Addressing the Clinical Importance of Equilibrative Nucleoside Transporters in Drug Discovery and Development.

The US Food and Drug Administration (FDA), European Medicines Agency (EMA), and Pharmaceuticals and Medical Devices Agency (PMDA) guidances on small-molecule drug-drug interactions (DDIs), with input from the International Transporter Consortium (ITC), recommend the evaluation of nine drug transporters. Although other clinically relevant drug uptake and efflux transporters have been discussed in ITC white papers, they have been excluded from further recommendation by the ITC and are not included in current regulatory guidances. These include the ubiquitously expressed equilibrative nucleoside transporters (ENT) 1 and ENT2, which have been recognized by the ITC for their potential role in clinically relevant nucleoside analog drug interactions for patients with cancer. Although there is comparatively limited clinical evidence supporting their role in DDI risk or other adverse drug reactions (ADRs) compared with the nine highlighted transporters, several in vitro and in vivo studies have identified ENT interactions with non-nucleoside/non-nucleotide drugs, in addition to nucleoside/nucleotide analogs. Some noteworthy examples of compounds that interact with ENTs include cannabidiol and selected protein kinase inhibitors, as well as the nucleoside analogs remdesivir, EIDD-1931, gemcitabine, and fialuridine. Consequently, DDIs involving the ENTs may be responsible for therapeutic inefficacy or off-target toxicity. Evidence suggests that ENT1 and ENT2 should be considered as transporters potentially involved in clinically relevant DDIs and ADRs, thereby warranting further investigation and regulatory consideration.

Deletion of equilibrative nucleoside transporter 2 disturbs energy metabolism and exacerbates disease progression in an experimental model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease, characterized by motor dysfunction and abnormal energy metabolism. Equilibrative nucleoside transporter 1 (ENT1) and ENT2 are the major nucleoside transporters in cellular plasma membrane of the brain. Yet, unlike ENT1 whose function has been better investigated in HD, the role of ENT2 in HD remains unclear. The present study aimed to investigate the impacts of ENT2 deletion on HD using a well-characterized mouse model (R6/2). Microarray analysis, quantitative real-time polymerase chain reaction, and immunostaining of ENT2 in postmortem human brain tissues were conducted. R6/2 mice with or without genetic deletion of ENT2 were generated. Motor functions, including rotarod performance and limb-clasping test, were examined at the age of 7 to 12 weeks. Biochemical changes were evaluated by immunofluorescence staining and immunoblotting at the age of 12 to 13 weeks. In regard to energy metabolism, levels of striatal metabolites were determined by liquid chromatography coupled with the fluorescence detector or quadrupole time-of-flight mass spectrometer. Mitochondrial bioenergetics was assessed by the Seahorse assay. The results showed that ENT2 protein was detected in the neurons and astrocytes of human brains and the levels in the postmortem brain tended to be higher in patients with HD. In mice, ENT2 deletion did not alter the phenotype of the non-HD controls. Yet, ENT2 deletion deteriorated motor function and increased the number of aggregated mutant huntingtin in the striatum of R6/2 mice. Notably, disturbed energy metabolism with decreased ATP level and increased AMP/ ATP ratio was observed in R6/2-Ent2-/- mice, compared with R6/2-Ent2+/+ mice, resulting in the activation of AMPK in the late disease stage. Furthermore, ENT2 deletion reduced the NAD+/NADH ratio and impaired mitochondrial respiration in the striatum of R6/2 mice. Taken together, these findings indicate the crucial role of ENT2 in energy homeostasis, in which ENT2 deletion further impairs mitochondrial bioenergetics and deteriorates motor function in R6/2 mice.

Remdesivir and EIDD-1931 Interact with Human Equilibrative Nucleoside Transporters 1 and 2: Implications for Reaching SARS-CoV-2 Viral Sanctuary Sites.

Equilibrative nucleoside transporters (ENTs) are present at the blood-testis barrier (BTB), where they can facilitate antiviral drug disposition to eliminate a sanctuary site for viruses detectable in semen. The purpose of this study was to investigate ENT-drug interactions with three nucleoside analogs, remdesivir, molnupiravir, and molnupiravir's active metabolite, ß-d-N4-hydroxycytidine (EIDD-1931), and four non-nucleoside molecules repurposed as antivirals for coronavirus disease 2019 (COVID-19). The study used three-dimensional pharmacophores for ENT1 and ENT2 substrates and inhibitors and Bayesian machine learning models to identify potential interactions with these transporters. In vitro transport experiments demonstrated that remdesivir was the most potent inhibitor of ENT-mediated [3H]uridine uptake (ENT1 IC50: 39 µM; ENT2 IC50: 77 µM), followed by EIDD-1931 (ENT1 IC50: 259 µM; ENT2 IC50: 467 µM), whereas molnupiravir was a modest inhibitor (ENT1 IC50: 701 µM; ENT2 IC50: 851 µM). Other proposed antivirals failed to inhibit ENT-mediated [3H]uridine uptake below 1 mM. Remdesivir accumulation decreased in the presence of 6-S-[(4-nitrophenyl)methyl]-6-thioinosine (NBMPR) by 30% in ENT1 cells (P = 0.0248) and 27% in ENT2 cells (P = 0.0054). EIDD-1931 accumulation decreased in the presence of NBMPR by 77% in ENT1 cells (P = 0.0463) and by 64% in ENT2 cells (P = 0.0132), which supported computational predictions that both are ENT substrates that may be important for efficacy against COVID-19. NBMPR failed to decrease molnupiravir uptake, suggesting that ENT interaction is likely inhibitory. Our combined computational and in vitro data can be used to identify additional ENT-drug interactions to improve our understanding of drugs that can circumvent the BTB. SIGNIFICANCE STATEMENT: This study identified remdesivir and EIDD-1931 as substrates of equilibrative nucleoside transporters 1 and 2. This provides a potential mechanism for uptake of these drugs into cells and may be important for antiviral potential in the testes and other tissues expressing these transporters.

Functional expression of equilibrative and concentrative nucleoside transporters in alveolar epithelial cells.

Equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs) mediate the cellular uptake of nucleosides and nucleobases across the plasma membrane and play important roles in the salvage pathways of nucleotide synthesis. However, information about nucleoside transport systems in the lung alveolar epithelial cells is limited. Therefore, in the present study, we examined the function and expression of nucleoside transporters using primary cultured alveolar type II cells and transdifferentiated type I-like cells. The uptake of uridine, a substrate for ENTs and CNTs, in type II and type I-like cells was time, temperature, and concentration dependent, and was inhibited by other nucleoside transporter substrates such as adenosine. Uridine uptake in both cells was insensitive to nanomolar concentrations of NBMPR, a potent ENT1 inhibitor, while it was inhibited by higher concentrations of NBMPR, suggesting that ENT2, but not ENT1, is involved in uridine uptake in these cells. Additionally, uridine uptake was higher in the presence of Na+ than in the absence of Na + and was partially inhibited by a CNT inhibitor phloridzin in these cells, suggesting that CNT is also involved in uridine uptake. In both cells, the mRNA expression of ENT1, ENT2, CNT2, and CNT3 was observed. Finally, the activity of uridine uptake was considerably higher in type II cells than in type I-like cells. In addition, the mRNA expression of ENT2, CNT2, and CNT3, but not ENT1, was lower in type I-like cells than in type II cells. These findings would help understand the functional roles of equilibrative and concentrative nucleoside transporters in alveolar epithelial cells.

Export of RNA-derived modified nucleosides by equilibrative nucleoside transporters defines the magnitude of autophagy response and Zika virus replication.

RNA contains a wide variety of posttranscriptional modifications covalently attached to its base or sugar group. These modified nucleosides are liberated from RNA molecules as the consequence of RNA catabolism and released into extracellular space, but the molecular mechanism of extracellular transport and its pathophysiological implications have been unclear. In the present study, we discovered that RNA-derived modified nucleosides are exported to extracellular space through equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2), with ENT1 showing higher preference for modified nucleosides than ENT2. Pharmacological inhibition or genetic deletion of ENT1 and ENT2 significantly attenuated export of modified nucleosides thereby resulting in their accumulation in cytosol. Using mutagenesis strategy, we identified an amino acid residue in ENT1 that is involved in the discrimination of unmodified and modified nucleosides. In ENTs-deficient cells, the elevated levels of intracellular modified nucleosides were closely associated with an induction of autophagy response as evidenced by increased LC3-II level. Importantly, we performed a screening of modified nucleosides capable of inducing autophagy and found that 1-methylguanosine (m1G) was sufficient to induce LC3-II levels. Pathophysiologically, defective export of modified nucleosides drastically induced Zika virus replication in an autophagy-dependent manner. In addition, we also found that pharmacological inhibition of ENTs by dilazep significantly induced Zika virus replication. Collectively, our findings highlight RNA-derived modified nucleosides as important signaling modulators that activate autophagy response and indicate that defective export of these modified nucleoside can have profound consequences for pathophysiology.

Differential Inhibition of Equilibrative Nucleoside Transporter 1 (ENT1) Activity by Tyrosine Kinase Inhibitors.

BACKGROUND AND OBJECTIVES: Equilibrative nucleoside transporter (ENT) 1 is a widely-expressed drug transporter, handling nucleoside analogues as well as endogenous nucleosides. ENT1 has been postulated to be inhibited by some marketed tyrosine kinase inhibitors (TKIs). To obtain insights into this point, the interactions of 24 TKIs with ENT1 activity have been analyzed. METHODS: Inhibition of ENT1 activity was investigated in vitro through quantifying the decrease of [3H]-uridine uptake caused by TKIs in HAP1 ENT2-knockout cells, exhibiting selective ENT1 expression. TKI effects towards ENT1-mediated transport were additionally characterized in terms of their in vivo relevance and of their relationship to TKI molecular descriptors. Putative transport of the TKI lorlatinib by ENT1/ENT2 was analyzed by LC-MS/MS. RESULTS: Of 24 TKIs, 12 of them, each used at 10 µM, were found to behave as moderate or strong inhibitors of ENT1, i.e., they decreased ENT1 activity by at least 35%. This inhibition was concentration-dependent for at least the strongest ones (IC50 less than 10 µM) and was correlated with some molecular descriptors, especially with atom-type E-state indices. Lorlatinib was notably a potent in vitro inhibitor of ENT1/ENT2 (IC50 values around 1.0-2.5 µM) and was predicted to inhibit these nucleoside transporters at relevant clinical concentrations, without, however, being a substrate for them. CONCLUSION: Our data unambiguously add ENT1 to the list of drug transporters inhibited by TKIs, especially by lorlatinib. This point likely merits attention in terms of possible drug-drug interactions, notably for nucleoside analogues, whose ENT1-mediated uptake into their target cells may be hampered by co-administrated TKIs such as lorlatinib.

Testicular disposition of clofarabine in rats is dependent on equilibrative nucleoside transporters.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5-year survival rate is high, some patients respond poorly to chemotherapy or have recurrence in locations such as the testis. The blood-testis barrier (BTB) can prevent complete eradication by limiting chemotherapeutic access and lead to testicular relapse unless a chemotherapeutic is a substrate of drug transporters present at this barrier. Equilibrative nucleoside transporter (ENT) 1 and ENT2 facilitate the movement of substrates across the BTB. Clofarabine is a nucleoside analog used to treat relapsed or refractory ALL. This study investigated the role of ENTs in the testicular disposition of clofarabine. Pharmacological inhibition of the ENTs by 6-nitrobenzylthioinosine (NBMPR) was used to determine ENT contribution to clofarabine transport in primary rat Sertoli cells, in human Sertoli cells, and across the rat BTB. The presence of NBMPR decreased clofarabine uptake by 40% in primary rat Sertoli cells (p = .0329) and by 53% in a human Sertoli cell line (p = .0899). Rats treated with 10 mg/kg intraperitoneal (IP) injection of the NBMPR prodrug, 6-nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), or vehicle, followed by an intravenous (IV) bolus 10 mg/kg dose of clofarabine, showed a trend toward a lower testis concentration of clofarabine than vehicle (1.81 ± 0.59 vs. 2.65 ± 0.92 ng/mg tissue; p = .1160). This suggests that ENTs could be important for clofarabine disposition. Clofarabine may be capable of crossing the human BTB, and its potential use as a first-line treatment to avoid testicular relapse should be considered.

ENT2 facilitates brain endothelial cell penetration and blood-brain barrier transport by a tumor-targeting anti-DNA autoantibody.

The blood-brain barrier (BBB) prevents antibodies from penetrating the CNS and limits conventional antibody-based approaches to brain tumors. We now show that ENT2, a transporter that regulates nucleoside flux at the BBB, may offer an unexpected path to circumventing this barrier to allow targeting of brain tumors with an anti-DNA autoantibody. Deoxymab-1 (DX1) is a DNA-damaging autoantibody that localizes to tumors and is synthetically lethal to cancer cells with defects in the DNA damage response. We found that DX1 penetrated brain endothelial cells and crossed the BBB, and mechanistic studies identify ENT2 as the key transporter. In efficacy studies, DX1 crosses the BBB to suppress orthotopic glioblastoma and breast cancer brain metastases. ENT2-linked transport of autoantibodies across the BBB has potential to be exploited in brain tumor immunotherapy, and its discovery raises hypotheses on actionable mechanisms of CNS penetration by neurotoxic autoantibodies in CNS lupus.

Amelioration of lipopolysaccharide-induced memory impairment in equilibrative nucleoside transporter-2 knockout mice is accompanied by the changes in glutamatergic pathways.

Neuroinflammation has been implicated in cognitive deficits in neurological and neurodegenerative diseases. Lipopolysaccharide (LPS)-induced neuroinflammation and the breakdown of the blood-brain barrier can be attenuated in mice with equilibrative nucleoside transporter-2 (ENT2/Ent2) deletion. The present study was aimed to investigate the role of ENT2 in cognitive and neuronal functions under physiological and inflammatory conditions, in terms of behavioral performance and synaptic plasticity in saline- and LPS-treated Ent2 knockout (KO) mice and their wild-type (WT) littermate controls. Repeated administrations of LPS significantly impaired spatial memory formation in Morris water maze and hippocampal-dependent long-term potentiation (LTP) in WT mice. The LPS-treated WT mice exhibited significant synaptic and neuronal damage in the hippocampus. Notably, the LPS-induced impairment in spatial memory and LTP performance were attenuated in Ent2 KO mice, along with the preservation of neuronal survival. The beneficial effects were accompanied by the normalization of excessive extracellular glutamate and aberrant downstream signaling of glutamate receptor activation, including the upregulation of phosphorylated p38 mitogen-activated protein kinase and the downregulation of phosphorylated cyclic adenosine monophosphate-response element-binding protein. There was no significant difference in behavioral outcome and all tested parameters between these two genotypes under physiological condition. These results suggest that ENT2 plays an important role in regulating inflammation-associated cognitive decline and neuronal damage.

Exosomes secreted from cancer-associated fibroblasts elicit anti-pyrimidine drug resistance through modulation of its transporter in malignant lymphoma.

The tumor microenvironment is deeply involved in the process of tumor growth and development. In this study, we focused on cancer-associated fibroblasts (CAFs) and their derived exosomes on the lymphoma microenvironment to uncover their clinical significance. CAFs were established from primary lymphoma samples, and exosomes secreted from CAFs were obtained by standard procedures. We then investigated the roles of CAFs and their derived exosomes in the survival and drug resistance of lymphoma cells. CAFs supported the survival of lymphoma cells through increased glycolysis, and the extent differed among CAFs. Exosomes were identified as a major component of the extracellular vesicles from CAFs, and they also supported the survival of lymphoma cells. The suppression of RAB27B, which is involved in the secretion of exosomes, using a specific siRNA resulted in reduced exosome secretion and decreased survival of lymphoma cells. Moreover, anti-pyrimidine drug resistance was induced in the presence of exosomes through the suppression of the pyrimidine transporter, equilibrative nucleoside transporter 2 (ENT2), and the suppression of ENT2 was significant in in vivo experiments and clinical samples. RNA sequencing analysis of miRNAs in exosomes identified miR-4717-5p as one of the most abundant miRNAs in the exosome, which suppressed the expression of ENT2 and induced anti-pyrimidine drug resistance in vitro. Our results suggest that exosomes including miR-4717-5p secreted from CAFs play a pivotal role in the lymphoma microenvironment, indicating that they are a promising therapeutic target.

Fluorouracil uptake in triple-negative breast cancer cells: Negligible contribution of equilibrative nucleoside transporters 1 and 2.

Equilibrative nucleoside transporters (ENTs) 1 and 2 reportedly accept fluorouracil as a substrate. Here, we evaluated ENT1/2 expression at the messenger RNA (mRNA), protein, and functional levels in a panel of four triple-negative breast cancer (TNBC) cell lines, BT-549, Hs578T, MDA-MB-231, and MDA-MB-435, and we examined the relationship of the observed profiles to fluorouracil sensitivity. Nitrobenzylthioinosine (NBMPR) at 0.1 µM inhibits only ENT1, while dipyridamole at 10 µM or NBMPR at 100 µM inhibits both ENT1 and ENT2. We found that the uptake of [3 H]uridine, a typical substrate of ENT1 and ENT2, was decreased to approximately 40% by 0.1 µM NBMPR. At 100 µM, NBMPR almost completely blocked the saturable uptake of [3 H]uridine, but this does not imply a functional role of ENT2, because 10 µM dipyridamole showed similar inhibition to 0.1 µM NBMPR. Expression of ENT1 mRNA was almost 1 order of magnitude higher than that of ENT2 in all TNBC cell lines. Liquid chromatography-tandem mass spectrometry(LC-MS/MS) LC-MS/MS-based targeted protein quantification showed that ENT1 protein levels were in the range of 9.3-30 fmol/µg protein in plasma membrane fraction of TNBC cell lines, whereas ENT2 protein was below the detection limit. [3 H]Fluorouracil uptake was insensitive to 0.1 µM NBMPR and 10 µM dipyridamole, suggesting a negligible contribution of ENT1 and ENT2 to fluorouracil uptake. The levels of ENT1 mRNA, ENT1 protein, ENT2 mRNA, and ENT1-mediated [3 H]uridine uptake in the four TNBC cell lines showed no correlation with fluorouracil sensitivity. These results indicate that neither ENT1 nor ENT2 contributes significantly to the fluorouracil sensitivity of TNBC cell lines.

Predicting Drug Interactions with Human Equilibrative Nucleoside Transporters 1 and 2 Using Functional Knockout Cell Lines and Bayesian Modeling.

Equilibrative nucleoside transporters (ENTs) 1 and 2 facilitate nucleoside transport across the blood-testis barrier (BTB). Improving drug entry into the testes with drugs that use endogenous transport pathways may lead to more effective treatments for diseases within the reproductive tract. In this study, CRISPR/CRISPR-associated protein 9 was used to generate HeLa cell lines in which ENT expression was limited to ENT1 or ENT2. We characterized uridine transport in these cell lines and generated Bayesian models to predict interactions with the ENTs. Quantification of [3H]uridine uptake in the presence of the ENT-specific inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBMPR) demonstrated functional loss of each transporter. Nine nucleoside reverse-transcriptase inhibitors and 37 nucleoside/heterocycle analogs were evaluated to identify ENT interactions. Twenty-one compounds inhibited uridine uptake and abacavir, nevirapine, ticagrelor, and uridine triacetate had different IC50 values for ENT1 and ENT2. Total accumulation of four identified inhibitors was measured with and without NBMPR to determine whether there was ENT-mediated transport. Clofarabine and cladribine were ENT1 and ENT2 substrates, whereas nevirapine and lexibulin were ENT1 and ENT2 nontransported inhibitors. Bayesian models generated using Assay Central machine learning software yielded reasonably high internal validation performance (receiver operator characteristic > 0.7). ENT1 IC50-based models were generated from ChEMBL; subvalidations using this training data set correctly predicted 58% of inhibitors when analyzing activity by percent uptake and 63% when using estimated-IC50 values. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can thereby circumvent the BTB through this transepithelial transport pathway in Sertoli cells. SIGNIFICANCE STATEMENT: This study is the first to predict drug interactions with equilibrative nucleoside transporter (ENT) 1 and ENT2 using Bayesian modeling. Novel CRISPR/CRISPR-associated protein 9 functional knockouts of ENT1 and ENT2 in HeLa S3 cells were generated and characterized. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can circumvent the blood-testis barrier through this transepithelial transport pathway in Sertoli cells.

Equilibrative Nucleoside Transporter 2: Properties and Physiological Roles.

Equilibrative nucleoside transporter 2 (ENT2) is a bidirectional transporter embedded in the biological membrane and is ubiquitously found in most tissue and cell types. ENT2 mediates the uptake of purine and pyrimidine nucleosides and nucleobase besides transporting a variety of nucleoside-derived drugs, mostly in anticancer therapy. Since high expression of ENT2 has been correlated with advanced stages of different types of cancers, consequently, this has gained significant interest in the role of ENT2 as a potential therapeutic target. Furthermore, ENT2 plays critical roles in signaling pathway and cell cycle progression. Therefore, elucidating the physiological roles of ENT2 and its properties may contribute to a better understanding of ENT2 roles beyond their transportation mechanism. This review is aimed at highlighting the main roles of ENT2 and at providing a brief update on the recent research.

Intracellular adenosine released from THP-1 differentiated human macrophages is involved in an autocrine control of Leishmania parasitic burden, mediated by adenosine A2A and A2B receptors.

Leishmania infected macrophages have conditions to produce adenosine. Despite its known immunosuppressive effects, no studies have yet established whether adenosine alter Leishmania parasitic burden upon macrophage infection. This work aimed at investigating whether endogenous adenosine exerts an autocrine modulation of macrophage response towards Leishmania infection, identifying its origin and potential pharmacological targets for visceral leishmaniasis (VL), using THP-1 differentiated macrophages. Adenosine deaminase treatment of infected THP-1 cells reduced the parasitic burden (29.1 ± 2.2%, P < 0.05). Adenosine A2A and A2B receptor subtypes expression was confirmed by RT-qPCR and by immunocytochemistry and their blockade with selective adenosine A2A and A2B antagonists reduced the parasitic burden [14.5 ± 3.1% (P < 0.05) and 12.3 ± 3.1% (P < 0.05), respectively; and 24.9 ± 2.8% (P < 0.05), by the combination of the two antagonists)], suggesting that adenosine A2 receptors are tonically activated in infected THP-1 differentiated macrophages. The tonic activation of adenosine A2 receptors was dependent on the release of intracellular adenosine through equilibrative nucleoside transporters (ENT1/ENT2): NBTI or dipyridamole reduced (~25%) whereas, when ENTs were blocked, adenosine A2 receptor antagonists failed to reduce and A2 agonists increase parasitic burden. Effects of adenosine A2 receptors antagonists and ENT1/2 inhibitor were prevented by L-NAME, indicating that nitric oxide production inhibition prevents adenosine from increasing parasitic burden. Results suggest that intracellular adenosine, released through ENTs, elicits an autocrine increase in parasitic burden in THP-1 macrophages, through adenosine A2 receptors activation. These observations open the possibility to use well-established ENT inhibitors or adenosine A2 receptor antagonists as new therapeutic approaches in VL.

Nucleoside Reverse Transcriptase Inhibitor Interaction with Human Equilibrative Nucleoside Transporters 1 and 2.

Equilibrative nucleoside transporters (ENTs) transport nucleosides across the blood-testis barrier (BTB). ENTs are of interest to study the disposition of nucleoside reverse-transcriptase inhibitors (NRTIs) in the human male genital tract because of their similarity in structure to nucleosides. HeLa S3 cells express ENT1 and ENT2 and were used to compare relative interactions of these transporters with selected NRTIs. Inhibition of [3H]uridine uptake by NBMPR was biphasic, with IC50 values of 11.3 nM for ENT1 and 9.6 µM for ENT2. Uptake measured with 100 nM NBMPR represented ENT2-mediated transport; subtracting that from total uptake represented ENT1-mediated transport. The kinetics of ENT1- and ENT2-mediated [3H]uridine uptake revealed no difference in Jmax (16.53 and 30.40 pmol cm-2 min-1) and an eightfold difference in Kt (13.6 and 108.9 µM). The resulting fivefold difference in intrinsic clearance (Jmax/Kt) for ENT1- and ENT2 transport accounted for observed inhibition of [3H]uridine uptake by 100 nM NBMPR. Millimolar concentrations of the NRTIs emtricitabine, didanosine, lamivudine, stavudine, tenofovir disoproxil, and zalcitabine had no effect on ENT transport activity, whereas abacavir, entecavir, and zidovudine inhibited both transporters with IC50 values of ∼200 µM, 2.5 mM, and 2 mM, respectively. Using liquid chromatography-tandem mass spectrometry and [3H] compounds, the data suggest that entecavir is an ENT substrate, abacavir is an ENT inhibitor, and zidovudine uptake is carrier-mediated, although not an ENT substrate. These data show that HeLa S3 cells can be used to explore complex transporter selectivity and are an adequate model for studying ENTs present at the BTB. SIGNIFICANCE STATEMENT: This study characterizes an in vitro model using S-[(4-nitrophenyl)methyl]-6-thioinosine to differentiate between equilibrative nucleoside transporter (ENT) 1- and ENT2-mediated uridine transport in HeLa cells. This provides a method to assess the influence of nucleoside reverse-transcriptase inhibitors on natively expressed transporter function. Determining substrate selectivity of the ENTs in HeLa cells can be effectively translated into the activity of these transporters in Sertoli cells that comprise the blood-testis barrier, thereby assisting targeted drug development of compounds capable of circumventing the blood-testis barrier.

Functional Analysis of the Role of Equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) in Adenine Transport in HepG2 Cells.

Equilibrative nucleobase transporter 1 (ENBT1/SLC43A3) has recently been identified as a purine-selective nucleobase transporter. Although it is highly expressed in the liver, its role in nucleobase transport has not been confirmed yet in hepatocytes or any relevant cell models. We, therefore, examined its role in adenine transport in the HepG2 cell line as a human hepatocyte model. The uptake of [3H]adenine in HepG2 cells was highly saturable, indicating the involvement of carrier-mediated transport. The carrier-mediated transport component, for which the Michaelis constant was estimated to be 0.268 µM, was sensitive to decynium-22, an ENBT1 inhibitor, with the half maximal inhibitory concentration of 2.59 µM, which was comparable to that of 2.30 µM for [3H]adenine uptake by ENBT1 in its transient transfectant human embryonic kidney 293 cells. Although equilibrative nucleoside transporter 1 (ENT1/SLC29A1) and ENT2/SLC29A2 are also known to be able to transport adenine, [3H]adenine uptake in HepG2 cells was not inhibited by the ENT1/2-specific inhibitor of either dipyridamole or nitrobenzylthioinosine. Finally, [3H]adenine uptake was extensively reduced by silencing of ENBT1 by RNA interference in the hepatocyte model. All these results, taken together, suggest the predominant role of ENBT1 in the uptake of adenine in HepG2 cells.

Blockade of equilibrative nucleoside transporter 1/2 protects against Pseudomonas aeruginosa-induced acute lung injury and NLRP3 inflammasome activation.

Pseudomonas aeruginosa infections are increasingly multidrug resistant and cause healthcare-associated pneumonia, a major risk factor for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Adenosine is a signaling nucleoside with potential opposing effects; adenosine can either protect against acute lung injury via adenosine receptors or cause lung injury via adenosine receptors or equilibrative nucleoside transporter (ENT)-dependent intracellular adenosine uptake. We hypothesized that blockade of intracellular adenosine uptake by inhibition of ENT1/2 would increase adenosine receptor signaling and protect against P. aeruginosa-induced acute lung injury. We observed that P. aeruginosa (strain: PA103) infection induced acute lung injury in C57BL/6 mice in a dose- and time-dependent manner. Using ENT1/2 pharmacological inhibitor, nitrobenzylthioinosine (NBTI), and ENT1-null mice, we demonstrated that ENT blockade elevated lung adenosine levels and significantly attenuated P. aeruginosa-induced acute lung injury, as assessed by lung wet-to-dry weight ratio, BAL protein levels, BAL inflammatory cell counts, pro-inflammatory cytokines, and pulmonary function (total lung volume, static lung compliance, tissue damping, and tissue elastance). Using both agonists and antagonists directed against adenosine receptors A2AR and A2BR, we further demonstrated that ENT1/2 blockade protected against P. aeruginosa -induced acute lung injury via activation of A2AR and A2BR. Additionally, ENT1/2 chemical inhibition and ENT1 knockout prevented P. aeruginosa-induced lung NLRP3 inflammasome activation. Finally, inhibition of inflammasome prevented P. aeruginosa-induced acute lung injury. Our results suggest that targeting ENT1/2 and NLRP3 inflammasome may be novel strategies for prevention and treatment of P. aeruginosa-induced pneumonia and subsequent ARDS.

Deletion of equilibrative nucleoside transporter-2 protects against lipopolysaccharide-induced neuroinflammation and blood-brain barrier dysfunction in mice.

Neuroinflammation is a common pathological feature of many brain diseases and is a key mediator of blood-brain barrier (BBB) breakdown and neuropathogenesis. Adenosine is an endogenous immunomodulator, whose brain extracellular level is tightly controlled by equilibrative nucleoside transporters-1 (ENT1) and ENT2. This study was aimed to investigate the role of ENTs in the modulation of neuroinflammation and BBB function. The results showed that mRNA level of Ent2 was significantly more abundant than that of Ent1 in the brain (hippocampus, cerebral cortex, striatum, midbrain, and cerebellum) of wild-type (WT) mice. Ent2-/- mice displayed higher extracellular adenosine level in the hippocampus than their littermate controls. Repeated lipopolysaccharide (LPS) treatment induced microglia activation, astrogliosis and upregulation of proinflammatory cytokines, along with aberrant BBB phenotypes (including reduced tight junction protein expression, pericyte loss, and immunoglobulin G extravasation) and neuronal apoptosis in the hippocampus of WT mice. Notably, Ent2-/- mice displayed significant resistance to LPS-induced neuroinflammation, BBB breakdown, and neurotoxicity. These findings suggest that Ent2 is critical for the modulation of brain adenosine tone and deletion of Ent2 confers protection against LPS-induced neuroinflammation and neurovascular-associated injury.

Effect of hypoxia on human equilibrative nucleoside transporters hENT1 and hENT2 in breast cancer.

Elevated proliferation rates in cancer can be visualized with positron emission tomography (PET) using 3'-deoxy-3'-l-[18F]fluorothymidine ([18F]FLT). This study investigates whether [18F]FLT transport proteins are regulated through hypoxia. Expression and function of human equilibrative nucleoside transporter (hENT)-1, hENT2, and thymidine kinase 1 (TK1) were studied under normoxic and hypoxic conditions, and assessed with [18F]FLT-PET in estrogen receptor positive (ER+)-MCF7, triple-negative MDA-MB231 breast cancer (BC) cells, and MCF10A cells (human mammary epithelial cells). Functional involvement of hENT2 [18F]FLT transport was demonstrated in all cell lines. In vitro [18F]FLT uptake was higher in MDA-MB231 than in MCF7: 242 ± 9 vs. 147 ± 18% radioactivity/mg protein after 60 min under normoxia. Hypoxia showed no significant change in radiotracer uptake. Protein analysis revealed increased hENT1 (P < 0.0963) in MDA-MB231. Hypoxia did not change expression of either hENT1, hENT2, or TK1. In vitro inhibition experiments suggested involvement of hENT1, hENT2, and human concentrative nucleoside transporters during [18F]FLT uptake into all cell lines. In vivo PET imaging revealed comparable tumor uptake in MCF7 and MDA-MB231 tumors over 60 min, reaching standardized uptake values of 0.96 ± 0.05 vs. 0.89 ± 0.08 (n = 3). Higher hENT1 expression in MDA-MB231 seems to drive nucleoside transport, whereas TK1 expression in MCF7 seems responsible for comparable [18F]FLT retention in ER+ tumors. Our study demonstrates that hypoxia does not significantly affect nucleoside transport as tested with [18F]FLT in BC.-Krys, D., Hamann, I., Wuest, M., Wuest, F. Effect of hypoxia on human equilibrative nucleoside transporters hENT1 and hENT2 in breast cancer.

Quantification of ENT1 and ENT2 Proteins at the Placental Barrier and Contribution of These Transporters to Ribavirin Uptake.

The aims of this study are to quantify the protein levels of nucleoside transporters in placental microvillous membranes (MVMs) and to clarify the contributions of these transporters to ribavirin uptake at the placental barrier. Placental MVMs of human and rat expressed equilibrative nucleoside transporter (ENT) 1 protein, whereas the expression of ENT2 protein was obscure. Maternal-to-fetal transfer of [3H]ribavirin in rats was much higher than that of [14C]sucrose. The uptake of [3H]ribavirin by rat placental trophoblast TR-TBT 18 d-1 cells, which functionally express both ENT1 and ENT2 proteins, was saturable, and was significantly inhibited by 0.1 µM nitrobenzylthioinosine, which selectively abolishes ENT1-mediated uptake. Dipyridamole at 10 µM is capable of inhibiting ENT2 as well as ENT1, but a degree of inhibition by 10 µM dipyridamole on [3H]ribavirin uptake was not much different from that by 0.1 µM nitrobenzylthioinosine (ENT1-specific inhibitor). Therefore, ENT2 may contribute little to [3H]ribavirin uptake by these cells. Rat ENT1 cRNA-injected oocytes showed increased [3H]ribavirin uptake compared with water-injected oocytes, while rat ENT2 cRNA-injected oocytes did not. In conclusion, ENT1 protein expressed in placental MVMs appears to play a predominant role in the uptake of ribavirin.