in affected patients (homozygotes), and approximately one-half normal levels are found in individuals heterozygous for ADA deficiency. The metabolism of purine nucleosides was studied in erythrocytes from normal individuals, four ADA-deficiency patients, and two heterozygous individuals. ADA deficiency in intake erythrocytes was confirmed by a very sensitive ammonia-liberation technique. Erythrocytic ADA activity in three heterozygous individuals (0.07,0.08, and 0.14 mumolar units/ml of packed cells) was between that of the four normal controls (0.20-0.37 mumol/ml) and the ADA-deficient patients (no activity). In vitro, adenosine was incorporated principally into IMP in the heterozygous and normal individuals but into the adenosine nucleotides in the ADa-deficient patients. Coformycin (3-beta-D-ribofuranosyl-6,7,8-trihydroimidazo[4,5-4] [1,3] diazepin-8 (R)-ol), a potent inhibitor of ADA, made possible incorporation of adenosine nucleotides in the ADA-deficient patients...

Conversion of 5'-deoxy-5'-methylthioadenosine and 5'-deoxy-5'-methylthioinosine to methionine in cultured human leukemic cells.

5'-Deoxy-5'-methylthioadenosine and 5'-deoxy-5'-methylthioinosine, which are metabolized to the methionine precursor, 5-methylthioribose-1-phosphate, by 5'-deoxy-5'-methylthioadenosine phosphorylase and purine nucleoside phosphorylase, respectively, can serve as sources of methionine for cultured HL-60 promyelocytic leukemia cells. CCRF-CEM T-cell leukemia cells, which lack 5'-deoxy-5'-methylthioadenosine phosphorylase, convert 5'-deoxy-5'-methylthioinosine (but not 5'-deoxy-5'-methylthioadenosine) to methionine; this conversion is blocked by purine nucleoside phosphorylase inhibitors. Therefore, the pathway for the conversion of 5-methylthioribose-1-phosphate to methionine is present in both human leukemic lines.

Inhibitors of purine nucleoside phosphorylase: effects of 9-deazapurine ribonucleosides and synthesis of 5'-deoxy-5'-iodo-9-deazainosine.

9-Deazapurine ribonucleosides constitute a new class of noncleavable purine nucleoside phosphorylase inhibitors that have at least 30-fold greater affinity for the enzyme than the corresponding C-nucleosides of the formycin B series. 9-Deazaguanosine, 9-deazainosine, and 5'-deoxy-5'-iodo-9-deazainosine competitively inhibited human erythrocytic purine nucleoside phosphorylase with Ki values of 29, 20, and 1.8 X 10(-7) M. The last compound is the most potent nucleoside inhibitor of the enzyme presently available and its synthesis is described. In contrast, 7,9-dideaza-7-thiainosine is a very weak inhibitor of the enzyme. When tested as an inhibitor of 2'-deoxyguanosine phosphorolysis in intact human erythrocytes and MOLT-3 human T-cell lymphoblastic leukemia cells, 5'-deoxy-5'-iodo-9-deazainosine was equipotent with 8-aminoguanosine (which is a precursor for 8-aminoguanine, Ki = 2 X 10(-7) M). Similarly, 5'-deoxy-5'-iodo-9-deazainosine and 8-aminoguanosine both potentiated the growth inhibition of human T-lymphocytic MOLT-3 cells by 2'-deoxyguanosine, reducing the 50% inhibitory concentration from approximately 2 X 10(-5) to approximately 2 X 10(-6) M.

Changes in nucleoside transport of HL-60 human promyelocytic cells during N,N-dimethylformamide induced differentiation.

The rate of nucleoside transport decreased profoundly in human promyelocytic leukemia HL-60 cells after myeloid differentiation was induced by 5-6 days of exposure to 0.8% N,N-dimethylformamide (DMF). The facilitated diffusion of 100 microM radiolabeled adenosine and 2'-deoxyadenosine, measured by rapid transport assays, decreased 10- to 20-fold. The transport of 2 microM coformycin or 2'-deoxycoformycin, which is mediated by the same mechanism and was monitored by the adenosine deaminase titration assay, decreased 29-fold. The reduction in nucleoside transport capacity after DMF treatment was confirmed by a 19-fold decrease in the number of specific binding sites per cell (from 24-30 X 10(4) to 1.2-1.7 X 10(4)) for [3H]-6-p-nitrobenzylthioinosine, a nucleoside transport inhibitor. The binding affinity of 6-p-nitrobenzylthioinosine was not altered significantly and nucleoside transport remained sensitive to the transport inhibitors, 6-p-nitrobenzylthioinosine, dipyridamole, and dilazep after DMF-induced maturation. Time-dependence studies showed that the rate of 100 microM deoxyadenosine transport was unchanged for the first 24 h of exposure to DMF but fell to about 36% of control rates at 24-26 h and then gradually decreased further to about 4-5% of control rates after 5-6 days. In contrast, transport rates of the purine bases were reduced only 2- to 3-fold in HL-60 cells after 5 days of DMF treatment. The rates of adenosine and deoxyadenosine transport were unchanged or reduced by no more than 2-fold after 5-6 days of exposure to 0.8% DMF in the following human tumor cell lines that are not inducible with DMF: ARH-77 (multiple myeloma), KG-1 (acute myelogenous), and K-562 (chronic myelogenous). Thus, changes in nucleoside transport may serve as an early, membrane-associated marker of differentiation of the HL-60 cell line.

Nonenzymatic glycosylation of erythrocytic proteins in normal and diabetic subjects. Enzymes of nucleoside and nucleotide metabolism.

A modification of the technique of Glyco-Gel affinity column chromatography has been employed to separate glycosylated proteins from nonglycosylated proteins of hemolysates. When glycosylation in hemolysates of 11 type I diabetic subjects was compared with that from 7 normal subjects, significant increases were found in glycosylation of hemoglobin (Hb) (12.1 +/- 6.0% versus 4.7 +/- 0.5%) and purine nucleoside phosphorylase (PNP) (5.3 +/- 3.0% versus 2.1 +/- 0.5%). However, no differences were found for nucleoside diphosphokinase (NDPK) (1.5 +/- 1.1% versus 1.0 +/- 0.4%) and adenylate kinase (AMPK) (0.5 +/- 0.4% versus 0.7 +/- 0.2%). Linear relationships were seen between glycosylated Hb and glycosylated PNP (r = 0.97) or glycosylated NDPK (r = 0.81). On incubation of hemolysates from normal individuals with high glucose (1500 mg/dl or 83 mM) and NaCNBH3 (20 mM), linear increases in the degrees of glycosylation were seen with time. After 18 h, the percentages of glycosylation of Hb, PNP, NDPK, and AMPK were increased from normal values to 31, 24, 11, and 3, respectively. When partially purified human erythrocytic PNP was incubated with various monosaccharides (20 mM) in the presence of NaCNBH3 for 6 h, glycosylation increases of 2-5-fold were seen in the order ribose greater than mannose greater than galactose greater than glucose.

Clinical, pharmacologic, and immunologic effects of 2'-deoxycoformycin.

Clinical, pharmacologic, and immunologic effects of 2'-deoxycoformycin (dCF) were evaluated in 15 patients with advanced malignancies. Toxicity was less severe with a low dose (4 mg/m2) of dCF, but this dose still resulted in suppression of cellular adenosine deaminase activity, skin test reactivity, and lymphocyte responses to mitogens. Improvement in cutaneous T cell lymphoma plaques was seen after dCF. Further investigations of antitumor efficacy with the use of this low dosage schedule should continue in patients with hematologic neoplasms, and additional preliminary studies of the combination of an adenosine deaminase inhibitor with an adenosine analog should also be considered.

Plasma adenosine deaminase2: a marker for human immunodeficiency virus infection.

Plasma concentrations of the two isoenzymes of adenosine deaminase (ADA, E.C. 3.5.4.4), adenosine deaminase1 (ADA1) and adenosine deaminase2 (ADA2), were measured in a cohort of ambulatory patients infected with the human immunodeficiency virus (HIV) and controls. A sensitive isoenzyme-specific radioisotopic assay system was developed for these studies. Among 22 HIV-infected patients, plasma ADA2 was significantly elevated as compared with 16 control subjects (p less than 0.01) and 6 uninfected subjects having a risk factor for HIV infection (p less than 0.01). Plasma ADA2 was not associated with the stage of disease as defined by clinical status (p greater than 0.05) or helper (CD4) lymphocyte count (p greater than 0.05). Available evidence suggests that elevated plasma ADA2 could be a useful surrogate marker for HIV infection that occurs early in the disease process.

Substrate specificities of 5'-deoxy-5'-methylthioadenosine phosphorylase from Trypanosoma brucei brucei and mammalian cells.

The separation by chromatofocusing of two distinct purine nucleoside cleaving activities from crude extracts of Trypanosoma brucei brucei is described. One catalyzes the reversible phosphorolysis of 5'-deoxy-5'-methylthioadenosine (MeSAdo) and adenosine (Ado) and was designated an MeSAdo/Ado phosphorylase, while the other catalyzes the hydrolysis of adenosine, inosine, and guanosine but not MeSAdo. The substrate specificity of trypanosomal MeSAdo/Ado phosphorylase differed from that of a mammalian MeSAdo phosphorylase (derived from murine Sarcoma 180 cells) in that it was able to phosphorolyze 2'-deoxyadenosine, 3'-deoxyadenosine and 2',3'-dideoxyadenosine. In addition, the trypanosomal phosphorylase was able to utilize the nucleoside analog, 6-methylpurine 2'-deoxyribonucleoside, as an alternative substrate, whereas the mammalian enzyme could not. Because of these differences, cytotoxic analogs of MeSAdo may be designed that are selectively activated by the trypanosomal MeSAdo/Ado phosphorylase.

Synthesis and biological evaluation of 6-amino-1H-pyrrolo[3,2-c]pyridin-4(5H)-one (3,7-dideazaguanine).

The synthesis of 6-amino-1H-pyrrolo[3,2-c]pyridin-4(5H)-one (3,7-dideazaguanine, 2) has been accomplished from 3-(ethoxycarbonyl)pyrrole-2-acetonitrile. In contrast to 3-deazaguanine, compound 2 did not show any antitumor, antiviral, or antibacterial properties. Furthermore, it was not a substrate for hypoxanthine-guanine phosphoribosyltransferase or purine nucleoside phosphorylase.

Inhibition of human and rat platelet aggregation by extracts of Mo-er (Auricularia auricula).

Hot water extracts of Mo-er (1 gm by 15 ml of water), an oriental food (Auricularia auricula), inhibit strongly both human and rat platelet ADP-induced aggregation. HPLC analysis of two varieties of Mo-er, A. auricula and A. polytricha (a black tree fungus), shows that they contain adenosine (Ado), 133 and 154 micrograms per gram of dry fungus, respectively. The inhibition of ADP-induced platelet aggregation by Mo-er extracts and by Ado was compared. Mo-er extracts caused a more rapid onset and a longer duration of inhibition that produced by equivalent amounts of Ado. Furthermore, Mo-er extract treated with adenosine deaminase to degrade the Ado retained the capacity to inhibit platelet aggregation. The inhibitory effects of Mo-er extracts of ADP-induced human platelet aggregation are greatly potentiated by the inhibitors of cyclic AMP phosphodiesterase such as oxagrelate (phthalazinol) and papaverine. The inhibition of platelet aggregation is only partially blocked by 2',5'-dideoxy-adenosine (DDA), an inhibitor of platelet adenylate cyclase and 5'-deoxy, 5'-methylthioadenosine (MTA), an antagonist of ADO receptors. ADP-induced rat platelet aggregation is strongly inhibited by Mo-er extracts, but not by Ado. This inhibition is not reversed by either DDA or MTA. These findings indicate that Mo-er extracts contain an agent (or agents) in addition to Ado, that blocks platelet aggregation by a mechanism that does not involve the platelet cyclic AMP system.

Platelet-activating factor (PAF)-induced platelet aggregation. Modulation by plasma adenosine and methylxanthines.

This study examined the role of plasma adenosine in the modulation of platelet-activating factor (PAF) activity on platelet aggregation and serotonin (5-HT) release in human platelet-rich plasma (PRP). In addition, the effects of methylxanthines (e.g. theophylline and caffeine) were studied on PAF-induced platelet aggregation in PRP isolated from blood samples from healthy subjects. Also, PAF-induced platelet aggregation was examined in PRP samples of patients receiving theophylline treatment. These studies demonstrate that plasma adenosine levels (0.1 to 0.3 microM) play a key role in negative modulation of PAF activity on platelet aggregation and 5-HT release. After depletion of plasma adenosine, the platelet-aggregating activity of PAF was increased greatly (> 10-fold). PAF at concentrations of 0.1 to 12 microM caused no 5-HT release in PRP containing normal amounts of adenosine (blood collected in the presence of 2'-deoxycoformycin and dilazep), whereas PAF at 0.1 microM caused 5-HT release (45%) in adenosine-depleted PRP, demonstrating that plasma adenosine is much more inhibitory of 5-HT release than platelet aggregation. The adenosine antagonists theophylline (50 microM), caffeine (50 microM) and a xanthine derivative, 3,7-dimethyl-l-propargylxanthine (DMPX, 10 microM) (a more specific adenosine A2 receptor antagonist), potentiated PAF activity on platelet aggregation in PRP samples containing adenosine. Also, patients receiving theophylline treatments showed significantly greater platelet aggregation induced by PAF in their PRP samples. PAF induced a rapid increase (80% in 15 sec) in intracellular Ca2+ mobilization, which was strongly inhibited by adenosine (IC50, 0.3 microM). Our studies suggest that agents that can increase plasma adenosine levels (e.g. inhibitors of adenosine uptake and adenosine metabolism) or methylxanthines may be useful in altering (inhibiting or enhancing, respectively) PAF actions on platelets and other tissues.

Differential incorporation of 2'-deoxyadenosine into human peripheral lymphocytes.

Human peripheral lymphocytes incubated with 2'-deoxycoformycin and 2'-deoxyadenosine (dAdo) reached a plateau in dATP accumulation after 4 hr that lasted for up to 24 hr. Total dATP accumulation did not exceed 15% of the control ATP concentration in the lymphocytes. In contrast, the human CCRF-CEM T lymphoblastic cell line and human erythrocytes showed a nearly linear pattern of dATP formation throughout the incubation period. By 6 hr the dATP concentration in the CCRF-CEM cells exceeded the control ATP concentration. A comparison of dATP accumulation in purified peripheral T and B lymphocytes indicated differences between these cells that favor greater dATP formation in the B lymphocytes. Incorporation studies with several adenosine analogs demonstrated that arabinosyladenine, 2-F-arabinosyladenine, tubercidin, formycin A, and 9-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl)adenine form corresponding amounts of analog triphosphate in the T and B cell-enriched lymphocytes. 9-(2'-Deoxy-2'-fluoro-beta-D-arabinofuranosyl)adenine (2'-F-araA) was the only compound to show an incorporation pattern similar to that observed with dAdo by forming analog triphosphate only in the B cell-enriched lymphocyte population. Nucleoside kinase measurements showed no significant differences in dAdo, adenosine, or 2'-deoxycytidine kinase activities between the T and B lymphocytes. The inability of the T cells to incorporate dAdo or the analog 2'-F-araA into their nucleotide pools may indicate the existence of a highly specific catabolic enzyme(s).

Nucleotidase activities of human peripheral lymphocytes.

Several B lymphoblastic cell lines are known to be relatively resistant to the combination of 2'-deoxyadenosine with an adenosine deaminase inhibitor. These cell lines are believed to have a greater capacity to dephosphorylate 2'-deoxyadenosine nucleotides, thus preventing excessive accumulation of potentially toxic metabolites. In this study, the 2'-deoxynucleoside 5'-monophosphate dephosphorylating activities of human peripheral lymphocytes were examined. Peripheral lymphocytes have at least three nucleotide 5'-monophosphate nucleotidases distinguished by different pH optimums, substrate preference, Mg2+ requirement, inhibitors, and molecular weights. Two of the enzymes appeared to be cytosolic, only one of which had significant substrate activity with dAMP. This enzyme had an acidic pH optimum (5.0), no Mg2+ requirement, was inhibited by tartrate, and demonstrated broad substrate specificity. The other cytosolic nucleotidase required Mg2+, had a pH optimum of 5.5 to 6.0, was activated by 2'-deoxyinosine, and demonstrated a substrate preference for 3'- and 5'-monophosphate 2'-deoxynucleosides of hypoxanthine, guanine, uracil, and thymine. The third enzyme, ecto 5'-nucleotidase, is associated with the cell membrane. Although the ecto 5'-nucleotidase activity was higher in the B lymphocytes, the cytosolic nucleotidases were similar in activity in the T and B lymphocytes.

5'-deoxy-5'-methylthioadenosine phosphorylase--II. Role of the enzyme in the metabolism and antineoplastic action of adenine-substituted analogs of 5'-deoxy-5'-methylthioadenosine.

The biological activities of several previously synthesized [J. A. Montgomery et al., J. med. Chem. 17, 1197 (1974)] adenine-substituted analogs of 5'-deoxy-5'-methylthio- or 5'-deoxy-5'-ethyl-thioadenosine, including the 2-fluoroadenine, 2-chloroadenine, 2,6-diaminopurine, 8-azaadenine, and 4-aminopyrazolo [3,4-d]pyrimidine-containing derivatives, have been reexamined. It is demonstrated that many of these analogs are cleaved to their respective free base analogs by 5'-deoxy-5'-methyl-thioadenosine phosphorylase (MTAPase), an enzyme associated with polyamine biosynthesis, and that this reaction is necessary for the cytotoxic action of these MTA analogs to be fully expressed. Evidence to support this includes: (1) the growth of two MTAPase-containing human colon carcinoma cell lines (the HCT-15 and DLD-1 lines) was inhibited by these analogs, whereas an MTAPase-deficient cell line, the CCRF-CEM human T-cell leukemia, was relatively insensitive to their cytotoxic action; (2) extracts of the MTAPase-containing colon carcinoma cell lines were able to cleave these analogs to their respective free base analogs; in contrast, extracts of MTAPase-deficient CCRF-CEM cells were unable to cleave these analogs; (3) intact colon carcinoma cells converted these MTA analogs to their corresponding 5'-phosphorylated analog nucleotides, whereas CCRF-CEM cells did not, at least to detectable levels; and (4) the MTA analog, 5'-deoxy-5'-ethylthio-4-aminopyrazolo [3,4-d]pyrimidine ribonucleoside, which is not a substrate of MTAPase, did not form analog nucleotides and was essentially noncytotoxic to all cell lines tested, whereas the corresponding adenine analog, 4-aminopyrazolo [3,4-d]pyrimidine, readily formed analog nucleotides and was highly cytotoxic to all the lines. It is postulated that the corresponding adenine analog 5'-phosphorylated nucleotides are the primary active metabolites of these MTA analogs, having been formed by the cleavage of these nucleosides to free adenine analogs by MTAPase, followed by the conversion of these base analogs to analog nucleotides by adenine phosphoribosyltransferase and the enzymes of adenine nucleotide phosphorylation. This pathway represents a novel drug-activation system for the synthesis of analog nucleotides and has the potential to be exploited chemotherapeutically.

Role of adenosine uptake and metabolism by blood cells in the antiplatelet actions of dipyridamole, dilazep and nitrobenzylthioinosine.

Adenosine (Ado, 10 microM) did not inhibit ADP-induced human platelet aggregation in whole blood. However, if the blood was preincubated with dipyridamole (10 microM), a potent inhibitor of the erythrocytic nucleoside transport system (NTS), Ado acted as a strong inhibitor of platelet aggregation. Similarly, Ado inhibited platelet aggregation in whole blood in the presence of other potent NTS inhibitors, dilazep (1 microM) and p-nitrobenzylthioinosine (NBMPR, 1 microM). RA 233 (10 microM), an analog of dipyridamole which is a potent inhibitor of platelet cAMP phosphodiesterase (PDE), did not evoke the Ado effect in whole blood. However, in platelet-rich plasma (PRP), RA 233 potentiated strongly Ado-mediated inhibition, whereas dipyridamole, dilazep and NBMPR were without activity. 5'-Methylthioadenosine (MTA), an Ado receptor antagonist, reversed the inhibition produced by a nucleoside transport system inhibitor plus Ado in whole blood. Dipyridamole (10 microM), dilazep (1 microM) or NBMPR (1 microM) blocked [14C]Ado (10 microM) uptake by blood cells in whole blood, whereas RA 233 (10 microM) was not effective. The combination of 2'-deoxycoformycin (dCF, 5 microM), a tight-binding inhibitor of adenosine deaminase (ADA), plus 5-iodotubercidin (ITu, 10 microM), a potent inhibitor of adenosine kinase (Ado kinase), gave comparable Ado-mediated inhibition of platelet aggregation in whole blood as was obtained when the blood was pretreated with dilazep. These studies suggest that the in vivo antiplatelet actions of drugs such as dipyridamole and dilazep result from their abilities to block erythrocytic Ado uptake and subsequent metabolism, thus elevating the extracellular steady-state concentration of the physiologically occurring, antiplatelet agent, Ado.

Adenosine metabolism in human whole blood. Effects of nucleoside transport inhibitors and phosphate concentration.

Adenosine (Ado, 10 microM) was metabolized in whole blood within 1 min, primarily to hypoxanthine and ATP. The concentration of Ado, the activities of adenosine deaminase (ADA) and Ado kinase, the Km values for Ado with ADA and Ado kinase, and the substrate inhibition of Ado kinase are factors that govern the Ado metabolism between deamination and phosphorylation. If ADA activity was blocked by 2'-deoxycoformycin (dCF, 5 microM), a tight-binding inhibitor of ADA, most of the Ado (96%) was incorporated into adenine nucleotides, whereas if Ado kinase activity was blocked with 5-iodotubercidin (10 microM), Ado was mainly (95%) metabolized into hypoxanthine. A high phosphate concentration (25 mM) caused marked increases in the formation of IMP. The nucleoside transport inhibitors dilazep (1 microM), dipyridamole (10 microM) and nitrobenzylthioinosine (NBMPR, 1 microM) strongly blocked cellular Ado metabolism. In the presence of nucleoside transport inhibitors, Ado which slowly enters the cell was metabolized principally by Ado kinase rather than ADA. Dilazep, NBMPR and dipyridamole were more effective in blocking Ado uptake and metabolism by erythrocytes suspended in a protein-free medium than by cells suspended in plasma.

Transport of deoxycoformycin in human erythrocytes. Measurement by adenosine deaminase titration and radioisotope assays.

The assay of residual adenosine deaminase (ADA) activity was used as a sensitive measure of the transport of deoxycoformycin (dCF) into human erythrocytes. Contrary to prior reports from this laboratory, the inactivation of intraerythrocytic ADA by dCF was linear rather than log-linear, with time. Linear inactivation rates were also seen when erythrocytes were preloaded with a 5-fold excess of calf intestinal ADA. The uptake of tritium-labeled dCF molecules and the rate of inactivation of ADA molecules showed an approximate 1:1 stoichiometry. The nucleoside transport inhibitors, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR) and dipyridamole, and the permeant, uridine, inhibited dCF transport with Ki values of 35 nM, 45 nM, and 340 microM respectively. The affinity of dCF for the nucleoside transporter was low with a Ki of approximately 10 mM for the inhibition of adenosine influx.

5'-Deoxy-5'-methylthioadenosine phosphorylase--III. Role of the enzyme in the metabolism and action of 5'-halogenated adenosine analogs.

5'-Deoxy-5'-halogenated adenosines are alternative substrates for 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAPase), an enzyme responsible for the metabolism of 5'-deoxy-5'-methylthioadenosine (MTA), a by-product of polyamine biosynthesis. The relative reactivity of these nucleosides with MTAPase from HL-60 human promyelocytic leukemia cells is MTA greater than 5'-deoxy-5'-fluoroadenosine (5'-FlAdo) greater than 5'-chloro-5'-deoxyadenosine (5'-ClAdo) greter than 5'-bromo-5'-deoxyadenosine (5'-BrAdo) greater than 5'-deoxy-5'-iodoadenosine (5'-IAdo). In MTAPase-containing cells, the adenine released from the 5'-halogenated adenosine was incorporated into adenine nucleotide pools; cleavage by (MTAPase appeared to be the rate-limiting step in this process. 5'-BrAdo and 5'-IAdo were growth inhibitors (EC50 values less than 10 microM) of MTAPase-containing cell lines (HL-60 human promyelocytic leukemia and the L5178Y murine lymphoblastic leukemia) but were much less active (EC50 values greater than 65 microM) against MTAPase-deficient cell lines (the CCRF-CEM human T cell leukemia and the L1210 murine leukemia). The full cytotoxicity of these compounds, therefore, appeared to be related to their phosphorolysis by MTAPase. Indirect evidence suggests that 5-halogenated ribose-1-phosphate derivatives of 5'-BrAdo or 5'-IAdo produced by the MTAPase reaction were the active metabolites of these 5'-halogenated adenosines.

C(2')-substituted purine nucleoside analogs. Interactions with adenosine deaminase and purine nucleoside phosphorylase and formation of analog nucleotides.

Four C(2')-substituted 2'-deoxyadenosines were examined as substrates for human erythrocytic adenosine deaminase and for formation of intracellular nucleotide analogs in human erythrocytes, lymphocytes and murine Sarcoma 180 cells: 9-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl)adenine, 9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)adenine, 9-(2'-azido-2'-deoxy-beta-D-ribofuranosyl)adenine (2'-N3-riboA) and 9-(2-azido-2'-deoxy-beta-D-arabinofuranosyl)adenine. All four adenosine analogs were substrates of human erythrocytic adenosine deaminase, but the corresponding inosine analogs (synthesized by the adenosine deaminase reaction) were highly resistant to cleavage by human erythrocytic purine nucleoside phosphorylase. Only 9-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl)hypoxanthine underwent very slow phosphorolysis, and no inhibition of inosine phosphorolysis was detected when a 30 microM concentration of any studied inosine analog was added to a reaction mixture containing 30 microM inosine (the Km concentration). Kinetic parameters were determined for the deamination of the adenosine analogs. The greatest affinity for adenosine deaminase was found with 2'-N3-ribo A (Ki = 2 microM), but the reaction velocity was highest with the F-substituted analogs. All four adenosine analogs formed triphosphate nucleotides after incubation with human erythrocytes, murine Sarcoma 180 cells, or human lymphocytes (tested only with the F analogs) in the presence of deoxycoformycin.

Inhibition of nucleoside transport by nitrobenzylthioformycin analogs.

The formycin analogs of nitrobenzylthioinosine and nitrobenzylthioguanosine were synthesized and evaluated as nucleoside transport inhibitors. These analogs have a potential therapeutic advantage over their parent compounds in that their C-nucleosidic linkages prevent them from being degraded to the immunosuppressive agents, 6-mercaptopurine and 6-thioguanine. 7-[(4-Nitrobenzyl)-thio]-3-(beta-D-ribofuranosyl)pyrazolo[4,3- d]pyrimidine (NBTF) and 5-amino-7-[(4-nitrobenzyl)thio]-3-(beta-D- ribofuranosyl)pyrazolo[4,3-d]pyrimidine (NBTGF) were inhibitors of nucleoside transport in human erythrocytes and HL-60 leukemia cells. The IC50 value for nitrobenzylthioinosine, NBTF and NBTGF with 10% erythrocyte suspensions were 18, 18 and 40 nM respectively. Specific binding studies with [3H]NBTF yielded a Kd of 3.4 nM with erythrocytes, approximately 10-fold higher than values reported for nitrobenzylthioinosine. NBTF and nitrobenzylthioinosine bound to HL-60 cells with Kd values of 8.1 and 0.81 nM respectively. The octanol/water partition coefficients of nitrobenzylthioinosine, NBTF and NBTGF were 3.5, 3.2, and 2.8 respectively. NBTF could be expected to be equipotent with nitrobenzylthioinosine in whole blood where inhibitor concentrations of 10(-7) to 10(-6) M are required in order to saturate erythrocytic binding sites; hence, it may exhibit the advantages inherent in a C-nucleoside.