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.     

Acquisition of human concentrative nucleoside transporter 2 (hcnt2) activity by gene transfer confers sensitivity to fluoropyrimidine nucleosides in drug-resistant leukemia cells

CEM-ARAC leukemia cells with resistance to cytarabine were shown to lack equilibrative transporter (hENT1) expression and activity. Stable transfer of hCNT2 cDNA into CEM-ARAC enabled Na(+)-dependent transport of purine and pyrimidine nucleoside analogs and provided a unique in vitro model for studying hCNT2. Analysis of [(3)H]uridine inhibitory activity by test substances in hCNT2 transfectant ARAC/D2 revealed structural requirements for interaction with hCNT2: 1) ribosyl and 2'-deoxyribosyl nucleosides were better inhibitors than 3'-deoxyribosyl, 2',3'-dideoxyribosyl or arabinosyl nucleosides; 2) uridine analogs with halogens at position 5 were better inhibitors than 5-methyluridine or thymidine; 3) 2-chloroadenosine was a better inhibitor than 2-chloro-2'-deoxyadenosine (cladribine); and 4) cytosine-containing nucleosides, 7-deazaadenosine and nucleobases were not inhibitors. Quantification of inhibitory capacity yielded K(i) values of 34-50 microM (5-halogenated uridine analogs, 2'-deoxyuridine), 82 microM (5-fluoro-2'-deoxyuridine), 197-246 microM (5-methyluridine < 5-bromo-2'-deoxyuridine < 5-iodo-2'-deoxyuridine), and 411 microM (5-fluoro-5'-deoxyuridine, capecitabine metabolite). Comparisons of hCNT2-mediated transport rates indicated halogenated uridine analogs were transported more rapidly than halogenated adenosine analogs, even though hCNT2 exhibited preference for physiologic purine nucleosides over uridine. Kinetics of hCNT2-mediated transport of 5-fluorouridine and uridine were similar (K(m) values, 43-46 microM). The impact of hCNT2-mediated transport on chemosensitivity was assessed by comparing antiproliferative activity of nucleoside analogs against hCNT2-containing cells with transport-defective, drug-resistant cells. Chemosensitivity was restored partially for cladribine, completely for 5-fluorouridine and 5-fluoro-2'-deoxyuridine, whereas there was little effect on chemosensitivity for fludarabine, 7-deazaadenosine, or cytarabine. These studies, which demonstrated hCNT2 uptake of halogenated uridine analogs, suggested that hCNT2 is an important determinant of cytotoxicity of this class of compounds in vivo.     

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.     

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.     

Ribavirin uptake by cultured human choriocarcinoma (BeWo) cells and Xenopus laevis oocytes expressing recombinant plasma membrane human nucleoside transporters

We investigated the mechanism of the transport of ribavirin (1-beta-D-ribofuranosyl-1,2,4-trizole-3-carboxamide) into placental epithelial cells using human choriocarcinoma (BeWo) cells and Xenopus oocytes expressing human nucleoside transporters. In BeWo cells, when a relatively low concentration (123 nM) of ribavirin was used, both Na(+)-dependent uptake and -independent uptake of ribavirin were observed. On the other hand, when a higher concentration (100 microM) of ribavirin was used, Na(+)-independent uptake was observed, but there was only a slight Na(+)-dependent uptake. In Xenopus oocytes, influxes of ribavirin mediated by hCNT2 (concentrative nucleoside transporter 2), hCNT3 (concentrative nucleoside transporter 3), hENT1 (equilibrative nucleoside transporter 1) and hENT2 (equilibrative nucleoside transporter 2) were saturable, and apparent K(m) values were 18.0 microM, 14.2 microM, 3.46 mM and 3.71 mM, respectively. These data indicate that hCNT2 and hCNT3 have higher affinity for ribavirin than do hENT1 and hENT2. Moreover, analysis by RT-PCR showed that BeWo cells express mRNA of hCNT3, hENT1 and hENT2. These results suggest that ribavirin is taken up by BeWo cells via both the high-affinity Na(+)-dependent transporter hCNT3 and the low-affinity Na(+)-independent transporters hENT1 and hENT2.     

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.     

Interaction of benzopyranone derivatives and related compounds with human concentrative nucleoside transporters 1, 2 and 3 heterologously expressed in porcine PK15 nucleoside transporter deficient cells. Structure-activity relationships and determinants of transporter affinity and selectivity

Unlike the major equilibrative nucleoside transporters, there is a dearth of potent specific inhibitors of concentrative nucleoside transporters (CNTs). We investigated the interaction of benzopyranone derivatives and related compounds with human (h) CNTs in newly established PK15NTD transfectant cells stably expressing hCNT1 or hCNT2, and previously established PK15NTD/hCNT3 cells. Flavones exhibited the highest inhibitory activity against hCNT2 and hCNT3, whereas the most potent selective inhibitor of hCNT1 was a coumarin derivative. hCNT3 was the only transporter that exhibited moderate sensitivity to the chalcones tested. The most active compound was 6-hydroxy-7-methoxyflavone, which was hCNT3-specific with an IC₅₀ of 0.57 ± 0.20 μM, and over 40-fold more potent than the standard CNT inhibitor, phloridzin (IC₅₀ of 25 ± 3.5 μM). The SAR (Structure-Activity Relationship) shows that high potency against all three hCNTs is conferred by the presence of hydroxyl substituents at both the 7- and 8-positions of flavones and isoflavones. CoMFA (Comparative Molecular Field Analysis) and CoMSIA (Comparative Molecular Similarity Indices Analysis) 3D-QSAR (three-Dimensional Quantitative Structure-Activity Relationship) modeling indicated that electrostatic and hydrophobic properties were the most influential for interactions between the flavonoids and hCNT1, while electrostatic, hydrophobic and hydrogen bond donor properties were predominate for interactions with hCNT2 and hCNT3. The 3D-QSAR results also suggested possible commonalities in hydrogen bonding interactions of flavonoids and nucleosides, suggesting similarities between the hCNT-binding sites of the two classes of compounds. We report the most potent and selective non-nucleoside CNT inhibitors to date; which may serve as research tools and/or leads for further inhibitor development.     

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.     

Nucleoside transport in primary cultured rabbit tracheal epithelial cells

The present study aimed at elucidating the mechanisms of nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) grown on a permeable filter support. Uptake of ³H-uridine, the model nucleoside substrate, from the apical fluid of primary cultured RTEC was examined with respect to its dependence on Na⁺, substrate concentration, temperature and its sensitivity to inhibitors, other nucleosides and antiviral nucleoside analogs. Apical ³H-uridine uptake in primary cultured RTEC was strongly dependent on an inward Na⁺ gradient and temperature. Ten micromolar nitro-benzyl-mercapto-purine-ribose (NBMPR) (an inhibitor of es-type nucleoside transport in the nanomolar range) did not further inhibit this process. ³H-uridine uptake from apical fluid was inhibited by basolateral ouabain (10 μM) and apical phloridzin (100 μM), indicating that uptake may involve a secondary active transport process. Uridine uptake was saturable with a K_m of 3.4 ± 1.8 μM and the V_max of 24.3 ± 5.2 pmoles/mg protein/30 s. Inhibition studies indicated that nucleoside analogs that have a substitution on the nucleobase competed with uridine uptake from apical fluid, but those with modifications on the ribose sugar including acyclic analogs were ineffective. The pattern of inhibition of apical ³H-uridine, ³H-inosine and ³H-thymidine uptake into RTEC cells by physiological nucleosides was consistent with multiple systems: A pyrimidine-selective transport system (CNT1); a broad nucleoside substrate transport system that excludes inosine (CNT4) and an equilibrative NBMPR-insensitive nucleoside transport system (ei type). These results indicate that the presence of apically located nucleoside transporters in the epithelial cells lining the upper respiratory tract can lead to a high accumulation of nucleosides in the trachea. At least one Na⁺-dependent, secondary, active transport process may mediate the apical absorption of nucleosides or analogous molecules.     

In vitro inhibition of human nucleoside transporters and uptake of azacitidine by an isocitrate dehydrogenase-2 inhibitor enasidenib and its metabolite AGI-16903

Abstract

1. The present study investigated inhibitory effects of enasidenib and its metabolite AGI-16903 on (a) recombinant human nucleoside transporters (hNTs) in hNT-producing Xenopus laevis oocytes, and (b) azacitidine uptake in a normal B-lymphoblast peripheral blood cell line (PBC) and acute myeloid leukemia (AML) cell lines.

2. Enasidenib inhibited hENT1, hENT2, hENT3, and hENT4 in oocytes with IC₅₀ values of 7, 63, 27, and 76?μM, respectively, but exhibited little inhibition of hCNT1-3. AGI-16903 exhibited little inhibition of any hNT produced in oocytes.

3. Azacitidine uptake was more than 2-fold higher in AML cells than in PBC. Enasidenib inhibited azacitidine uptake into OCI-AML2, TF-1 and PBC cells in a concentration-dependent manner with IC₅₀ values of 0.27, 1.7, and 1.0?µM in sodium-containing transport medium, respectively.

4. IC₅₀ values shifted approximately 100-fold higher when human plasma was used as the incubation medium (27?µM in OCI-AML2, 162?µM in TF-1, and 129?µM in PBC), likely due to high human plasma protein binding of enasidenib (98.5% bound).

5. Although enasidenib inhibits hENTs and azacitidine uptake in vitro, plasma proteins attenuate this inhibitory effect, likely resulting in no meaningful in vivo effects in humans.     

SLC28 genes and concentrative nucleoside transporter (CNT) proteins

The human concentrative nucleoside transporter (hCNT) protein family has three members, hCNT1, 2, and 3, encoded by SLC28A1, A2, and A3 genes, respectively. hCNT1 and hCNT2 translocate pyrimidine- and purine-nucleosides, respectively, by a sodium-dependent mechanism, whereas hCNT3 shows broad substrate selectivity and the unique ability of translocating nucleosides both in a sodium- and a proton-coupled manner. hCNT proteins are also responsible for the uptake of most nucleoside-derived antiviral and anticancer drugs. Thus, hCNTs are key pharmacological targets. This review focuses on several crucial aspects of hCNT biology and pharmacology: protein structure–function, structural determinants for transportability, pharmacogenetics of hCNT-encoding genes, role of hCNT proteins in nucleoside-based therapeutics, and finally hCNT physiology.     

The role of human nucleoside transporters in cellular uptake of 4'-thio-beta-D-arabinofuranosylcytosine and beta-D-arabinosylcytosine

4'-Thio-beta-D-arabinofuranosyl cytosine (TaraC) is in phase I development for treatment of cancer. In human equilibrative nucleoside transporter (hENT) 1-containing CEM cells, initial rates of uptake (10 microM; picomoles per microliter of cell water per second) of [3H]TaraC and [3H]1-beta-D-arabinofuranosyl cytosine (araC) were low (0.007 +/- 003 and 0.034 +/- 0.003, respectively) compared with that of [3H]uridine (0.317 +/- 0.048), a highactivity hENT1 permeant. In hENT1- and hENT2-containing HeLa cells, initial rates of uptake (10 microM; picomoles per cell per second) of [3H]TaraC, [3H]araC, and [3H]deoxycytidine were low (0.30 +/- 0.003, 0.42 +/- 0.03, and 0.51 +/- 0.11, respectively) and mediated primarily by hENT1 (approximately 74, approximately 65, and approximately 61%, respectively). In HeLa cells with recombinant human concentrative nucleoside transporter (hCNT) 1 or hCNT3 and pharmacologically blocked hENT1 and hENT2, transport of 10 microM[3H]TaraC and [3H]araC was not detected. The apparent affinities of recombinant transporters (produced in yeast) for a panel of cytosine-containing nucleosides yielded results that were consistent with the observed low-permeant activities of TaraC and araC for hENT1/2 and negligible permeant activities for hCNT1/2/3. During prolonged drug exposures of CEM cells with hENT1 activity, araC was more cytotoxic than TaraC, whereas coexposures with nitrobenzylthioinosine (to pharmacologically block hENT1) yielded identical cytotoxicities for araC and TaraC. The introduction by gene transfer of hENT2 and hCNT1 activities, respectively, into nucleoside transport-defective CEM cells increased sensitivity to both drugs moderately and slightly. These results demonstrated that nucleoside transport capacity (primarily via hENT1, to a lesser extent by hENT2 and possibly by hCNT1) is a determinant of pharmacological activity of both drugs.     

Transient Expression of a Purine-Selective Nucleoside Transporter (SPNTint) in a Human Cell Line (HeLa)

Purpose. The goal of this study was to develop a mammalian expression system for the cloned rat intestinal, Na⁺-dependent, purine-selective nucleoside transporter (SPNT_int) and to study the interactions of nucleosides and nucleoside analogs with this transporter. Methods. Lipofection was used to transfect HeLa cells with a mammalian expression vector (pcDNA3) containing the cDNA insert encoding SPNT_int. Nucleoside transport activity was measured using [³H] inosine, [³H]uridine, [³H]-dideoxyinosine (ddl), and [³H]-2-chloro-2-deoxyadenosine (2CdA) as model substrates. Results. Expression of SPNT_int was observed between 36 and 90 h post-transfection, with maximal expression at 66 h. At 66 h, Na⁺-stimulated uptake of [³H]inosine in cells transiently transfected with SPNT_int was approximately threefold greater than that in cells transfected with empty vector (p < 0.05). The Na⁺-stimulated uptake of both inosine and uridine was saturable (K_m = 28.1 ± 7.1 M and 20.6 ± 5.6 M, respectively) in the transfected cells and was significantly inhibited by the naturally occurring nucleosides (1 mM) inosine and uridine and to a lesser extent by thymidine. The nucleoside analogs ddl (IC₅₀ = 46 M) and 2CdA (IC₅₀ =.13 M) also significantly inhibited the Na⁺-stimulated uptake of [³H]inosine. A Na⁺-stimulated uptake of [³H]2CdA was observed suggesting that 2CdA is also a permeant of SPNT_int. Conclusions. HeLa cells transiently transfected with SPNT_int represent a useful tool to study the kinetics and interactions of drugs with SPNT_int.     

Electrophysiological characterization and modeling of the structure activity relationship of the human concentrative nucleoside transporter 3 (hCNT3)

We characterized the electrophysiology, kinetics, and quantitative structure-activity relationship (QSAR) of the human concentrative nucleoside transporter 3 (hCNT3) expressed in Xenopus laevis oocytes by measuring substrate-induced inward currents using a two-microelectrode voltage-clamp system. At membrane potentials between -30 and -150 mV, sodium activation of gemcitabine transport was sigmoidal, with a K0.5 of 8.5+/-0.3 mM for Na+ and a Hill coefficient of 2.2+/-0.25 independent of membrane potential. We measured the Imax and K0.5 for substrate at -50 mV for the nucleoside analog drugs gemcitabine (638+/-58 nA, 59.7+/-17.5 microM), ribavirin (546+/-37 nA, 61.0+/-13.2 microM), AZT (420+/-4 nA, 310+/-9 microM), and 3-deazauridine (506+/-30 nA, 50.8+/-9.90 microM). K0.5 and Imax for substrate were dependent on membrane potential (both increasing as the membrane became more hyperpolarized) for all four drugs. hCNT3 also exhibited pre-steady-state currents. The quantitative structure-activity relationship (QSAR) was examined using comparative molecular field analysis and comparative molecular similarity indices analysis of the inward currents induced by 27 nucleoside analogs with substitutions at both the ribose and the nucleobase. Two statistically significant QSAR models identified electrostatic interaction as the major force in hCNT3 transport and attributed a critical role to the 3'-hydroxyl position of hCNT3 substrates. Steric hindrance at the 3-position and positive charge at the 5-position of the pyrimidine ring were favorable for transport. Two hCNT3 pharmacophore models revealed the minimal features required for hCNT3 transport as two hydrogen bond acceptors at 3'-OH and 5'-O and the hydrophobic center occupied by the base ring.     

Interactions with selected drug renal transporters and transporter-mediated cytotoxicity in antiviral agents from the group of acyclic nucleoside phosphonates

Members of acyclic nucleoside phosphonates (ANPs) possess antiviral and antiproliferative activities. However, several clinically important ANPs may cause renal injury, most likely due to their active accumulation in the renal tubular cells. The goal of this study was to investigate in vitro relationships between the affinity of several structurally related potent ANPs to selected human transporters and their cytotoxicity. SLC (solute carrier family) transporters (hOAT1, hOCT2, hCNT2, hCNT3) and ABC (ATP-binding cassette) transporters (MDR1, BCRP), which are typically expressed in the kidney, were included in the study. The transport and toxic parameters of the tested compounds were compared to those of two clinically approved ANPs, adefovir and tenofovir. Transport studies with transiently transfected cells were used as the main method in the experiments. Most of the ANPs studied showed the potency to interact with hOAT1. GS-9191, a double prodrug of PMEG, displayed an affinity for hOAT1 comparable with that of adefovir and tenofovir. No significant interaction of the tested ANPs with hOCT2, hCNT2 and hCNT3 was observed. Only GS-9191 was found to be a strong inhibitor for both MDR1 and BCRP. PMEO-DAPy showed the potency to interact with MDR1. Most of the tested substances caused a significant decrease in cellular viability in the cells transfected with hOAT1. Only with the exclusion of GS-9191, a relatively lipophilic compound, did the in vitro cytotoxicity of the ANPs closely correspond to their potential to interact with hOAT1. The increased cytotoxicity of the studied ANPs found in OAT1 transfected cells was effectively reduced by OAT inhibitors probenecid and quercetin. The higher cytotoxicity of the compounds with affinity to hOAT1 proved in the inhibitory experiments evidences that ANPs are not only inhibitors but also substrates of hOAT1. Any clear relationship between the potency of ANPs to inhibit the studied efflux transporters and their cytotoxicity was not demonstrated. In conclusion, the study documented that among the studied transporters hOAT1 seems to be the decisive determinant for renal handling in most of the tested ANPs. This transporter may also play an important role in the mechanism of their potential cytotoxic effects. These facts are in good accordance with previous findings in the clinically used ANPs.     

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.     

Cytotoxic activity of gemcitabine in cultured cell lines derived from histologically different types of bladder cancer: Role of thymidine kinase 2

World wide incidence of bladder cancer is rising with nearly 13,760 deaths attributed to bladder cancer in 2007 in the USA. Tumor types of the urothelium include transitional cell carcinomas, squamous cell carcinomas, and adenocarcinomas. This study was undertaken to determine gemcitabine's efficacy against bladder cancer cell lines of different origins (HTB2, a papilloma; HTB3, a squamous cell carcinoma; and HTB4, a transitional cell carcinoma). Roles of nucleoside transporters and key enzymes in gemcitabine pharmacology were examined on the premise that cells originating from different types of bladder cancer exhibit different levels and/or types of nucleoside transporters and enzymes and thus may respond differently to gemcitabine. HTB2 cells had the highest transport efficiency and were also most responsive to gemcitabine. HTB3 and HTB4 cells had similar transport efficiencies, but exhibited different sensitivities to gemcitabine (HTB4 > HTB3). The highest accumulation of [³H]gemcitabine was in HTB2 cells and the lowest was in HTB3 cells. Sequencing experiments revealed no mutations either in coding exons or intron-exon boundaries of the hENT1 genes of the three cell lines. HTB3 cells exhibited high thymidine kinase 2 (TK2) activity whereas HTB2 and HTB4 cells lacked detectable TK2 activity and pretreatment of HTB3 but not of HTB2 and HTB4 cells with extracellular thymidine resulted in enhanced sensitivity to gemcitabine. Our results highlight the importance of hENT1 and TK2 activities in response to gemcitabine. Elevated TK expression in squamous cell carcinomas warrants further study and offers new insights into rational treatment strategies based on bladder cancer phenotype.     

Interaction of Nucleoside Analogues with the Sodium–Nucleoside Transport System in Brush Border Membrane Vesicles from Human Kidney

The therapeutic efficacy of nucleosides and nucleoside analogues as antitumor, antiviral, antiparasitic, and antiarrhythmic agents has been well documented. Pharmacokinetic studies suggest that many of these compounds are actively transported in the kidney. The goal of this study was to determine if therapeutically relevant nucleosides or analogues interact with the recently characterized Na⁺-driven nucleoside transport system of the brush border membrane of the human kidney. Brush border membrane vesicles (BBMV) were prepared from human kidney by divalent cation precipitation and differential centrifugation. The initial Na⁺-driven ³H-uridine uptake into vesicles was determined by rapid filtration. The effect of several naturally occurring nucleosides (cytidine, thymidine, adenosine), a pyrimidine base (uracil), a nucleotide (UMP), and several synthetic nucleoside analogues [zidovudine (AZT), cytarabine (Ara-C), and dideoxycytidine (ddC)] on Na⁺–uridine transport was determined. At a concentration of 100 µM the naturally occurring nucleosides, uracil, and UMP significantly inhibited Na+-uridine transport, whereas the three synthetic nucleoside analogues did not. Adenosine competitively inhibited Na+-uridine uptake with a K _i of 26.4 µM (determined by constructing a Dixon plot). These data suggest that naturally occurring nucleosides are substrates of the Na⁺–nucleoside transport system in the renal brush border membrane, whereas synthetic nucleoside analogues with modifications on the ribose ring are not. The K _i of adenosine is higher than clinically observed concentrations and suggests that the system may play a physiologic role in the disposition of this nucleoside.     

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.     

3-Hydroxy-2'-methoxy-6-methylflavone: a potent anxiolytic with a unique selectivity profile at GABA(A) receptor subtypes

Genetic and pharmacological studies have demonstrated that α2- and α4-containing GABA_A receptors mediate the anxiolytic effects of a number of agents. Flavonoids are a class of ligands that act at GABA_A receptors and possess anxiolytic effects in vivo. Here we demonstrate that the synthetic flavonoid, 3-hydroxy-2′-methoxy-6-methylflavone (3-OH-2′MeO6MF) potentiates GABA-induced currents at recombinant α1/2β2, α1/2/4/6β1–3γ2L but not α3/5β1–3γ2L receptors expressed in Xenopus oocytes. The enhancement was evident at micromolar concentrations (EC₅₀ values between 38 and 106 μM) and occurred in a flumazenil-insensitive manner. 3-OH-2′MeO6MF displayed preference for β2/3- over β1-containing receptors with the highest efficacy observed at α2β2/3γ2L, displaying a 4–11-fold increase in efficacy over α2β1γ2L and α1/4/6-containing subtypes. In contrast, 3-OH-2′MeO6MF acted as a potent bicuculline-sensitive activator, devoid of potentiation effects at extrasynaptic α4β2/3δ receptors expressed in oocytes. The affinity of 3-OH-2′MeO6MF for α4β2/3δ receptors (EC₅₀ values between 1.4 and 2.5 μM) was 10-fold higher than at α4β1δ GABA_A receptors. 3-OH-2′MeO6MF acted as a full agonist at α4β2/3δ (105% of the maximal GABA response) but as a partial agonist at α4β1δ (61% of the maximum GABA response) receptors. In mice, 3-OH-2′MeO6MF (1–100 mg/kg i.p.) induced anxiolytic-like effects in two unconditioned models of anxiety: the elevated plus maze and light/dark paradigms. No sedative or myorelaxant effects were detected using holeboard, actimeter and horizontal wire tests and only weak barbiturate potentiating effects on the loss of righting reflex test. Taken together, these data suggest that 3-OH-2′MeO6MF is an anxiolytic without sedative and myorelaxant effects acting through positive allosteric modulation of the α2β2/3γ2L and direct activation of α4β2/3δ GABA_A receptor subtypes.