Interactions of nucleoside analogs, caffeine, and nicotine with human concentrative nucleoside transporters 1 and 2 stably produced in a transport-defective human cell line

Pharmacologically important drugs were examined as potential inhibitors or permeants of human concentrative nucleoside transporters 1 (hCNT1)- and 2 (hCNT2)-producing stable transfectants by assessing their abilities to inhibit uridine transport. hCNT1 exhibited high affinities for uridine analogs (5-fluorouridine, 2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, and 5-fluoro-5'-deoxyuridine) with K(i) values of 22 to 33 microM, whereas hCNT2 exhibited moderate affinities for 5-fluoro-2'-deoxyuridine, high affinities for 2'-deoxyuridine and 5-fluorouridine, and low affinity for 5-fluoro-5'-deoxyuridine. The uridine analogs were transported at 2-fold higher rates (at 10 microM) by hCNT1 than by hCNT2. Enantiomeric configuration and the 3'-hydroxyl group of the ribose ring were important determinants for interaction with hCNTs, whereas the 2'-hydroxyl group was less important. Both transporters bound N(6)-(p-aminobenzyl)adenosine with affinities similar to those of adenosine (K(i) = 28-39 microM). Other adenosine receptor ligands, including caffeine, bound better to hCNT1 than to hCNT2 (K(i) = 46 versus 103 microM, respectively), whereas 2-chloroadenosine bound better to hCNT2 than to hCNT1 (K(i) = 37 and 101 microM, respectively). There was a greater than 3-fold difference in binding affinities between hCNT1 and hCNT2 for nicotine (K(i) = 63 versus 227 microM). However, direct measurements of nicotine and caffeine uptake rates (10 microM) failed to demonstrate mediated uptake by either transporter. Although hCNT1 bound several adenosine analogs relatively well, it did not transport 2-chloro-2'-deoxyadenosine (cladribine) or 2-fluoro-9-beta-d-arabinofuranosyladenine (fludarabine), whereas hCNT2 transported both, albeit with low activities. The results indicated that although hCNT1 and hCNT2 possess some overlap in transport of several uridine and adenosine analogs, they also exhibit distinct differences in capacity to interact with some adenosine receptor ligands, adenosine-based drugs, and nicotine.     

Molecular requirements of the human nucleoside transporters hCNT1, hCNT2, and hENT1

Concentrative nucleoside transporters (CNTs) and equilibrative nucleoside transporters (ENTs) are important in physiological and pharmacological activity and disposition of nucleosides and nucleoside drugs. A better understanding of the structural requirements of inhibitors for these transporters will aid in designing therapeutic agents. To define the relative and unified structural requirements of nucleoside analogs for interaction with hCNT1, hCNT2, and hENT1, we applied an array of structure-activity techniques. Unique pharmacophore models for each respective nucleoside transporter were generated. These models reveal that hCNT2 affinity is dominated by hydrogen bonding features, whereas hCNT1 and hENT1 displayed mainly electrostatic and steric features. Hydrogen bond formation over 3'-OH is essential for all nucleoside transporters. Inhibition of nucleoside transporters by a series of uridine and adenosine analogs and a variety of drugs was analyzed by comparative molecular field analysis. Cross-validated r2 (q2) values were 0.65, 0.52, and 0.74 for hCNT1, hCNT2, and hENT1, respectively. The predictive quality of the models was further validated by successful prediction of the inhibition of a set of test compounds. Addition of a hydroxyl group around the 2-position of purine (or 3-position of pyrimidine) may increase inhibition to hCNT2 transporter; addition of hydroxyl group around the 2,7-position of purine (or the 3,5-position of pyrimidine) would increase the inhibition to hENT1 transporter. Utilization of these models should assist the design of high-affinity nucleoside transporter inhibitors and substrates for both anticancer and antiviral therapy.     

A comparison of the transportability, and its role in cytotoxicity, of clofarabine, cladribine, and fludarabine by recombinant human nucleoside transporters produced in three model expression systems

2-Chloro-9-(2'-deoxy-2'-fluoro-beta-d-arabinofuranosyl)adenine (Cl-F-ara-A, clofarabine), a purine nucleoside analog with structural similarity to 2-chloro-2'-deoxyadenosine (Cl-dAdo, cladribine) and 9-beta-d-arabinofuranosyl-2-fluoroadenine (F-ara-A, fludarabine), has activity in adult and pediatric leukemias. Mediated transport of the purine nucleoside analogs is believed to occur through the action of two structurally unrelated protein families, the equilibrative nucleoside transporters (ENTs) and the concentrative nucleoside transporters (CNTs). The current work assessed the transportability of Cl-F-ara-A, Cl-dAdo, and F-ara-A in cultured human leukemic CEM cells that were either nucleoside transport-defective or possessed individual human nucleoside transporter types and in Xenopus laevis oocytes and Saccharomyces cerevisiae yeast that produced individual recombinant human nucleoside transporter types. Cells producing hENT1 or hCNT3 exhibited the highest uptake of Cl-F-ara-A, whereas nucleoside transport-deficient cells and cells producing hCNT1 lacked uptake altogether. When Cl-F-ara-A transport rates by hENT1 were compared with those of Cl-dAdo and F-ara-A, Cl-dAdo had the highest efficiency of transport, although Cl-F-ara-A showed the greatest accumulation during 5-min exposures. In cytotoxicity studies with the CEM lines, Cl-F-ara-A was more cytotoxic to cells producing hENT1 than to the nucleoside transport-deficient cells. The efficiency of Cl-F-ara-A transport by oocytes with recombinant transporters was hCNT3 > hENT2 > hENT1 > hCNT2; no transport was observed with hCNT1. Affinity studies with recombinant transporters produced in yeast showed that hENT1, hENT2, and hCNT3 all had higher affinities for Cl-F-ara-A than for either Cl-dAdo or F-ara-A. These results suggest that the nature and activity of the plasma membrane proteins capable of inward transport of nucleosides are important determinants of Cl-F-ara-A activity in human cells.     

Interaction of fused-pyrimidine nucleoside analogs with human concentrative nucleoside transporters: High-affinity inhibitors of human concentrative nucleoside transporter 1

Human concentrative nucleoside transporters (hCNTs) mediate electrogenic secondary active transport of physiological nucleosides and nucleoside drugs into cells. Six fused-pyrimidine ribonucleosides and one 2′-deoxynucleoside were assessed for their abilities to inhibit [³H]uridine transport in the yeast Saccharomyces cerevisiae producing recombinant hCNT1, hCNT2 or hCNT3. Six of the analogs inhibited hCNT1 with K_i values < 1 μM whereas only two analogs inhibited hCNT3 with K_i values < 1 μM and none inhibited hCNT2. To assess if the inhibitory analogs were also permeants, currents evoked were measured in oocytes of Xenopus laevis producing recombinant hCNT1, hCNT2 or hCNT3. Significant inward currents, indicating permeant activity, were generated with (i) three of the analogs in hCNT1-producing oocytes, (ii) none of the analogs in hCNT2-producing oocytes and (iii) all of the analogs in hCNT3-producing oocytes. Four were not, or were only very weakly, transported by hCNT1. The thienopyrimidine 2′-deoxynucleoside (dMeThPmR, 3) and ribonucleoside (MeThPmR, 4) were the most active inhibitors of uridine transport in hCNT1-producing oocytes and were an order of magnitude more effective than adenosine, a known low-capacity transport inhibitor of hCNT1. Neither was toxic to cultured human leukemic CEM cells, and both protected CEM cell lines with hCNT1 but not with hENT1 against gemcitabine cytotoxicity. In summary, dMeThPmR (3) and MeThPmR (4) were potent inhibitors of hCNT1 with negligible transportability and little apparent cytotoxicity, suggesting that pending further evaluation for toxicity against normal cells, they may have utility in protecting normal hCNT1-producing tissues from toxicities resulting from anti-cancer nucleoside drugs that enter via hCNT1.     

Antiviral activity, antimetabolic activity, and cytotoxicity of 3'-substituted deoxypyrimidine nucleosides

The antiviral activity, effect on cellular DNA and RNA synthesis, and cytotoxicity toward mammalian cells of 5-fluoro-2'-deoxyuridine, 5-methoxymethyl-2'-deoxyuridine, 2'-deoxythymidine, and their corresponding 3'-p-nitrophenylphosphate and 3'-p-aminophenylphosphate derivatives were determined. The 3'-p-aminophenylphosphate-2'-deoxy-5-methoxymethyluridine derivative was as potent as 5-methoxy-methyl-2'-deoxyuridine in inhibiting herpes simplex viruses; however, 3'-p-aminophenylphosphate-2'-deoxy-5-fluorouridine was less potent than 5-fluoro-2'-deoxyuridine in inhibiting viral replication. The results suggest that the deoxypyrimidine ribonucleoside kinase has bulk tolerance for substituents at the 3-position of the ribofuranose moiety. The effect on cellular DNA and RNA synthesis and cytotoxicity toward mammalian cells were monitored by studying the incorporation of radioactive precursors. 5-Methoxymethyl-2'-deoxyuridine and 3'-p-aminophenylphosphate-2'-deoxy-5-methoxymethyluridine failed to inhibit DNA or RNA synthesis. 5-Fluoro-2'-deoxyuridine and 3'-p-aminophenylphosphate-2'-deoxy-5-fluorouridine decreased incorporation of [3H]deoxyuridine by 50% at 1.0 and 40 microM, respectively. Cytotoxicity (microscopic lesions using monolayer cells) on exposure to 5-methoxymethyl-2'-deoxyuridine, 3'-p-aminophenylphosphate-2'-deoxy-5-methoxymethyluridine, 5-fluoro-2'-deoxyuridine, and 3'-p-aminophenylphosphate-2'-deoxy-5-fluorouridine was observed at 3800, 1600, 1.6, and 110 microM, respectively.     

Uridine binding motifs of human concentrative nucleoside transporters 1 and 3 produced in Saccharomyces cerevisiae

An extensive series of structural analogs of uridine that differed in substituents in the sugar and/or base moieties were subjected to inhibitor-sensitivity assays in a yeast expression system to define uridine structural determinants for inhibitors of human concentrative nucleoside transporters 1 and 3 (hCNT1 and hCNT3). The production of recombinant hCNT1 and hCNT3 in a nucleoside-transporter deficient strain of yeast was confirmed by immunoblotting, and uridine transport parameters (Km, Vmax) were determined by defining the concentration dependence of initial rates of uptake of [3H]uridine by intact yeast. The Ki values of uridine analogs were obtained from inhibitory-effect curves and converted to binding energies. hCNT1 and hCNT3 recognized uridine through distinguishable binding motifs. hCNT1 was sensitive to modifications at C(3), less sensitive at C(5') or N(3), and much less sensitive at C(2'). hCNT3 was sensitive to modifications at C(3'), but much less sensitive at N(3), C(5') or C(2'). The changes of binding energy between transporter proteins and different uridine analogs suggested that hCNT1 formed hydrogen bonds (H-bonds) with C(3')-OH, C(5')-OH, or N(3)-H of uridine, but not with C(2')-OH, whereas hCNT3 formed H-bonds to C(3')-OH, but not to N(3)-H, C(5')-OH, and C(2')-OH. Both transporters barely tolerated modifications at C(3') or inversion of configurations at C(2')orC(3'). The binding profiles identified in this study can be used to predict the potential transportability of nucleoside analogs, including anticancer or antiviral nucleoside drugs, by hCNT1 and hCNT3.     

Inhibition of poly(ADP-ribose)polymerase activity by nucleoside analogs of thymidine.

The poly ADP-ribosylation of proteins catalyzed by poly(ADP-ribose)polymerase (PARP) is involved in a number of important cellular metabolic activities. We evaluated various analogs of deoxythymidine and deoxyuridine as inhibitors of PARP. Most of these compounds have antiviral and/or anticancer activities. The structural requirements for these nucleoside analogs to be inhibitors of PARP were determined. The compounds evaluated had various substitutions on the 2-, 4- and/or 5-position of the pyrimidine ring, as well as on the 2'-, 3'- and/or 5'-position of the pentose moiety. Inhibition of PARP was strongly dependent on the size of the alkyl or halogen substituent on the 5-position of the pyrimidine ring. Whereas the 5-position of the pyrimidine ring could be varied, alteration of the 2- or 4-position drastically decreased the inhibition of PARP. Kinetic analysis was performed with concentrations of 1-10 microM NAD+. The Ki values for many compounds were five to seven times lower than the Ki for 3-aminobenzamide, a previously described potent inhibitor of PARP. Compounds with combined substituents at both the 5-position of the pyrimidine ring and the 3'- or 5'-position of deoxyribose generally were potent inhibitors of PARP, as for example 3'-amino-2', 3'-dideoxy-(E)-5-(2-bromovinyl)uridine (Ki = 0.7 microM), or 5'-azido-2',5'-dideoxy-5-ethyluridine (Ki = 0.8 microM). The 5-halogenated analogs had Ki values of 18, 35, 110 and greater than 1000 microM for 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-chloro-2'-deoxyuridine, and 5-fluoro-2'-deoxyuridine, respectively, and the 5-alkyl analogs had Ki values of 45, 2.2, 7, 16 and 180 microM for 5-methyl-2'-deoxyuridine, 5-ethyl-2'-deoxyuridine, 5-propyl-2'-deoxyuridine, 5-butyl-2'-deoxyuridine and 5-pentyl-2'-deoxyuridine, respectively. Two other compounds with substituents in the 5-position of the pyrimidine moiety also had potent activities: (E)-5-(2-bromovinyl)-2'-deoxyuridine (Ki = 6 microM) and 5-trifluoromethyl-2'-deoxyuridine (Ki = 1.6 microM). Compounds substituted in the 2'-, 3'- and/or 5'-position of the deoxyribose moiety were investigated and 5'-azido-5'-deoxythymidine, 5'-amino-5'-deoxythymidine, 3'-azido-3'-deoxythymidine and 3'-deoxythymidine (d2T) and Ki values of 12, 16, 18 and 30 microM, respectively.     

Homogeneous uridine kinase from Ehrlich ascites tumor: substrate specificity and inhibition by bisubstrate analogs

Uridine kinase has been purified to homogeneity from Ehrlich ascites tumor cells. For the phosphate acceptor site, the enzyme shows substrate specificity only for ribopyrimidine nucleosides and is active with various analogs that have limited structural alterations; both endocyclic and exocyclic substituents can be acceptable. Of nucleosides that have been used in the chemotherapy of cancer, 5-fluorouridine, 6-azauridine, and 3-deazauridine are good substrates, whereas arabinosylcytosine is a poor substrate. No analogs are better substrates than the physiological substrates uridine and cytidine. 5', 5' -P1, P4-Bisnucleoside oligophosphate bisubstrate analogs (e.g., Ap4U, Ap5U) were synthesized and tested as inhibitors. The most effective compound was Ap4U; with a Ki of 197 microM, it bound more tightly than ATP but no better than uridine. Ap3A, Ap4A, and Ap5A were also tested, with the result that both Ap4A and Ap4U were most effective, suggesting that this size of bisubstrate analog most closely approaches the spacing of the catalytic site.     

Role of human nucleoside transporters in the cellular uptake of two inhibitors of IMP dehydrogenase, tiazofurin and benzamide riboside

Benzamide riboside (BR) and tiazofurin (TR) are converted to analogs of NAD that inhibit IMP dehydrogenase (IMPDH), resulting in cellular depletion of GTP and dGTP and inhibition of proliferation. The current work was undertaken to identify the human nucleoside transporters involved in cellular uptake of BR and TR and to evaluate their role in cytotoxicity. Transportability was examined in Xenopus laevis oocytes and Saccharomyces cerevisiae that produced individual recombinant human concentrative nucleoside transporter (CNT) and equilibrative nucleoside transporter (ENT) types (hENT1, hENT2, hCNT1, hCNT2, or hCNT3). TR was a better permeant than BR with a rank order of transportability in oocytes of hCNT3 > hENT1 > hENT2 > hCNT2 > hCNT1. The concentration dependence of inhibition of [(3)H]uridine transport in S. cerevisiae by TR exhibited lower K(i) values than BR: hCNT3 (5.4 versus 226 microM), hENT2 (16 versus 271 microM), hENT1 (57 versus 168 microM), and hCNT1 (221 versus 220 microM). In cytotoxicity experiments, BR was more cytotoxic than TR to cells that were either nucleoside transport-defective or -competent, and transport-competent cells were more sensitive to both drugs. Exposure to nitrobenzylmercaptopurine ribonucleoside conferred resistance to BR and TR cytotoxicity to hENT1-containing CEM cells, thereby demonstrating the importance of transport capacity for manifestation of cytoxicity. A breast cancer cell line with mutant p53 exhibited 9-fold higher sensitivity to BR than the otherwise similar cell line with wild-type p53, suggesting that cells with mutant p53 may be potential targets for IMPDH inhibitors. Further studies are warranted to determine whether this finding can be generalized to other cell types.     

Identification and quantitation of 5-fluorouridine in rat urine after administration of 5-fluorouracil or 5-fluoro-2'-deoxyuridine

The ribonucleoside 5-fluorouridine was identified in 24-hr urine samples from rats after ip administration of 5-fluorouracil (200 mg/kg) or 5-fluoro-2'-deoxyuridine (380 mg/kg) by means of thin-layer chromatography, high-pressure liquid chromatography, capillary gas-liquid chromatography and gas-liquid chromatography-mass spectrometry techniques, and by comparison with the synthetic compound. By use of a specific extraction procedure, 5-fluorouridine was then quantitatively determined by capillary gas-liquid chromatography with a nitrogen-selective detector. These measurements indicate that not more than 0.2% of the administered dose was excreted as 5-fluorouridine; lower urinary metabolite levels were found after treatment with 5-fluorouracil than after 5-fluoro-2'-deoxyuridine administration. Following an ip dose of either drug, variable amounts of 2'-deoxyuridine and thymidine were detected in the urine samples.     

Heterogeneity of nucleoside transport in mammalian cells. Two types of transport activity in L1210 and other cultured neoplastic cells

The characteristics of nucleoside transport were examined in L1210 murine leukemia cells and five other cultured neoplastic cells. The initial rates of uridine, adenosine, and thymidine transport in L1210 cells were only partially inhibited by 1 microM nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport in other cells. The IC50 for NBMPR inhibition of uridine transport was 5 nM, but 20% of the activity remained insensitive to concentrations as high as 3 microM. Uridine uptake in the presence of 1 microM NBMPR was saturable and was inhibited by other nucleosides, suggesting the participation of an NBMPR-insensitive transport mechanism. There appeared to be little difference in the specificity of NBMPR-sensitive and -insensitive transport for the physiological nucleosides. Uridine, adenosine, and thymidine were all substrates for both mechanisms, and the Km values for total and NBMPR-insensitive uridine transport were the same (250 microM). Furthermore, little difference was found in the ability of several other nucleosides to inhibit total or NBMPR-insensitive uridine transport. In both cases, adenosine was the most effective inhibitor while cytidine and deoxycytidine were the least effective. The two transport processes did, however, differ from each other in their sensitivity to p-mercuribenzenesulfonate (pMBS). NBMPR-insensitive uridine transport was inhibited by pMBS with an IC50 less than 25 microM, while the IC50 for NBMPR-sensitive transport was greater than 400 microM. Cloning of the parent L1210 cell line indicated that both NBMPR-sensitive and -insensitive transport occurred in the same cell. Both types of uridine transport activity were also observed in three other cell lines (RPMI 6410, L5178Y, and P388), while two lines, S49 and Walker 256, exhibited only NBMPR-sensitive and -insensitive transport, respectively. The level of NBMPR-insensitive transport was an important determinant in the ability of NBMPR to inhibit uridine uptake over prolonged periods (10 min), with as little as 20% NBMPR-insensitive transport sufficient to render uptake over 10 min virtually insensitive to NBMPR. The existence of these two types of nucleoside transport activity in mammalian cells may have important implications in the chemotherapeutic use of transport inhibitors in combination with cytotoxic nucleosides or with inhibitors of pyrimidine and purine biosynthesis.     

Mouse equilibrative nucleoside transporter 2 (mENT2) transports nucleosides and purine nucleobases differing from human and rat ENT2

Several mammalian nucleoside transporters have been identified at the molecular level. Human and rat equilibrative nucleoside transporter 2 (hENT2 and rENT2, respectively) was previously reported to have the dual ability of transporting both nucleosides and nucleobases. In the present study, we characterized the transport of a variety of nucleosides and nucleobases via recombinant mouse ENT2 (mENT2). Cloned mENT2 mediated the uptake of nucleosides and purine nucleobases, but not pyrimidine nucleobases. The mENT2-mediated uptake of adenosine was significantly inhibited by nucleosides and nucleobases, irrespective of purine and pyrimidine. The K(m) values for the uptake of nucleosides and purine nucleobases mediated by mENT2 varied between 1.24 and 16.3 microM, and the transport clearances of adenosine and hypoxanthine via the transporter were greater than those of other substrates. Therefore, we concluded that mENT2 is nucleoside and purine nucleobase transporter, and pyrimidine nucleobases are blockers for the transporter, differing from hENT2 and rENT2 that were reported to also transport pyrimidine nucleobases.     

Identification and functional analysis of variants in the human concentrative nucleoside transporter 2, hCNT2 (SLC28A2) in Chinese, Malays and Indians

The human concentrative nucleoside transporter (hCNT2), also known as SLC28A2, plays an important role in the cellular uptake across intestinal membrane of some naturally occurring nucleosides and nucleoside analogs. This study aims to determine the genetic variability of hCNT2 (SLC28A2) in three major Asian ethnic groups residing in Singapore: Chinese, Malay and Indian, and functionally characterize the variants of hCNT2. Healthy participants (n=96) from each group were screened for genetic variations in the exons of hCNT2 (SLC28A2) using denaturing high performance liquid chromatography and sequencing analyses. A total of 23 polymorphisms were identified in the exonic and flanking intronic regions, and ethnic differences in single nucleotide polymorphism frequencies were evident. Five novel nonsynonymous variants (L12R, R142H, E172D, E385K, M612T) were constructed by mutagenesis and functionally characterized in U-251 cells. Expression of these variants in U-251 cells revealed that all except E385K can uptake various substrates of hCNT2: inosine, ribavirin and uridine.     

Adenosine transporters in bloodstream forms of Trypanosoma brucei brucei: substrate recognition motifs and affinity for trypanocidal drugs

Adenosine influx by Trypanosoma brucei brucei P1 and P2 transporters was kinetically characterized. The P1 transporter displayed a higher affinity and capacity for adenosine (K(m) = 0.38 +/- 0.10 microM, V(max) = 2.8 +/- 0.4 pmol x 10(7) cells(-1) x s(-1)) than the P2 transporter (K(m) = 0.92 +/- 0.06 microM, V(max) = 1.12 +/- 0.08 4 pmol x 10(7) cells(-1) x s(-1)). To formulate a structure-activity relationship for the interaction of adenosine with the transporters, a series of analogs were evaluated as potential inhibitors of adenosine transport, and the K(i) values were converted to binding energy. The P1 transporter was found to be selective inhibited by purine nucleosides (K(i) approximately 1 microM for inosine and guanosine), but nucleobases and pyrimidines had little effect on P1-mediated transport. The P1 transporter appears to form hydrogen bonds with N3 and N7 of the purine ring as well as with the 3' and 5' hydroxyl groups of the ribose moiety, with apparent bond energies of 12.8 to 15.8 kJ/mol. The P2 transporter, in contrast, had high-affinity (K(i) = 0.2-4 microM) for 6-aminopurines, including adenine, 2'-deoxyadenosine, and tubercidin, but not for any oxopurines. The main interaction of adenosine with the P2 transporter is suggested to be via hydrogen bonds to N1 and the 6-amino group. Additional pi-pi interactions of the purine ring and electrostatic interactions with N9 may also be important. The predicted substrate recognition motif of P2, but not of P1, corresponds to parts of the melaminophenylarsenical and diamidine molecules, confirming the potent inhibition observed with these trypanocides for P2-mediated adenosine transport (K(i) = 0.4-2.4 microM).     

Membrane permeation characteristics of 5'-modified thymidine analogs.

The membrane permeation characteristics of 5'-deoxythymidine (5'-ddThd) and 5'-azido-5'-deoxythymidine (5'-N3-5'-ddThd) were investigated in human erythrocytes, with an inhibitor-stop assay, at 20 degrees. Uptake of both nucleoside analogs occurred without metabolism, was nonconcentrative, and was partially inhibited by nucleosides or inhibitors of nucleoside transport at micromolar permeant concentrations. At higher permeant concentrations (greater than 1.0 mM), the influx rate of each analog was linearly dependent on concentration and insensitive to inhibition by nucleosides, inhibitors of nucleoside transport, and nucleobases. Kinetic analyses using nonlinear regression revealed that a saturable component of 5'-ddThd influx (Km = 200 microM) was competitively inhibited by thymidine (dThd) (Ki = 86 microM) or 5-iodo-2'-deoxyuridine (Ki = 84 microM). Similarly, a saturable component of 5'-N3-5'-ddThd influx (Km = 220 microM) was competitively inhibited by 2-chloroadenosine (Ki = 18 microM). The Ki values for these nucleoside inhibitors were similar to their reported Km values as permeants of the nucleoside transporter. Both 5'-ddThd and 5'-N3-5'-ddThd competitively inhibited the influx of dThd (Km = 60 microM), with similar Ki values (150 and 200 microM, respectively). We conclude that these two 5'-modified dThd analogs enter human erythrocytes both by nonfacilitated diffusion and by the nucleoside transporter. The absence of the 5'-hydroxyl group of dThd (5'-ddThd) resulted in a large increase in the octanol/buffer partition coefficient, in an ability to permeate human erythrocytes by nonfacilitated diffusion, and in a 3-fold diminished binding to the nucleoside transporter. The 5'-azido group (5'-N3-5'-ddThd) resulted in an additional 1.4-fold increase in the octanol/buffer partition coefficient and in a 2-fold increase in the rate of nonfacilitated diffusion.     

Synthesis and biological activity of various 3'-azido and 3'-amino analogues of 5-substituted pyrimidine deoxyribonucleosides

Various new 5-substituted 3'-azido- and 3'-amino derivatives of 2'-deoxyuridine and 2'-deoxycytidine have been synthesized and biologically evaluated. Among these compounds, 3'-amino-2',3'-dideoxy-5-fluorouridine (3), 3'-amino-2',3'-dideoxycytidine (7a), and 3'-amino-2',3'-dideoxy-5-fluorocytidine (7c) were found to be the most active against murine L1210 and sarcoma 180 neoplastic cells in vitro, with an ED50 of 15 and 1 microM, 0.7 and 4 microM, and 10 and 1 microM, respectively. The 3'-azido derivatives, 2 and 6c, were less active in comparison with their 3'-amino counterparts. In addition, the 5-fluoro-3'-amino nucleosides, 3 and 7c, were tested against L1210 leukemia bearing CDF1 mice. Our preliminary findings indicate that compound 7c (6 X 200 mg/kg) was as active as the positive control, 5-fluorouracil (6 X 20 mg/kg), yielding a T/C X 100 of 146 and 129, respectively. However, 3 was found to be inactive in this experiment.     

Differential effects of 2,2'-anhydro-5-ethyluridine, a uridine phosphorylase inhibitor, on the antitumor activity of 5-fluorouridine and 5-fluoro-2'-deoxyuridine

2,2'-Anhydro-5-ethyluridine (ANEUR), a potent inhibitor of uridine phosphorylase, markedly potentiated the antitumor activity of fluorouridine (FUR) against murine mammary adenocarcinoma 755 in BDF1 mice and human colon adenocarcinoma LS174T in athymic-nude mice. Whereas ANEUR annihilated the antitumor activity of 5-fluoro-2'-deoxyuridine (FUdR) and 5'-deoxy-5-fluorouridine (DFUR) in the murine adenocarcinoma 755 system, it did not alter the antitumor activity of FUdR in the human adenocarcinoma LS174T system. In vitro, ANEUR proved inhibitory to the phosphorolytic cleavage of both FUR and FUdR by uridine phosphorylase, and this could explain why in vivo conversion of FUR and FUdR to 5-fluorouracil was suppressed. FUR can be held directly responsible for the antitumor effects observed in the murine adenocarcinoma 755 system, whereas in the activity against human adenocarcinoma LS174T may be mediated by both FUR and FUdR.     

Selective abolishment of pyrimidine nucleoside kinase activity of herpes simplex virus type 1 thymidine kinase by mutation of alanine-167 to tyrosine

Herpes simplex virus type 1 (HSV-1) encodes a thymidine kinase (TK) that markedly differs from mammalian nucleoside kinases in terms of substrate specificity. It recognizes both pyrimidine 2'-deoxynucleosides and a variety of purine nucleoside analogs. Based on a computer modeling study and in an attempt to modify this specificity, an HSV-1 TK mutant enzyme containing an alanine-to-tyrosine mutation at amino acid position 167 was constructed. Compared with wild-type HSV-1 TK, the purified mutant HSV-1 TK(A167Y) enzyme was heavily compromised in phosphorylating pyrimidine nucleosides such as (E)-5-(2-bromovinyl)-2'-deoxyuridine and the natural substrate dThd, whereas its ability to phosphorylate the purine nucleoside analogs ganciclovir (GCV) and lobucavir was only reduced approximately 2-fold. Moreover, a markedly decreased competition of natural pyrimidine nucleosides (i.e., thymidine) with purine nucleoside analogs for phosphorylation by HSV-1 TK(A167Y) was observed. Human osteosarcoma cells transduced with the wild-type HSV-1 TK gene were extremely sensitive to the cytostatic effects of antiherpetic pyrimidine [i.e., (E)-5-(2-bromovinyl)-2'-deoxyuridine] and purine (i.e., GCV) nucleoside analogs. Transduction with the HSV-1 TK(A167Y) gene sensitized the osteosarcoma cells to a variety of purine nucleoside analogs, whereas there was no measurable cytostatic activity of pyrimidine nucleoside analogs. The unique properties of the A167Y mutant HSV-1 TK may give this enzyme a therapeutic advantage in an in vivo setting due to the markedly reduced dThd competition with GCV for phosphorylation by the HSV-1 TK.     

Na(+)-dependent, concentrative nucleoside transport in rat macrophages. Specificity for natural nucleosides and nucleoside analogs, including dideoxynucleosides, and comparison of nucleoside transport in rat, mouse and human macrophages.

The effects of natural nucleosides and various analogs thereof on Na(+)-dependent, concentrative transport of formycin B by cultured rat macrophages were investigated. Concentrative transport is the sole nucleoside transport system of these cells. The results indicated that uridine, 5'-fluorouridine, all natural purine nucleosides, 2-chloroadenosine and 5'-deoxyadenosine are efficient substrates for the transporter. None of nine other pyrimidine nucleosides was transported. 3'-Deoxy-adenosine, 2',3'-dideoxyadenosine, 8-azidoadenosine, tubercidin, 5'-methylthioadenosine 6-mercaptopurine riboside and adenosine arabinoside were either poor substrates or not transported significantly. The substrate activity of some of the natural nucleosides and the lack of substrate activity of 3'-deoxyadenosine, 2',3'-dideoxyadenosine, 8-azidoadenosine and 2',3'-dideoxycytidine were confirmed by direct uptake measurements. No significant concentrative nucleoside transport was detected in cultured human monocytes/macrophages, whereas mouse macrophages possessed both concentrative and equilibrative nucleoside transporters.     

Uridine binding and transportability determinants of human concentrative nucleoside transporters

Human concentrative nucleoside transporters 1, 2, and 3 (hCNT1, hCNT2, and hCNT3) exhibit different functional characteristics, and a better understanding of their permeant selectivities is critical for development of nucleoside analog drugs with optimal pharmacokinetic properties. In this study, the sensitivity of a high-throughput yeast expression system used previously for hCNT1 and hCNT3 was improved and used to characterize determinants for interaction of uridine (Urd) with hCNT2. The observed changes of binding energy between hCNT2 and different Urd analogs suggested that it interacts with C3'-OH, C5'-OH, and N3-H of Urd. The C2' and C5 regions of Urd played minor but significant roles for Urd-hCNT2 binding, possibly through Van der Waals interactions. Because the yeast assay only provided information about potential transportability, the permeant selectivities of recombinant hCNT1, hCNT2, and hCNT3 produced in Xenopus laevis oocytes were investigated using a two-electrode voltage clamp assay. hCNT1-mediated transport was sensitive to modifications of the N3, C3', and C5' positions of Urd. hCNT2 showed some tolerance for transporting Urd analogs with C2' or C5 modifications, little tolerance for N3 modifications, and no tolerance for any modifications at C3' or C5' of Urd. Although hCNT3 was sensitive to C3' modifications, it transported a broad range of variously substituted Urd analogs. The transportability profiles identified in this study, which reflected the binding profiles well, should prove useful in the development of anticancer and antiviral therapies with nucleoside drugs that are permeants of members of the hCNT protein family.