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The prognosis of pancreatic cancer is poor with the overall 5-year survival rate of less than 5% changing minimally over the past decades and future projections predicting it developing into the second leading cause of cancer related mortality within the next decade. Investigations into the mechanisms of pancreatic cancer development, progression and acquired chemoresistance have been constant for the past few decades, thus resulting in the identification of human nucleoside transporters and factors affecting cytotoxic uptake via said transporters. This review summaries the aberrant expression and role of human nucleoside transports in pancreatic cancer, more specifically human equilibrative nucleoside transporter 1/2 (hENT1, hENT2), and human concentrative nucleoside transporter 1/3 (hCNT1, hCNT3), while briefly discussing the connection and importance between these nucleoside transporters and mucins that have also been identified as being aberrantly expressed in pancreatic cancer. The review also discusses the incidence, current diagnostic techniques as well as the current therapeutic treatments for pancreatic cancer. Furthermore, we address the importance of chemoresistance in nucleoside analogue drugs, in particular, gemcitabine and we discuss prospective therapeutic treatments and strategies for overcoming acquired chemoresistance in pancreatic cancer by the enhancement of human nucleoside transporters as well as the potential targeting of mucins using a combination of mucolytic compounds with cytotoxic agents.

[Image: see text] The dihydroorotate dehydrogenase (DHODH) inhibitor brequinar failed all clinical trials for solid tumors. To investigate mechanisms to increase brequinar’s efficacy, we employed a combination strategy to simultaneously inhibit the nucleotide salvage pathways. Brequinar is synergistic with the equilibrative nucleoside transporter (ENT) inhibitor dipyridamole, but not the concentrative nucleoside transporter inhibitor phlorizin. This synergy carries over to ENT1/2 inhibition, but not ENT4. Our previously described brequinar analogue 41 was also synergistic with dipyridamole as were the FDA-approved DHODH inhibitors leflunomide and teriflunomide but the latter required much higher concentrations than brequinar. Therefore, a combination of brequinar and ENT inhibitors presents a potential anti-cancer strategy in select tumors.

Some women take medication during pregnancy to address a variety of clinical conditions. Because of ethical and logistical concerns, it is impossible to determine fetal drug exposure, and therefore fetal risk, during pregnancy. Hence, alternative approaches need to be developed to predict maternal-fetal drug exposure throughout pregnancy. To do so, we previously developed and verified a maternal-fetal physiologically based pharmacokinetic model, which can predict fetal exposure to drugs that passively cross the placenta. However, many drugs are actively transported by the placenta (e.g., human immunodeficiency virus protease inhibitors). To extend our maternal-fetal physiologically based pharmacokinetic model to these actively transported drugs, we determined the gestational age-dependent changes in the protein abundance of placental transporters. Total cellular membrane fractions from first trimester (T1; n = 15), second trimester (T2; n = 19), and term (n = 15) human placentae obtained from uncomplicated pregnancies were isolated by ultracentrifugation. Transporter protein abundance was determined by targeted quantitative proteomics using liquid chromatography tandem mass specrometry. We observed that breast cancer resistance protein and P-glycoprotein abundance significantly decreased from T1 to term by 55% and 69%, respectively (per gram of tissue). Organic anion-transporting polypeptide (OATP) 2B1 abundance significantly decreased from T1 to T2 by 32%. In contrast, organic cation transporter (OCT) 3 and organic anion transporter 4 abundance significantly increased with gestational age (2-fold from T1 to term, 1.6-fold from T2 to term). Serotonin transporter and norepinephrine transporter did not change with gestational age. The abundance of bile salt export pump, multidrug resistance-associated protein 1-5, Na+-taurocholate cotransporting polypeptide, OATP1B1, OATP1B3, OCTN1-2, concentrative nucleoside transporter 1-3, equilibrative nucleoside transporter 2, and multidrug and toxin extrusion 1 could not be quantified. These data can be incorporated into our maternal-fetal physiologically based pharmacokinetic model to predict fetal exposure to drugs that are actively transported across the placenta. SIGNIFICANCE STATEMENT: We quantified the protein abundance of key placental uptake and efflux transporters [organic cation transporter (OCT) 3, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP)] across gestational ages (first trimester, second trimester, and term) using quantitative targeted proteomics. We observed that the protein abundance of P-gp and BCRP decreased, whereas that of OCT3 increased with gestational age. Incorporating the protein abundance determined in this study into maternal-fetal physiologically based pharmacokinetic model can help us better predict fetal drug exposure to substrates of these transporters.

Background Abdominal aortic aneurysm (AAA) is a focal aortic dilatation progressing towards rupture. Non-invasive AAA-associated cell proliferation biomarkers are not yet established. We investigated the feasibility of the cell proliferation radiotracer, fluorine-18-fluorothymidine ([18F]FLT) with positron emission tomography/computed tomography (PET/CT) in a progressive pre-clinical AAA model (angiotensin II, AngII infusion). Methods and Results Fourteen-week-old apolipoprotein E-knockout (ApoE−/−) mice received saline or AngII via osmotic mini-pumps for 14 (n = 7 and 5, respectively) or 28 (n = 3 and 4, respectively) days and underwent 90-minute dynamic [18F]FLT PET/CT. Organs were harvested from independent cohorts for gamma counting, ultrasound scanning, and western blotting. [18F]FLT uptake was significantly greater in 14- (n = 5) and 28-day (n = 3) AAA than in saline control aortae (n = 5) (P 18F]FLT uptake in 14-day AAA (n = 9) compared to saline-infused aortae (n = 4) (P r = 0.71, P n = 3 each) (all P Conclusions [18F]FLT uptake is increased during the active growth phase of the AAA model compared to saline control mice and late-stage AAA.

Nucleoside homeostasis, which is mediated by transporters and channels, is essential for all life on Earth. In Escherichia coli, NupG mediates the transport of nucleosides and was deemed to be the prototype of the nucleoside proton symporter (NHS) family and the major facilitator superfamily. To date, the substrate recognition and transport mechanisms of NHS transporters are still elusive. Here, we report two crystal structures of NupG (WT and D323A NupG) resolved at 3.0 A. Both structures reveal an identical inward-open conformation. Together with molecular docking and molecular dynamics simulations and in vitro uridine-binding assays, we found that the uridine binding site, which locates in the central cavity between N and C domains of NupG, is constituted by R136, T140, F143, Q225, N228, Q261, E264, Y318, and F322. Moreover, we found that D323 is very important for substrate binding via in vitro uridine-binding assays using D323 mutations, although it does not have a direct contact with uridine. Our structural and biochemical data therefore provide an important framework for the mechanistic understanding of nucleoside transporters of the NHS family.

Aim Gemcitabine is a frontline agent for locally-advanced and metastatic pancreatic ductal adenocarcinoma (PDAC), but neither gemcitabine alone nor in combination produces durable remissions of this tumor type. We developed three PDAC patient-derived xenograft (PDX) models with gemcitabine resistance (gemR) acquired in vivo, with which to identify mechanisms of resistance relevant to drug exposure in vivo and to evaluate novel therapies. Methods Mice bearing independently-derived PDXs received 100 mg/kg gemcitabine once or twice weekly. Tumors initially responded, but regrew on treatment and were designated gemR. We used immunohistochemistry to compare expression of proteins previously associated with gemcitabine resistance [ribonucleotide reductase subunit M1 (RRM1), RRM2, human concentrative nucleoside transporter 1 (hCNT1), human equilibrative nucleoside transporter 1 (hENT1), cytidine deaminase (CDA), and deoxycytidine kinase (dCK)] in gemR and respective gemcitabine-naive parental tumors. Results Parental and gemR tumors did not differ in tumor cell morphology, amount of tumor-associated stroma, or expression of stem cell markers. No consistent pattern of expression of the six gemR marker proteins was observed among the models. Increases in RRM1 and CDA were consistent with in vitro-derived gemR models. However, rather than the expected decreases of hCNT1, hENT1, and dCK, gemR tumors expressed no change in or higher levels of these gemR marker proteins than parental tumors. Conclusion These models are the first PDAC PDX models with gemcitabine resistance acquired in vivo. The data indicate that mechanisms identified in models with resistance acquired in vitro are unlikely to be the predominant mechanisms when resistance is acquired in vivo. Ongoing work focuses on characterizing unidentified mechanisms of gemR and on identifying agents with anti-tumor efficacy in these gemR models.

Concentrative nucleoside transporters (CNTs), members of the solute carrier (SLC) 28 transporter family, facilitate the salvage of nucleosides and therapeutic nucleoside derivatives across the plasma membrane. Despite decades of investigation, the structures of human CNTs remain unknown. We determined the cryogenic electron microscopy (cryo-EM) structure of human CNT (hCNT) 3 at an overall resolution of 3.6 Å. As with its bacterial homologs, hCNT3 presents a trimeric architecture with additional N-terminal transmembrane helices to stabilize the conserved central domains. The conserved binding sites for the substrate and sodium ions unravel the selective nucleoside transport and distinct coupling mechanism. Structural comparison of hCNT3 with bacterial homologs indicates that hCNT3 is stabilized in an inward-facing conformation. This study provides the molecular determinants for the transport mechanism of hCNTs and potentially facilitates the design of nucleoside drugs.

Background and purpose The concentrative nucleoside transporter 2 (CNT2) mediates the uptake of both natural nucleosides and nucleoside-derived drugs. Therefore, it is important both physiologically and pharmacologically. However, CNT2 expression is significantly repressed in colorectal cancer (CRC). Here, we have elucidated the mechanism(s) underlying CNT2 repression in CRC. Experimental approach Repression of CNT2 in tumour samples from patients with CRC was identified using Western blot and RT-qPCR. The histone acetylation state at the CNT2 promoter region was then evaluated with chromatin immunoprecipitation and trichostatin A (TSA) treatment. To find the key enzyme responsible for hypoacetylation at the CNT2 promoter region, siRNA knockdown and RT-qPCR were used. Effects of combining HDAC inhibitors and cladribine were studied in HCT15 and HT29 cells. Key results Histone deacetylase 7 was significantly up-regulated in CRC, leading to histone hypoacetylation at the CNT2 promoter region, especially at sites H3K9Ac, H3K18Ac and H4Ac. This hypoacetylation condensed the chromatin structure and reduced CNT2 expression. All these effects were reversed by treatment with TSA, a histone deacetylase inhibitor. In HCT15 and HT29 cells, inhibition of histone deacetylase increased cell uptake and decreased IC50 for cladribine. Conclusions and implications Histone hypoacetylation due to increased levels of histone deacetylase 7 results in CNT2 repression in CRC tumour tissue and could lead to decreased uptake of and consequent resistance to nucleoside anti-cancer agents. Such resistance could be overcome by combining inhibitors of histone deacetylase with the nucleoside anti-cancer agent.

Cladribine Tablets (MAVENCLAD (R)) are used to treat relapsing multiple sclerosis (MS). The recommended dose is 3.5 mg/kg, consisting of 2 annual courses, each comprising 2 treatment weeks 1 month apart. We reviewed the clinical pharmacology of Cladribine Tablets in patients with MS, including pharmacokinetic and pharmacometric data. Cladribine Tablets are rapidly absorbed, with a median time to reach maximum concentration (T-max) of 0.5 h (range 0.5-1.5 h) in fasted patients. When administered with food, absorption is delayed (median T-max 1.5 h, range 1-3 h), and maximum concentration (C-max) is reduced by 29% (based on geometric mean). Area under the concentration-time curve (AUC) is essentially unchanged. Oral bioavailability of cladribine is approximately 40%, pharmacokinetics are linear and time-independent, and volume of distribution is 480-490 L. Plasma protein binding is 20%, independent of cladribine plasma concentration. Cladribine is rapidly distributed to lymphocytes and retained (either as parent drug or its phosphorylated metabolites), resulting in approximately 30- to 40-fold intracellular accumulation versus extracellular concentrations as early as 1 h after cladribine exposure. Cytochrome P450-mediated biotransformation of cladribine is of minor importance. Cladribine elimination is equally dependent on renal and non-renal routes. In vitro studies indicate that cladribine efflux is minimally P-glycoprotein (P-gp)-related, and clinically relevant interactions with P-gp inhibitors are not expected. Cladribine distribution across membranes is primarily facilitated by equilibrative nucleoside transporter (ENT)1, concentrative nucleoside transporter (CNT)3 and breast cancer resistance protein (BCRP), and there is no evidence of any cladribine-related effect on heart rate, atrioventricular conduction or cardiac repolarisation (QTc interval prolongation). Cladribine Tablets are associated with targeted lymphocyte reduction and durable efficacy, with the exposure-effect relationship showing the recommended dose is appropriate in reducing relapse risk.

Combination antiretroviral drug treatments depend on 3'-deoxy-nucleoside analogs such as 3'-azido-3'-deoxythymidine (AZT) and 2'3'-dideoxyinosine (DDI). Despite being effective in inhibiting human immunodeficiency virus replication, these drugs produce a range of toxicities, including myopathy, pancreatitis, neuropathy, and lactic acidosis, that are generally considered as sequelae to mitochondrial damage. Although cell surface-localized nucleoside transporters, such as human equilibrative nucleoside transporter 2 (hENT2) and human concentrative nucleoside transporter 1 (hCNT1), are known to increase the carrier-mediated uptake of 3'-deoxy-nucleoside analogs into cells, another ubiquitously expressed intracellular nucleoside transporter (namely, hENT3) has been implicated in the mitochondrial transport of 3'-deoxy-nucleoside analogs. Using site-directed mutagenesis, generation of chimeric hENTs, and 3H-permeant flux measurements in mutant/chimeric RNA-injected Xenopus oocytes, here we identified the molecular determinants of hENT3 that dictate membrane translocation of 3'-deoxy-nucleoside analogs. Our findings demonstrated that whereas hENT1 had no significant transport activity toward 3'-deoxy-nucleoside analogs, hENT3 was capable of transporting 3'-deoxy-nucleoside analogs similar to hENT2. Transport analyses of hENT3-hENT1 chimeric constructs demonstrated that the N-terminal half of hENT3 is primarily responsible for the hENT3-3'-deoxy-nucleoside analog interaction. In addition, mutagenic studies identified that 225D and 231L in the N-terminal half of hENT3 partially contribute to the ability of hENT3 to transport AZT and DDI. The identification of the transporter segment and amino acid residues that are important in hENT3 transport of 3'-deoxy-nucleoside analogs may present a possible mechanism for overcoming the adverse toxicities associated with 3'-deoxy-nucleoside analog treatment and may guide rational development of novel nucleoside analogs.

Trifluridine (FTD) exhibits anticancer activities after its oral administration despite its hydrophilic nature. It was previously reported that concentrative nucleoside transporter (CNT) 1 mediates the apical uptake of FTD in human small intestinal epithelial cells (HIECs). In the present study, FTD was also identified as a substrate for equilibrative nucleoside transporter (ENT) 1 and ENT2 in transporter gene-transfected cells. An immunocytochemical analysis revealed that ENT1 was expressed at the basolateral and apical membranes of HIECs. Cellular accumulation increased in the presence of S-(4-nitrobenzyl)-6-thioinosine (NBMPR), an ENT selective inhibitor. Cytotoxicity in HIEC monolayers at low FTD concentrations was increased by NBMPR, and this may have been due to inhibition of the ENT-mediated basolateral transport of FTD by NBMPR. These results suggest that ENTs reduce the intestinal cytotoxicity of FTD by facilitating its basolateral efflux. On the other hand, the intracellular accumulation and cytotoxicity of FTD in HIECs were decreased at higher concentrations of FTD by NBMPR, and this may have been due to the NBMPR inhibition of the apical uptake of FTD, which has been suggested to be mediated by CNTs and ENTs. In conclusion, ENTs were responsible for intestinal transepithelial permeation by mediating the basolateral efflux of FTD after its uptake by CNT1 from the apical side, resulting in decreases in its intracellular accumulation and intestinal toxicity in humans. Equilibrative nucleoside transporters may also partially contribute to the low-affinity uptake of FTD across the apical membrane along with high-affinity CNT1.

The human SLC28 family of concentrative nucleoside transporter (CNT) proteins has three members: hCNT1, hCNT2, and hCNT3. Na+-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 transports both pyrimidine and purine nucleosides utilizing Na+ and/or H+ electrochemical gradients. Escherichia coli CNT family member NupC resembles hCNT1 in permeant selectivity but is H+-coupled. Using heterologous expression in Xenopus oocytes and the engineered cysteine-less hCNT3 protein hCNT3(C−), substituted cysteine accessibility method analysis with the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate was performed on the transport domain (interfacial helix 2, hairpin 1, putative transmembrane domain (TM) 7, and TM8), as well as TM9 of the scaffold domain of the protein. This systematic scan of the entire C-terminal half of hCNT3(C−) together with parallel studies of the transport domain of wild-type hCNT1 and the corresponding TMs of cysteine-less NupC(C−) yielded results that validate the newly developed structural homology model of CNT membrane architecture for human CNTs, revealed extended conformationally mobile regions within transport-domain TMs, identified pore-lining residues of functional importance, and provided evidence of an emerging novel elevator-type mechanism of transporter function.

Membrane transporters move substrates across the membrane by alternating access of their binding sites between the opposite sides of the membrane. An emerging model of this process is the elevator mechanism, in which a substrate-binding transport domain moves a large distance across the membrane. This mechanism has been characterized by a transition between two states, but the conformational path that leads to the transition is not yet known, largely because the available structural information has been limited to the two end states. Here we present crystal structures of the inward-facing, intermediate, and outward-facing states of a concentrative nucleoside transporter from Neisseria wadsworthii. Notably, we determined the structures of multiple intermediate conformations, in which the transport domain is captured halfway through its elevator motion. Our structures present a trajectory of the conformational transition in the elevator model, revealing multiple intermediate steps and state-dependent conformational changes within the transport domain that are associated with the elevator-like motion.

Humans possess three members of the cation-coupled concentrative nucleoside transporter CNT (SLC 28) family, hCNT1-3: hCNT1 is selective for pyrimidine nucleosides but also transports adenosine, hCNT2 transports purine nucleosides and uridine, and hCNT3 transports both pyrimidine and purine nucleosides. hCNT1/2 transport nucleosides using the transmembrane Na+ electrochemical gradient, while hCNT3 is both Na+- and H+-coupled. By producing recombinant hCNT3 in Xenopus laevis oocytes, we have used radiochemical high performance liquid chromatography (HPLC) analysis to investigate the metabolic fate of transported [3H] or [14C] pyrimidine and purine nucleosides once inside cells. With the exception of adenosine, transported nucleosides were generally subject to minimal intracellular metabolism. We also used radiochemical HPLC analysis to study the mechanism by which adenosine functions as a low Km, low Vmax permeant of hCNT1. hCNT1-producing oocytes were pre-loaded with [3H] uridine, after which efflux of accumulated radioactivity was measured in transport medium alone, or in the presence of extracellular non-radiolabelled adenosine or uridine. hCNT1-mediated [3H]-efflux was stimulated by extracellular uridine, but inhibited by extracellular adenosine, with >95% of the radioactivity exiting cells being unmetabolized uridine, consistent with a low transmembrane mobility of the hCNT1/adenosine complex. Humans also possess four members of the equilibrative nucleoside transporter ENT (SLC 29) family, hENT1-4. Of these, hENT1 and hENT2 transport both nucleosides and nucleobases into and out of cells, but their relative contributions to nucleoside and nucleobase homeostasis and, in particular, to adenosine signaling via purinoreceptors, are not known. We therefore used HPLC to determine plasma nucleoside and nucleobase concentrations in wild-type, mENT1-, mENT2- and mENT1/mENT2-knockout (KO) mice, and to compare the findings with knockout of mCNT3. Results demonstrated that ENT1 was more important than ENT2 or CNT3 in determining plasma adenosine concentrations, indicated modest roles of ENT1 in the homeostasis of other nucleosides, and suggested that none of the transporters is a major participant in handling of nucleobases.

Background The human concentrative nucleoside transporter 1 (hCNT1) a product of the SLC28A1 gene is one of the three concentrative nucleoside transporters, with a substrate specificity for physiological pyrimidine nucleosides. It has recently been implicated in tumor suppression. We have unraveled a splice variant RNA transcript that is overexpressed in some tumor tissues and some cancer cells. This study established that observation. Methods We examined several clones of hCNT1 generated from RT-PCR of total RNA from human kidney tissue purchased from Ambion. The resulting cDNA clones were then sequenced, and a variant that retained intron 4, and skipped some exons fully or partly, specifically exons 5 and 13 were completely missed and only part of exon 6 was spliced. Tissue expression analysis by PCR indicated a similar distribution of expression of RNA of the splice variant hCNT1-IR as that of the dominant variant hCNT1, particularly in the small intestine, kidney and liver. Further, analysis of various tumor samples with PCR primers designed from this novel hCNT1 splice variant (hCNT1-IR) revealed interestingly that it is overexpressed in some cancer tissues relative to normal tissues, particularly kidney, liver and pancreatic cancers. Conclusion We have identified a novel intron retaining and exon skipping splice variant of the hCNT1 nucleoside transporter, and designated it hCNT1-IR, which has a similar tissue expression distribution as the normal hCNT1 variant, but unlike the normal transcript, hCNT1-IR is overexpressed in some cancers and may serve as a potential cancer biomarker.

Na+-d-glucose cotransporter 1 (SGLT1) is rate-limiting for glucose absorption in the small intestine. Shortly after intake of glucose-rich food, SGLT1 abundance in the luminal membrane of the small intestine is increased. This upregulation occurs via glucose-induced acceleration of the release of SGLT1-containing vesicles from the trans-Golgi network (TGN), which is regulated by a domain of protein RS1 (RSC1A1) named RS1-Reg. Dependent on phosphorylation, RS1-Reg blocks release of vesicles containing SGLT1 or concentrative nucleoside transporter 1. The hypothesis has been raised that RS1-Reg binds to different receptor proteins at the TGN, which trigger release of vesicles with different transporters. To identify the presumed receptor proteins, two-hybrid screening was performed. Interaction with ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme of polyamine synthesis, was observed and verified by immunoprecipitation. Binding of RS1-Reg mutants to ODC1 was characterized using surface plasmon resonance. Inhibition of ODC1 activity by RS1-Reg mutants and the ODC1 inhibitor difluoromethylornithine (DFMO) was measured in the absence and presence of glucose. In addition, short-term effects of DFMO, RS1-Reg mutants, the ODC1 product putrescine, and/or glucose on SGLT1 expressed in oocytes of Xenopus laevis were investigated. High-affinity binding of RS1-Reg to ODC1 was demonstrated, and evidence for a glucose binding site in ODC1 was provided. Binding of RS1-Reg to ODC1 inhibits the enzymatic activity at low intracellular glucose, which is blunted at high intracellular glucose. The data suggest that generation of putrescine by ODC1 at the TGN stimulates release of SGLT1-containing vesicles. This indicates a biomedically important role of ODC1 in regulation of glucose homeostasis.

Specialized nucleoside transporter (NT) proteins are required for passage of nucleosides and hydrophilic nucleoside analogues across biological membranes. Physiologic nucleosides serve as central salvage metabolites in nucleotide biosynthesis, and nucleoside analogues are used as chemotherapeutic agents in the treatment of cancer and antiviral diseases. The nucleoside adenosine modulates numerous cellular events via purino-receptor cell signalling pathways. Human NTs are divided into two structurally unrelated protein families: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family. Human CNTs are inwardly directed Na+-dependent nucleoside transporters found predominantly in intestinal and renal epithelial and other specialized cell types. Human ENTs mediate bidirectional fluxes of purine and pyrimidine nucleosides down their concentration gradients and are ubiquitously found in most, possibly all, cell types. Both protein families are evolutionarily old: CNTs are present in both eukaryotes and prokaryotes; ENTs are widely distributed in mammalian, lower vertebrate and other eukaryote species. This mini-review describes a 30-year collaboration with Professor Stephen Baldwin to identify and understand the structures and functions of these physiologically and clinically important transport proteins.

OBJECTIVES The testes are a potential viral sanctuary site for HIV-1 infection. Our study aims to provide insight into the expression and localization of key drug transporters and metabolic enzymes relevant to ART in this tissue compartment. METHODS We characterized gene and protein expression of 12 representative drug transporters and two metabolic enzymes in testicular tissue samples obtained from uninfected (n = 8) and virally suppressed HIV-1-infected subjects on ART (n = 5) and quantified antiretroviral drug concentrations in plasma and testicular tissues using LC/MS/MS from HIV-1-infected subjects. RESULTS Our data demonstrate that key ABC drug transporters (permeability glycoprotein, multidrug-resistance protein 1, 2 and 4, and breast cancer resistance protein), solute carrier transporters (organic anion transporting polypeptides 1B1 and 2B1, organic anion transporter 1, concentrative nucleoside transporter 1, equilibrative nucleoside transporter 2) and cytochrome P450 metabolic enzymes (CYP3A4 and CYP2D6) previously shown to interact with many commonly used antiretroviral drugs are expressed at the mRNA and protein level in the testes of both subject groups and localize primarily at the blood-testis barrier, with no significant differences between the two groups. Furthermore, we observed that PIs known to be substrates for ATP-binding cassette membrane transporters, displayed variable testicular tissue penetration, with darunavir concentrations falling below therapeutic values. In contrast, the NRTIs emtricitabine, lamivudine and tenofovir displayed favourable tissue penetration, reaching concentrations comparable to plasma levels. We also demonstrated that nuclear receptors, peroxisome proliferator-activated receptors α and γ exhibited higher gene expression in the testicular tissue compared with pregnane X receptor and constitutive androstane receptor, suggesting a potential regulatory pathway governing drug transporter and metabolic enzyme expression in this tissue compartment. CONCLUSIONS Our data suggest the testes are a complex pharmacological compartment that can restrict the distribution of certain antiretroviral drugs and potentially contribute to HIV-1 persistence.

To test the hypothesis that inhibitors of human concentrative nucleoside transporter 2 (hCNT2) suppress increases in serum urate levels derived from dietary purines, we previously identified adenosine derivative 1 as a potent hCNT2 inhibitor (IC50 = 0.64 μM), but further study was hampered due to its poor solubility. Here we describe the results of subsequent research to identify more soluble and more potent hCNT2 inhibitors, leading to the discovery of the benzimidazole nucleoside 22, which is the most potent hCNT2 inhibitor (IC50 = 0.062 μM) reported to date. Compound 22 significantly suppressed the increase in plasma uric acid levels after oral administration of purine nucleosides in rats. Because compound 22 was poorly absorbed orally in rats (F = 0.51%), its pharmacologic action was mostly limited to the gastrointestinal tract. These findings suggest that inhibition of hCNT2 in the gastrointestinal tract can be a promising approach for the treatment of hyperuricemia.

Tenofovir-associated renal toxicity is influenced by several factors, including plasma exposure and genetic variants in transporter-encoding genes. Tenofovir plasma exposure has been associated with a polymorphism in SLC28A2 gene (encoding the concentrative nucleoside transporter 2, CNT2): particularly, SLC28A2 124 CT/TT genotype patients show higher plasma tenofovir concentrations, compared to CC group. In literature, substrate studies are lacking; for this reason, our aim was to understand if tenofovir and tenofovir-alafenamide are CNT2 substrates. We performed an in vitro study using CNT2 expressing MDCKII cells. We observed that tenofovir and tenofovir-alafenamide are not substrates of CNT2. Tenofovir-alafenamide influx pathway remains to be clarified.

Equilibrative ( SLC29A) and concentrative ( SLC28A) nucleoside transporters contribute to proper placental development and mediate uptake of nucleosides/nucleoside-derived drugs. We analyzed placental expression of SLC28A mRNA during gestation. Moreover, we studied in choriocarcinoma-derived BeWo cells whether SLC29A and SLC28A mRNA levels can be modulated by activity of adenylyl cyclase, retinoic acid receptor activation, CpG islands methylation, or histone acetylation, using forskolin, all- trans-retinoic acid, 5-azacytidine, and sodium butyrate/sodium valproate, respectively. We found that expression of SLC28A1, SLC28A2, and SLC28A3 increases during gestation and reveals considerable interindividual variability. SLC28A2 was shown to be a dominant subtype in the first-trimester and term human placenta, while SLC28A1 exhibited negligible expression in the term placenta only. In BeWo cells, we detected mRNA of SLC28A2 and SLC28A3. Levels of the latter were affected by 5-azacytidine and all- trans-retinoic acid, while the former was modulated by sodium valproate (but not sodium butyrate), all- trans-retinoic acid, 5-azacytidine, and forskolin that caused 25-fold increase in SLC28A2 mRNA; we documented by analysis of syncytin-1 that the observed changes in SLC28A expression do not correlate with the morphological differentiation state of BeWo cells. Upregulated SLC28A2 mRNA was reflected in elevated uptake of [3H]-adenosine, high-affinity substrate of concentrative nucleoside transporter 2. Using KT-5720 and inhibitors of phosphodiesterases, we subsequently confirmed importance of cAMP/protein kinase A pathway in SLC28A2 regulation. On the other hand, SLC29A genes exhibited constitutive expression and none of the tested compounds increased SLC28A1 expression to detectable levels. In conclusion, we provide the first evidence that methylation status and activation of retinoic acid receptor affect placental SLC28A2 and SLC28A3 transcription and substrates of concentrative nucleoside transporter 2 might be taken up in higher extent in placentas with overactivated cAMP/protein kinase A pathway and likely in the term placenta.

Searching novel hypouricemic agents of high efficacy and safety has attracted a great attention. Previously, we reported the hypouricemic effect of Ganoderma applanatum, but its bioactives, was not referred. Herein, we report the hypouricemic effect of 2,5-dihydroxyacetophenone (DHAP), a compound screened from Ganoderma applanatum computationally. Serum parameters, such as uric acid (SUA), xanthine oxidase (XOD) activity, blood urea nitrogen (BUN), and creatinine were recorded. Real-time reverse transcription PCR (RT-PCR) and Western blot were exploited to assay RNA and protein expressions of organic anion transporter 1 (OAT1), glucose transporter 9 (GLUT9), uric acid transporter 1 (URAT1), and gastrointestinal concentrative nucleoside transporter 2 (CNT2). DHAP at 20, 40, and 80 mg/kg exerted excellent hypouricemic action on hyperuricemic mice, reducing SUA from hyperuricemic control (407 ± 31 μmol/L, p < 0.01) to 180 ± 29, 144 ± 13, and 139 ± 31 μmol/L, respectively. In contrast to the renal toxic allopurinol, DHAP showed some kidney-protective effects. Moreover, its suppression on XOD activity, in vivo and in vitro, suggested that XOD inhibition may be a mechanism for its hypouricemic effect. Given this, its binding mode to XOD was explored by molecular docking and revealed that three hydrogen bonds may play key roles in its binding and orientation. It upregulated OAT1 and downregulated GLUT9, URAT1, and CNT2 too. In summary, its hypouricemic effect may be mediated by regulation of XOD, OAT1, GLUT9, URAT1, and CNT2.

Cellular uptake adapts rapidly to physiologic demands by changing transporter abundance in the plasma membrane. The human gene RSC1A1 codes for a 67-kDa protein named RS1 that has been shown to induce downregulation of the sodium-D-glucose cotransporter 1 (SGLT1) and of the concentrative nucleoside transporter 1 (CNT1) in the plasma membrane by blocking exocytosis at the Golgi. Injecting RS1 fragments into Xenopus laevis oocytes expressing SGLT1 or CNT1 and measuring the expressed uptake of α-methylglucoside or uridine 1 hour later, we identified a RS1 domain (RS1-Reg) containing multiple predicted phosphorylation sites that is responsible for this post-translational downregulation of SGLT1 and CNT1. Dependent on phosphorylation, RS1-Reg blocks the release of SGLT1-containing vesicles from the Golgi in a glucose-dependent manner or glucose-independent release of CNT1-containing vesicles. We showed that upregulation of SGLT1 in the small intestine after glucose ingestion is promoted by glucose-dependent disinhibition of the RS1-Reg-blocked exocytotic pathway of SGLT1 between meals. Mimicking phosphorylation of RS1-Reg, we obtained a RS1-Reg variant that downregulates SGLT1 in the brush-border membrane at high luminal glucose concentration. Because RS1 mediates short-term regulation of various transporters, we propose that the RS1-Reg-navigated transporter release from Golgi represents a basic regulatory mechanism of general importance, which implies the existence of receptor proteins that recognize different phosphorylated forms of RS1-Reg and of complex transporter-specific sorting in the trans-Golgi. RS1-Reg-derived peptides that downregulate SGLT1 at high intracellular glucose concentrations may be used for downregulation of glucose absorption in small intestine, which has been proposed as strategy for treatment of type 2 diabetes.

Here bicyclo[3.1.0]hexane locked deoxycytidine (S-MCdC, N-MCdC), and deoxyadenosine analogs (S-MCdA and N-MCdA) were examined as substrates for purified preparations of human deoxynucleoside kinases: dCK, dGK, TK2, TK1, the ribonucleoside kinase UCK2, two NMP kinases (CMPK1, TMPK) and a NDP kinase. dCK can be important for the first step of phosphorylation of S-MCdC in cells, but S-MCdCMP was not a substrate for CMPK1, TMPK, or NDPK. dCK and dGK had a preference for the S-MCdA whereas N-MCdA was not a substrate for dCK, TK1, UCK2, TK2, dGK nucleoside kinases. The cell growth experiments suggested that N-MCdC and S-MCdA could be activated in cells by cellular kinases so that a triphosphate metabolite was formed. List of abbreviations: ddC, 2', 3'-didioxycytosine, Zalcitabine; 3TC, β-L-(-)-2',3'-dideoxy-3'-thiacytidine, Lamivudine; CdA, 2-cloro-2'-deoxyadenosine, Cladribine; AraA, 9-β-D-arabinofuranosyladenine; hCNT 1-3, human Concentrative Nucleoside Transporter type 1, 2 and 3; hENT 1-4, human Equilibrative Nucleoside Transporter type 1, 2, 3, and 4.

Gemcitabine is widely used for pancreatic, lung, and bladder cancer. However, drug resistance against gemcitabine is a large obstacle to effective chemotherapy. Nucleoside transporters, nucleoside and nucleotide metabolic enzymes, and efflux transporters have been reported to be involved in gemcitabine resistance. Although most of the resistant factors are supposed to be related to each other, it is unclear how one factor can affect the other one. In this study, we established gemcitabine-resistant pancreatic cancer cell lines. Gemcitabine resistance in these cells is caused by two major processes: a decrease in gemcitabine uptake and overexpression of ribonucleotide reductase large subunit (RRM1). Knockdown of RRM1, but not the overexpression of concentrative nucleoside transporter 1 (CNT1), could completely overcome the gemcitabine resistance. RRM1 knockdown in gemcitabine-resistant cells could increase the intracellular accumulation of gemcitabine by increasing the nucleoside transporter expression. Furthermore, a synergistic effect was observed between hydroxyurea, a ribonucleotide reductase (RR) inhibitor, and gemcitabine on the gemcitabine-resistant cells. Here we indicate that RR is one of the most promising targets to overcome gemcitabine resistance in gemcitabine-resistant cells with dual resistant factors.

Purine-rich foods have long been suspected as a major cause of hyperuricemia. We hypothesized that inhibition of human concentrative nucleoside transporter 2 (hCNT2) would suppress increases in serum urate levels derived from dietary purines. To test this hypothesis, the development of potent hCNT2 inhibitors was required. By modifying adenosine, an hCNT2 substrate, we successfully identified 8-aminoadenosine derivatives as a new class of hCNT2 inhibitors. Compound 12 moderately inhibited hCNT2 (IC50 = 52 ± 3.8 μM), and subsequent structure-activity relationship studies led to the discovery of compound 48 (IC50 = 0.64 ± 0.19 μM). Here we describe significant findings about structural requirements of 8-aminoadenosine derivatives for exhibiting potent hCNT2 inhibitory activity.

Extracellular adenosine concentrations are regulated by a panel of membrane transporters which, in most cases, mediate its uptake into cells. Adenosine transporters belong to two gene families encoding Equilibrative and Concentrative Nucleoside Transporter proteins (ENTs and CNTs, respectively). The lack of appropriate pharmacological tools targeting every transporter subtype has introduced some bias on the current knowledge of the role of these transporters in modulating adenosine levels. In this regard, ENT1, for which pharmacology is relatively well-developed, has often been identified as a major player in purinergic signaling. Nevertheless, other transporters such as CNT2 and CNT3 can also contribute to purinergic modulation based on their high affinity for adenosine and concentrative capacity. Moreover, both transporter proteins have also been shown to be under purinergic regulation via P1 receptors in different cell types, which further supports its relevance in purinergic signaling. Thus, several transporter proteins regulate extracellular adenosine levels. Moreover, CNT and ENT proteins are differentially expressed in tissues but also in particular cell types. Accordingly, transporter-mediated fine tuning of adenosine levels is cell and tissue specific. Future developments focusing on CNT pharmacology are needed to unveil transporter subtype-specific events.

To evaluate the role of nucleoside transporters in the nose-to-brain uptake of [18F]fluorothymidine (FLT), an equilibrative nucleoside transporter (ENT1,2) and concentrative nucleoside transporter (CNT1–3) substrate, using PET to measure local tissue concentrations. Anesthetized Sprague-Dawley rats were administered FLT by intranasal (IN) instillation or tail-vein injection (IV). NBMPR (nitrobenzylmercaptopurine riboside), an ENT1 inhibitor, was administered either IN or intraperitoneally (IP). Dynamic PET imaging was performed for up to 40 min. A CT was obtained for anatomical co-registration and attenuation correction. Time-activity curves (TACs) were generated for the olfactory bulb (OB) and remaining brain, and the area-under-the-curve (AUC) for each TAC was calculated to determine the total tissue exposure of FLT. FLT concentrations were higher in the OB than in the rest of the brain following IN administration. IP administration of NBMPR resulted in increased OB and brain FLT exposure following both IN and IV administration, suggesting that NBMPR decreases the clearance rate of FLT from the brain. When FLT and NBMPR were co-administered IN, there was a decrease in the OB AUC while an increase in the brain AUC was observed. The decrease in OB exposure was likely the result of inhibition of ENT1 uptake activity in the nose-to-brain transport pathway. FLT distribution patterns show that nucleoside transporters, including ENT1, play a key role in the distribution of transporter substrates between the nasal cavity and the brain via the OB.

rCNT2 is a purine-preferring concentrative nucleoside transporter implicated in the regulation of extracellular adenosine levels and purinergic signaling. This study addressed the analysis of the CNT2 C-terminus tail as a domain likely to be implicated in transporter sorting. The topological mapping of this segment revealed that Cys(615) and Cys(649) are important residues for the proper trafficking of CNT2 to the plasma membrane. The inhibition of protein disulfide isomerase (PDI) and ER glycosidase I and II impaired rCNT2 trafficking to the cell surface, similarly to Cys(615) and Cys(649) mutants. The present work suggests these two cysteine residues are relevant for the proper sorting of the transporter and its functional performance.

2'-Deoxyadenosine 5'-monophosphate (dAMP), a deoxyribonucleotide found in DNA, affects intestinal cell growth. The molecular mechanisms underlying gastrointestinal absorption of foreign DNA ingested along with food has hardly been investigated. The aim of this study was to investigate the mechanism underlying intestinal absorption of dAMP. The uptake of [H-3]dAMP by Caco-2 cells was Na+- and pH-dependent and was inhibited by various nucleosides. In contrast, nitrobenzylthioinosine (NMBPR), an equilibrative nucleoside transporter inhibitor, showed little inhibitory effects on [H-3]dAMP uptake. Additionally, human concentrative nucleoside transporter (CNT) 3, transiently expressed in COS-7 cells, mediated the uptake of [3H]dAMP. A kinetic study revealed that the K-m value of CNT3-mediated uptake of dAMP (59.6 mu M) was close to that of 2'-deoxyadenosine (dAdo) (56.3 mu M), whereas the dAMP V-max (15.6 pmol.mg protein(-1)min(-1)) was 500-fold lesser than the dAdo V-max (7782 pmol.mg protein-1min-1). Further, [3H]dAMP uptake was greater in COS-7 cells expressing ecto-5'-nucleotidase/CD73 with CNT3 than in those expressing CNT3 alone. These data suggest that, although dAMP is a substrate of CNT3, it is dephosphorylated to dAdo by CD73 and is efficiently absorbed as dAdo from the intestinal lumen.

This study aimed to investigate the mechanisms underlying the anti-proliferative effects of the ethanolic Cimicifuga racemosa extract BNO-1055 on prostate cells and evaluate its therapeutic potential. BNO-1055 dose-dependently attenuated cellular uptake and incorporation of thymidine and BrdU and significantly inhibited cell growth after long-time exposure. Similar results were obtained using saponin-enriched sub-fractions of BNO-1055. These inhibitory effects of BNO-1055 could be mimicked using pharmacological inhibitors and isoform-specific siRNAs targeting the equilibrative nucleoside transporters ENT1 and ENT2. Moreover, BNO-1055 attenuated the uptake of clinically relevant nucleoside analogs, e.g. the anti-cancer drugs gemcitabine and fludarabine. Consistent with inhibition of the salvage nucleoside uptake pathway BNO-1055 potentiated the cytotoxicity of the de novo nucleotide synthesis inhibitor 5-FU without significantly altering its uptake. Collectively, these data show for the first time that the anti-proliferative effects of BNO-1055 result from hindered nucleoside uptake due to impaired ENT activity and demonstrate the potential therapeutic use of BNO-1055 for modulation of nucleoside transport.

Nucleoside transporters play key roles in the physiology of nucleosides and nucleobases in mammals and also contribute to the pharmacological actions produced by their analogs. Several mammalian nucleoside transporters have been identified at the molecular level. The aim of this study was to examine the expression of the major nucleoside transporter isoforms in the mouse testis and ovary. Equilibrative Nucleoside Transporter 1 (ENT1) and ENT2 were expressed in the mouse testis and ovary, which was consistent with the general consideration that these transporters are ubiquitous in mouse tissues. The expression of Concentrative Nucleoside Transporter 2 (CNT2) and CNT3, but not CNT1 was detected in the mouse testis. These three CNT isoforms were also expressed in the ovary. Therefore, we concluded that the mouse testis and ovary simultaneously exhibit multiple transport processes for nucleosides and bases and also that these transporters are responsible for the homeostatic control of biological levels of nucleosides and bases in these tissues.

Objective: The nucleoside transporter family is an emerging target for cancer, viral and cardiovascular diseases. Due to the difficulty in the expression, isolation and crystallization of membrane proteins, there is a lack of structural information on any of the mammalian and for that matter the human proteins. Thus the objective of this study was to build homology models for the three cloned concentrative nucleoside transporters hCNT1, hCNT2 and hCNT3 and validate them for screening towards the discovery of much needed inhibitors and probes. Methods: The recently reported crystal structure of the Vibrio cholerae concentrative nucleoside transporter (vcCNT), has satisfactory similarity to the human CNT autologs and was thus used as a template to build homology models of all three hCNTs. The Schrodinger modeling suite was used for the exercise. External validation of the homology models was carried out by docking a set of recently reported known hCNT1 nucleoside class inhibitors at the putative binding site using induced fit docking (IDF) methodology with the Glide docking program. Then hCNT1 homology model was subsequently used to conduct a virtual screening of a 360,000 compound library and 172 compounds were obtained and biologically evaluated for hCNT 1, 2 and 3 inhibitory potency and selectivity. Results: Good quality homology models were obtained for all three hCNTs as indicated by interrogation for various structural parameters and also external validated by docking of known inhibitors. The IDF docking results showed good correlations between IDF scores and inhibitory activities; particularly for hCNT1. From the top 0.1% of compounds ranked by virtual screening with the hCNT1 homology model, the 172 compounds selected and tested for against hCNT1, hCNT2 and hCNT3, yielded 14 new inhibitors (hits) of (i.e., 8% success rate). The most active compound exhibited an IC50 of 9.05 μM, which shows a greater than 25-fold higher potency than phlorizin the standard CNT inhibitor (IC50 of 250 μM). Conclusion: We successfully undertook a homology modeling and validation for all human concentrative nucleoside transporters (hCNT 1, 2 and 3). The proof-of-concept that these models are promising from virtual screening to identify potent and selective inhibitors was also obtained using the hCNT1 model. Thus we identified a novel potent hCNT1 inhibitor that is more potent and more selective than the standard inhibitor phlorizin. The other hCNT1 hits also mostly exhibited selectivity. These homology models should be useful for virtual screening to identify novel hCNT inhibitors, as well as for optimization of hCNT inhibitors.

Nucleosides play key roles in biology as precursors for salvage pathways of nucleotide synthesis. Prokaryotes import nucleosides across the cytoplasmic membrane by proton- or sodium-driven transporters belonging to the Concentrative Nucleoside Transporter (CNT) family or the Nucleoside:H(+) Symporter (NHS) family of the Major Facilitator Superfamily. The high resolution structure of a CNT from Vibrio cholerae has recently been determined, but no similar structural information is available for the NHS family. To gain a better understanding of the molecular mechanism of nucleoside transport, in the present study the structures of two conformations of the archetypical NHS transporter NupG from Escherichia coli were modelled on the inward- and outward-facing conformations of the lactose transporter LacY from E. coli, a member of the Oligosaccharide:H(+) Symporter (OHS) family. Sequence alignment of these distantly related proteins (∼ 10% sequence identity), was facilitated by comparison of the patterns of residue conservation within the NHS and OHS families. Despite the low sequence similarity, the accessibilities of endogenous and introduced cysteine residues to thiol reagents were found to be consistent with the predictions of the models, supporting their validity. For example C358, located within the predicted nucleoside binding site, was shown to be responsible for the sensitivity of NupG to inhibition by p-chloromercuribenzene sulphonate. Functional analysis of mutants in residues predicted by the models to be involved in the translocation mechanism, including Q261, E264 and N228, supported the hypothesis that they play important roles, and suggested that the transport mechanisms of NupG and LacY, while different, share common features.

Entecavir (ETV), a nucleoside analog with high efficacy against hepatitis B virus, is recommended as a first-line antiviral drug for the treatment of chronic hepatitis B. However, scant information is available on the use of ETV in pregnancy. To better understand the safety of ETV in pregnant women, we aimed to demonstrate whether ETV could permeate placental barrier and the underlying mechanism. Our study showed that small amount of ETV could permeate across placenta in mice. ETV accumulation in activated or nonactivated BeWo cells (treated with or without forskolin) was sharply reduced in the presence of 100 µM of adenosine, cytidine, and in Na+ free medium, indicating that nucleoside transporters possibly mediate the uptake of ETV. Furthermore, ETV was proved to be a substrate of concentrative nucleoside transporter (CNT) 2 and CNT3, of organic cation transporter (OCT) 3, and of breast cancer resistance protein (BCRP) using transfected cells expressing respective transporters. The inhibition of ETV uptake in primary human trophoblast cells further confirmed that equilibrative nucleoside transporter (ENT) 1/2, CNT2/3, OCT3, and organic cation/carnitine transporter (OCTN) 2 might be involved in ETV transfer in human placenta. Therefore, ETV uptake from maternal circulation to trophoblast cells was possibly transported by CNT2/3, ENT1/2, and OCTN2, whereas ETV efflux from trophoblast cells to fetal circulation was mediated by OCT3, and efflux from trophoblast cells to maternal circulation might be mediated by BCRP, multidrug resistance-associated protein 2, and P-glycoprotein. The information obtained in the present study may provide a basis for the use of ETV in pregnancy.

To virtual screen the compound of Chicory combined with the concentrative nucleoside transporter 2 (CNT2) in molecular docking technology.The homology model of hCNT2 was produced, and then the Vina software was employed to virtual screen the Chicory compound combined with CNT2. Compared with 7,8,3'-trihydroxyflavone, a CNT2 inhibitors, 23 score higher chicory compounds were hit.Meanwhile, the ten top compounds have been revealed that play important role in decrease the uric level. The bioactivity to CNT2 needs to be investigatedin experiment. CNT2 may be a potential target of chicory, which decreases the absorption of purine nucleoside in intestinal tract.

Nucleoside transporter (NT) and nucleic-related enzyme (NRE) play key roles in the physiology of nucleosides and the pharmacology of its analogs in mammals. In this study, we examined the effect of fluoxetine, a selective serotonin reuptake inhibitor, and pergolide, a dopamine D receptor agonist, on the expression of NTs and NREs in mouse brain. It was confirmed by the detection of corresponding mRNAs that three equilibrative nucleoside transporter (ENT1-3) isoforms, concentrative nucleoside transporter 2 (CNT2), CNT3, adenosine kinase (AK), and apyrase, but not CNT1, were expressed in brain tissue. Based on an assessment by mRNA determination, the cerebral expression of CNT2 was found to be increased by administration of fluoxetine and pergolide to mice. Furthermore, pergolide increased the expression of ENT2. However, fluoxetine and pergolide had no significant effect on the expression of mRNA for other NTs, AK, and apyrase. Therefore, we concluded that the expression of several NT isoforms, but not NREs, in mouse brain was affected by treatment with fluoxetine and pergolide.

The concentrative nucleoside transporter CNT1 and equilibrated nucleoside transporter ENT1 mediate the cellular uptake of naturally occurring pyrimidine and purine nucleosides and many structurally diverse anticancer and antiviral nucleoside analogs, thereby regulating drug responses or toxicity at the target site. The objectives of this study were to analyze interindividual variations in the cellular accumulation of gemcitabine and to examine the correlation between the uptake of gemcitabine and expression levels of CNT1 and ENT1 transporters. Gemcitabine was a substrate for both CNT1 and ENT1 with higher affinity to CNT1 than to ENT1. The difference in gemcitabine uptake was 4.8-fold in peripheral blood mononuclear cells (PBMCs) from 10 subjects. Among these, the CNT1- and ENT1-mediated uptake of gemcitabine was 14.3- and 16.5-folds, respectively. CNT1-mediated gemcitabine uptake showed a higher correlation with the CNT1 expression level than did ENT1-mediated uptake with ENT1 expression level. In conclusion, CNT1 seemed to be a major contributing factor to gemcitabine uptake in PBMCs and showed 14.3-fold inter-individual variations. However, ENT1-mediated uptake of gemcitabine might compensate for the total uptake of gemcitabine; therefore, the variation in the apparent accumulation of gemcitabine was smaller than that of the individual transporters.

Nucleosides are required for DNA and RNA synthesis, and the nucleoside adenosine has a function in a variety of signalling processes. Transport of nucleosides across cell membranes provides the major source of nucleosides in many cell types and is also responsible for the termination of adenosine signalling. As a result of their hydrophilic nature, nucleosides require a specialized class of integral membrane proteins, known as nucleoside transporters (NTs), for specific transport across cell membranes. In addition to nucleosides, NTs are important determinants for the transport of nucleoside-derived drugs across cell membranes. A wide range of nucleoside-derived drugs, including anticancer drugs (such as Ara-C and gemcitabine) and antiviral drugs (such as zidovudine and ribavirin), have been shown to depend, at least in part, on NTs for transport across cell membranes. Concentrative nucleoside transporters, members of the solute carrier transporter superfamily SLC28, use an ion gradient in the active transport of both nucleosides and nucleoside-derived drugs against their chemical gradients. The structural basis for selective ion-coupled nucleoside transport by concentrative nucleoside transporters is unknown. Here we present the crystal structure of a concentrative nucleoside transporter from Vibrio cholerae in complex with uridine at 2.4 A. Our functional data show that, like its human orthologues, the transporter uses a sodium-ion gradient for nucleoside transport. The structure reveals the overall architecture of this class of transporter, unravels the molecular determinants for nucleoside and sodium binding, and provides a framework for understanding the mechanism of nucleoside and nucleoside drug transport across cell membranes.

ααα-Trifluorothymidine (TFT), an anticancer nucleoside analog, is a potent thymidylate synthase inhibitor. TFT exerts its antitumor activity primarily by inducing DNA fragmentation after incorporation of the triphosphate form of TFT into the DNA. Although an oral combination of TFT and a thymidine phosphorylase inhibitor has been clinically developed, there is little information regarding TFT absorption. Therefore, we investigated TFT absorption in the rat small intestine. After oral administration of TFT in rats, more than 75% of the TFT was absorbed. To identify the uptake transport system, uptake studies were conducted by using everted sacs prepared from rat small intestines. TFT uptake was saturable, significantly reduced under Na(+)-free conditions, and strongly inhibited by the addition of an endogenous pyrimidine nucleoside. From these results, we suggested the involvement of concentrative nucleoside transporters (CNTs) in TFT absorption into rat small intestine. In rat small intestines, the mRNAs coding for rat CNT1 (rCNT1) and rCNT2, but not for rCNT3, were predominantly expressed. To investigate the roles of rCNT1 and rCNT2 in TFT uptake, we conducted uptake assays by using Xenopus laevis oocytes injected with rCNT1 complementary RNA (cRNA) and rCNT2 cRNA. TFT uptake by X. laevis oocytes injected with rCNT1 cRNA, and not rCNT2 cRNA, was significantly greater than that by water-injected oocytes. In addition, in situ single-pass perfusion experiments performed using rat jejunum regions showed that thymidine, a substrate for CNT1, strongly inhibited TFT uptake. In conclusion, TFT is absorbed via rCNT1 in the intestinal lumen in rats.

The plasma membrane distribution and related biological activity of nucleoside transporter proteins (NTs) were investigated in human syncytiotrophoblast from term placenta using a variety of approaches, including nucleoside uptake measurements into vesicles from selected plasma membrane domains, NT immunohistochemistry, and subcellular localization (basal, heavy, and light apical membranes as well as raft-enriched membranes from the apical domain). In contrast with other epithelia, in this epithelium, we have identified the high-affinity pyrimidine-preferring human concentrative nucleoside transporter (hCNT) 1 as the only hCNT-type protein expressed at both the basal and apical membranes. hCNT1 localization in lipid rafts is also dependent on its subcellular localization in the apical plasma membrane, suggesting a complex cellular and regional expression. Overall, this result favors the view that the placenta is a pyrimidine-preferring nucleoside sink from both maternal and fetal sides, and hCNT1 plays a major role in promoting pyrimidine salvage and placental growth. This finding may be of pharmacological relevance, because hCNT1 is known to interact with anticancer nucleoside-derived drugs and other molecules, such as nicotine and caffeine, for which a great variety of harmful effects on placental and fetal development, including intrauterine growth retardation, have been reported.