Improved syntheses of 5'-S-(2-aminoethyl)-6-N-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), analogues, and fluorescent probe conjugates: analysis of cell-surface human equilibrative nucleoside transporter 1 (hENT1) levels for prediction of the antitumor efficacy of gemcitabine.

5'-S-(2-aminoethyl)-6-N-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), 5'-S-(2-acetamidoethyl)-6-N-[(4-substituted)benzyl]-5'-thioadenosine analogues, 5'-S-[2-(6-aminohexanamido)]ethyl-6-N-(4-nitrobenzyl)-5'-thioadenosine (SAHENTA), and related compounds were synthesized by S(N)Ar displacement of fluoride from 6-fluoropurine intermediates with 4-(substituted)benzylamines. Conjugation of the pendant amino groups of SAENTA and SAHENTA with fluorescein-5-yl isothiocyanate (FITC) gave fluorescent probes that bound at nanomolar concentrations specifically to human equilibrative nucleoside transporter 1 (hENT1) produced in recombinant form in model expression systems and in native form in cancer cell lines. Transporter binding effects were studied and the ability of the probes to predict the potential antitumor efficacy of 2'-deoxy-2',2'-difluorocytidine (gemcitabine) was demonstrated.

Mutation of Trp29 of human equilibrative nucleoside transporter 1 alters affinity for coronary vasodilator drugs and nucleoside selectivity.

hENT1 (human equilibrative nucleoside transporter 1) is inhibited by nanomolar concentrations of various structurally distinct coronary vasodilator drugs, including dipyridamole, dilazep, draflazine, soluflazine and NBMPR (nitrobenzylmercaptopurine ribonucleoside). When a library of randomly mutated hENT1 cDNAs was screened using a yeast-based functional complementation assay for resistance to dilazep, a clone containing the W29G mutation was identified. Multiple sequence alignments revealed that this residue was highly conserved.

Residues 334 and 338 in transmembrane segment 8 of human equilibrative nucleoside transporter 1 are important determinants of inhibitor sensitivity, protein folding, and catalytic turnover.

Equilibrative nucleoside transporters (ENTs) are important for the metabolic salvage of nucleosides and the cellular uptake of antineoplastic and antiviral nucleoside analogs. Human equilibrative nucleoside transporter 1 (hENT1) is inhibited by nanomolar concentrations of structurally diverse compounds, including dipyridamole, dilazep, nitrobenzylmercaptopurine ribonucleoside (NBMPR), draflazine, and soluflazine. Random mutagenesis and screening by functional complementation for inhibitor-resistant mutants in yeast revealed mutations at Phe-334 and Asn-338.

Characterization of the transport mechanism and permeant binding profile of the uridine permease Fui1p of Saccharomyces cerevisiae.

The uptake of Urd into the yeast Saccharomyces cerevisiae is mediated by Fui1p, a Urd-specific nucleoside transporter encoded by the FUI1 gene and a member of the yeast Fur permease family, which also includes the uracil, allantoin, and thiamine permeases. When Fui1p was produced in a double-permease knock-out strain (fur4Deltafui1Delta) of yeast, Urd uptake was stimulated at acidic pH and sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone. Electrophysiological analysis of recombinant Fui1p produced in Xenopus oocytes demonstrated that Fui1p-mediated Urd uptake was dependent on proton cotransport with a 1:1 stoichiometry.

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.

Cysteine-accessibility analysis of transmembrane domains 11-13 of human concentrative nucleoside transporter 3.

hCNT3 (human concentrative nucleoside transporter 3) is a nucleoside-sodium symporter that transports a broad range of naturally occurring purine and pyrimidine nucleosides as well as anticancer nucleoside drugs. To understand its uridine binding and translocation mechanisms, a cysteine-less version of hCNT3 was constructed and used for cysteine-accessibility and permeant-protection assays. Cysteine-less hCNT3, with 14 endogenous cysteine residues changed to serine, displayed wild-type properties in a yeast expression system, indicating that endogenous cysteine residues are not essential for hCNT3-mediated nucleoside transport.

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.

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.

Identification and functional characterization of variants in human concentrative nucleoside transporter 3, hCNT3 (SLC28A3), arising from single nucleotide polymorphisms in coding regions of the hCNT3 gene.

Introduction: Human concentrative nucleoside transporter 3, hCNT3 (SLC28A3), which mediates transport of purine and pyrimidine nucleosides and a variety of antiviral and anticancer nucleoside drugs, was investigated to determine if there are single nucleotide polymorphisms in the coding regions of the hCNT3 gene.

Methods And Results: Ninety-six DNA samples from Caucasians (Coriell Panel) were sequenced and sixteen variants in exons and flanking intronic regions were identified, of which five were coding variants; three of these were non-synonymous (S5N, L131F, Y513F) and were further investigated for functional alterations of the resulting recombinant proteins in Saccharomyces cerevisiae and Xenopus laevis oocytes. In yeast, immunostaining and fluorescence quantitation of the reference (wild-type) and variant CNT3 proteins showed similar levels of expression.

Uridine recognition motifs of human equilibrative nucleoside transporters 1 and 2 produced in Saccharomyces cerevisiae.

The sugar moiety of nucleosides has been shown to play a major role in permeant-transporter interaction with human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2). To better understand the structural requirements for interactions with hENT1 and hENT2, a series of uridine analogs with sugar modifications were subjected to an assay that tested their abilities to inhibit [3H]uridine transport mediated by recombinant hENT1 and hENT2 produced in Saccharomyces cerevisiae. hENT1 displayed higher affinity for uridine than hENT2.