Search

Neuroinflammation plays a critical role in the neurological recovery of spinal cord injury (SCI). Adenosine can modulate neuroinflammation, whose uptake is mediated by nucleoside transporters. This study aimed to investigate the roles of equilibrative nucleoside transporter 1 (Ent1) in the inflammatory responses and functional recovery of SCI. Spinal cord contusion at the T10 dorsal portion was induced in mice to cause partial paralysis of the hindlimbs. Genetic deletion and pharmacological inhibition of Ent1 were used to evaluate the role of Ent1 in SCI. The outcomes were evaluated in terms of the Basso Mouse Scale (BMS), gait analysis, astrogliosis, microgliosis, and cytokine levels on day 14 post-injury. As a result, Ent1 deletion reduced neuroinflammation and improved the BMS score (4.88 ± 0.35 in Ent1

Rapamycin and FK506 are macrocyclic natural products with an extraordinary mode of action—they form binary complexes with FKBP through a shared FKBP-binding domain before forming ternary complexes with their respective targets, mTOR and calcineurin, respectively. Inspired by this, we sought to build a rapamycin-like macromolecule library to target new cellular proteins by replacing the effector domain of rapamycin with a combinatorial library of oligopeptides. We developed a robust macrocyclization method using ring-closing metathesis and synthesized a 45,000-compound library of hybrid macrocycles that are named rapafucins using optimized FKBP-binding domains. Screening of the rapafucin library in human cells led to the discovery of rapadocin, an inhibitor of nucleoside uptake. Rapadocin is a potent, isoform-specific and FKBP-dependent inhibitor of the equilibrative nucleoside transporter 1 and is efficacious in an animal model of kidney ischemia reperfusion injury. Together, these results demonstrate that rapafucins are a new class of chemical probes and drug leads that can expand the repertoire of protein targets well beyond mTOR and calcineurin., Graphical abstract

Cladribine is a nucleoside analog that is phosphorylated in its target cells (B and T-lymphocytes) to its active triphosphate form (2-chlorodeoxyadenosine triphosphate). Cladribine tablets 10 mg (Mavenclad

Objectives Neuroinflammation and apoptosis are two key factors contributing to early brain injury (EBI) after subarachnoid hemorrhage (SAH) and are strongly associated with a poor prognosis. Recently, equilibrative nucleoside transporter 1 (ENT1) was emerged to accelerate the severity of inflammation and cell apoptosis in several nervous system diseases, including cerebral ischemia, neurodegeneration and epilepsy. However, no study has yet elaborated the expression levels and effects of ENT1 in EBI after SAH. Methods Sprague-Dawley rats were subjected to SAH by endovascular perforation. Nitrobenzylthioinosine (NBTI) was intranasally administered at 0.5 h after SAH. The protein expression levels of ENT1, NLRP3, Bcl2, Bax, ACS, Caspase-1, IL-1 were detected by western blot. The modified Garcia score and beam balance score were employed to evaluate the neurologic function of rats following SAH. In addition, hematoxylin-eosin, fluoro-jade C and TdT-mediated dUTP nick-end labeling staining were then used to evaluate brain tissue damage and neuronal apoptosis. Results Analysis indicated that endogenous levels of ENT1 were significantly upregulated at 24-hour post-SAH, accompanied by NLRP3 inflammasome activation and Bcl2 decline. The administration of NBTI, an inhibitor of ENT1, at a dose of 15 mg/kg, ameliorated neurologic deficits and morphologic lesions at both 24 and 72 h after SAH. Moreover, ENT1 inhibition efficiently mitigated neuronal degeneration and cell apoptosis. In addition, NBTI at 15 mg/kg observably increased Bcl2 content and decreased Bax level. Furthermore, suppression of ENT1 notably reduced the expression levels of NLRP3, apoptosis associated speck like protein containing CARD, caspase-1 and IL-1β. Conclusions NBTI relieved SAH-induced EBI partly through ENT1/NLRP3/Bcl2 pathway.

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.

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, β-d-N(4)-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 [(3)H]uridine uptake (ENT1 IC(50): 39 μM; ENT2 IC(50): 77 μM), followed by EIDD-1931 (ENT1 IC(50): 259 μM; ENT2 IC(50): 467 μM), whereas molnupiravir was a modest inhibitor (ENT1 IC(50): 701 μM; ENT2 IC(50): 851 μM). Other proposed antivirals failed to inhibit ENT-mediated [(3)H]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.

Inhibitors of dihydroorotate dehydrogenase (DHODH), a key enzyme for de novo synthesis of pyrimidine nucleotides, have failed in clinical trials for various cancers despite robust efficacy in preclinical animal models. To probe for druggable mediators of DHODH inhibitor resistance, we performed a combination screen with a small molecule library against pancreatic cancer cell lines that are highly resistant to the DHODH inhibitor brequinar (BQ). The screen revealed that CNX-774, a preclinical Bruton tyrosine kinase (BTK) inhibitor, sensitizes resistant cell lines to BQ. Mechanistic studies showed that this effect is independent of BTK and instead results from inhibition of equilibrative nucleoside transporter 1 (ENT1) by CNX-774. We show that ENT1 mediates BQ resistance by taking up extracellular uridine, which is salvaged to generate pyrimidine nucleotides in a DHODH-independent manner. In BQ-resistant cell lines, BQ monotherapy slowed proliferation and caused modest pyrimidine nucleotide depletion, whereas combination treatment with BQ and CNX-774 led to profound cell viability loss and pyrimidine starvation. We also identify N-acetylneuraminic acid accumulation as a potential marker of the therapeutic efficacy of DHODH inhibitors. In an aggressive, immunocompetent pancreatic cancer mouse model, combined targeting of DHODH and ENT1 dramatically suppressed tumor growth and prolonged mouse survival. Overall, our study defines CNX-774 as a previously uncharacterized ENT1 inhibitor and provides strong proof of concept support for dual targeting of DHODH and ENT1 in pancreatic cancer.

Riboflavin transporter deficiency 2 (RTD2) is a rare neurological disorder caused by mutations in the Solute carrier family 52 member 2 (Slc52a2) gene encoding human riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed and mediates tissue distribution of riboflavin, a water-soluble vitamin that, after conversion into FMN and FAD, plays pivotal roles in carbohydrate, protein, and lipid metabolism. The 3D structure of RFVT2 has been constructed by homology modeling using three different templates that are equilibrative nucleoside transporter 1 (ENT1), Fucose: proton symporter, and glucose transporter type 5 (GLUT5). The structure has been validated by several approaches. All known point mutations of RFVT2, associated with RTD2, have been localized in the protein 3D model. Six of these mutations have been introduced in the recombinant protein for functional characterization. The mutants W31S, S52F, S128L, L312P, C325G, and M423V have been expressed in E. coli, purified, and reconstituted into proteoliposomes for transport assay. All the mutants showed impairment of function. The Km for riboflavin of the mutants increased from about 3 to 9 times with respect to that of WT, whereas Vmax was only marginally affected. This agrees with the improved outcome of most RTD2 patients after administration of high doses of riboflavin.

Ticagrelor is an oral antiplatelet drug that can reversibly bind to the platelet P2Y12 receptor. Ticagrelor is metabolized mainly by CYP3A4 and produces a rapid blood concentration-dependent platelet inhibitory effect. Unlike other P2Y12 receptor antagonists, many clinical features of ticagrelor are not related to P2Y12 receptor antagonism. This review aims to gather existing literature on the clinical effects of ticagrelor after inhibiting adenosine uptake. The current study reviewed literature related to the effects of ticagrelor on adenosine metabolism. The review also examined the drug's biological effects and clinical characteristics to see how it could be used in a clinical setting. Many studies have shown that ticagrelor can inhibit equilibrative nucleoside transporter 1 (ENT1). This inhibition leads to intracellular adenosine uptake, increased adenosine half-life and plasma concentration levels and an enhanced adenosine-mediated biological effect. Based on the studies reviewed, it was found that ticagrelor essentially inhibits adenosine absorption of adenosine into cells through ENT1, which increases the concentration in the blood and subsequently increases the protection of the heart muscle by adenosine. It also prevents platelet aggregation, and extends the biological effects of coronary arteries. Moreover, it leads to a lower mortality rate in acute coronary syndrome (ACS) patients.

International audience; 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 [H-3]-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 mu M, 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 mu M) 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 mu M) 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.

The study aimed to evaluate the clinical outcomes of tailored adjuvant chemotherapy according to human equilibrative nucleoside transporter 1 (hENT1) expression in resected pancreatic ductal adenocarcinoma (PDA).Patients who underwent pancreatectomy for PDA were enrolled prospectively. According to intra-tumoral hENT1 expression, the high hENT1 (≥50%) group received gemcitabine and the low hENT1 (50%) group received 5-fluorouracil plus folinic acid (5-FU/FA). The propensity score-matched control consisted of patients who received hENT1-independent adjuvant chemotherapy. The primary outcome was recurrence free survival (RFS) and the secondary outcomes were overall survival (OS) and toxicities.Between May 2015 and June 2017, we enrolled 44 patients with resected PDA. During a median follow-up period of 28.5 months, the intention-to-treat population showed much longer median RFS [22.9 (95% CI, 11.3-34.5) vs. 10.9 (95% CI, 6.9-14.9) months, P = 0.043] and median OS [36.2 (95% CI, 26.5-45.9) vs. 22.1 (95% CI, 17.7-26.6) months, P = 0.001] compared to the controls. Among 5 patients in the low hENT1 group who discontinued treatment, 2 patients receiving 5-FU/FA discontinued treatment due to drug toxicities (febrile neutropenia and toxic epidermal necrolysis).Tailored adjuvant chemotherapy based on hENT1 staining provides excellent clinical outcomes among patients with resected PDA.clinicaltrials.gov identifier: NCT02486497.

Our goal was to measure the absolute differential abundance of key drug transporters in human epileptogenic brain tissue and to compare them between patients and at various distances from the epileptogenic zone within the same patient. Transporter protein abundance was quantified in brain tissue homogenates from patients who underwent epilepsy surgery, using targeted proteomics, and correlations with clinical and tissue characteristics were assessed. Fourteen brain samples (including four epileptogenic hippocampal samples) were collected from nine patients. Among the quantifiable drug transporters, the abundance (median, range) ranked: breast cancer resistance protein (ABCG2/BCRP; 0.55, 0.01-3.26 pmol/g tissue) > P-glycoprotein (ABCB1/MDR1; 0.30, 0.02-1.15 pmol/g tissue) > equilibrative nucleoside transporter 1 (SLC29A1/ENT1; 0.06, 0.001-0.35 pmol/g tissue). The ABCB1/ABCG2 ratio (mean 0.27, range 0.08-0.47) was comparable with literature values from nonepileptogenic brain tissue (mean 0.5-0.8). Transporter abundance was lower in the hippocampi than in the less epileptogenic neocortex of the same patients. ABCG2/BCRP and ABCB1/MDR1 expression strongly correlated with that of glucose transporter 1 (SLC2A1/GLUT1) (r = 0.97, p < 0.001; r = 0.90, p < 0.01, respectively). Low transporter abundance was found in patients with overt vascular pathology, whereas the highest abundance was seen in a sample with normally appearing blood vessels. In conclusion, drug transporter abundance highly varies across patients and between epileptogenic and less epileptogenic brain tissue of the same patient. The strong correlation in abundance of ABCB1/MDR1, ABCG2/BCRP, and SLC2A1/GLUT1 suggests variation in the content of the functional vasculature within the tissue samples. The epileptogenic tissue can be depleted of key drug transport mechanisms, warranting consideration when selecting treatments for patients with drug-resistant epilepsy.

Equilibrativenucleoside transporters (ENTs) participate in the pharmacokinetics and disposition of nucleoside analog drugs. Understanding drug interactions with the ENTs may inform and facilitate the development of new drugs, including chemotherapeutics and antivirals that require access to sanctuary sites such as the male genital tract. This study created three-dimensional pharmacophores for ENT1 and ENT2 substrates and inhibitors using K(t) and IC(50) data curated from the literature. Substrate pharmacophores for ENT1 and ENT2 are distinct, with partial overlap of hydrogen bond donors, whereas the inhibitor pharmacophores predominantly feature hydrogen bond acceptors. Mizoribine and ribavirin mapped to the ENT1 substrate pharmacophore and proved to be substrates of the ENTs. The presence of the ENT-specific inhibitor 6-S-[(4-nitrophenyl)methyl]-6-thioinosine (NBMPR) decreased mizoribine accumulation in ENT1 and ENT2 cells (ENT1, ∼70% decrease, P = 0.0046; ENT2, ∼50% decrease, P = 0.0012). NBMPR also decreased ribavirin accumulation in ENT1 and ENT2 cells (ENT1: ∼50% decrease, P = 0.0498; ENT2: ∼30% decrease, P = 0.0125). Darunavir mapped to the ENT1 inhibitor pharmacophore and NBMPR did not significantly influence darunavir accumulation in either ENT1 or ENT2 cells (ENT1: P = 0.28; ENT2: P = 0.53), indicating that darunavir’s interaction with the ENTs is limited to inhibition. These computational and in vitro models can inform compound selection in the drug discovery and development process, thereby reducing time and expense of identification and optimization of ENT-interacting compounds. SIGNIFICANCE STATEMENT: This study developed computational models of human equilibrative nucleoside transporters (ENTs) to predict drug interactions and validated these models with two compounds in vitro. Identification and prediction of ENT1 and ENT2 substrates allows for the determination of drugs that can penetrate tissues expressing these transporters.

Background High-grade meningiomas are aggressive tumors with high morbidity and mortality rates that frequently recur even after surgery and adjuvant radiotherapy. However, limited information is currently available on the biology of these tumors, and no alternative adjuvant treatment options exist. Although we previously demonstrated that high-grade meningioma cells were highly sensitive to gemcitabine in vitro and in vivo, the underlying molecular mechanisms remain unknown. Methods We examined the roles of hENT1 (human equilibrative nucleoside transporter 1) and dCK (deoxycytidine kinase) in the gemcitabine sensitivity and growth of meningioma cells in vitro. Tissue samples from meningiomas (26 WHO grade I and 21 WHO grade II/III meningiomas) were immunohistochemically analyzed for hENT1 and dCK as well as for Ki-67 as a marker of proliferative activity. Results hENT1 and dCK, which play critical roles in the intracellular transport and activation of gemcitabine, respectively, were responsible for the high gemcitabine sensitivity of high-grade meningioma cells and were strongly expressed in high-grade meningiomas. hENT1 expression was required for the proliferation and survival of high-grade meningioma cells and dCK expression. Furthermore, high hENT1 and dCK expression levels correlated with stronger tumor cell proliferative activity and shorter survival in meningioma patients. Conclusions The present results suggest that hENT1 is a key molecular factor influencing the growth capacity and gemcitabine sensitivity of meningioma cells and also that hENT1, together with dCK, may be a viable prognostic marker for meningioma patients as well as a predictive marker of their responses to gemcitabine.

Rapadocin is a novel rapamycin-inspired polyketide–tetrapeptide hybrid macrocycle that possesses highly potent and isoform-specific inhibitory activity against the human equilibrative nucleoside transporter 1 (hENT1). Rapadocin contains an epimerizable chiral center in phenylglycine and an olefin group, and can thus exist as a mixture of four stereoisomers. Herein, we report the first total synthesis of the four stereoisomers of rapadocin using two different synthetic strategies and the assignment of their structures. The inhibitory activity of each of the four synthetic isomers on both hENT1 and hENT2 was determined. It was found that the stereochemistry of phenylglycine played a more dominant role than the configuration of the olefin in the activity of rapadocin. These findings will guide the future design and development of rapadocin analogs as new modulators of adenosine signaling., Rapadocin is a novel rapamycin-inspired polyketide–tetrapeptide hybrid macrocycle that possesses highly potent and isoform-specific inhibitory activity against the human equilibrative nucleoside transporter 1 (hENT1).

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 [(3)H]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 IC(50) 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 IC(50)-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-IC(50) 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.

Epilepsy is a chronic nervous system disease. Excessive increase of the excitatory neurotransmitter glutamate in the body results in an imbalance of neurotransmitters and excessive excitation of neurons, leading to epileptic seizures. Long-term recurrent seizures lead to behavior and cognitive changes, and even increase the risk of death by 2- to 3-fold relative to the general population. Adenosine A1 receptor (A1R), a member of the adenosine system, has notable anticonvulsant effects, and adenosine levels are controlled by the type 1 equilibrative nucleoside transporter (ENT1); in addition the p38 MAPK signaling pathway is involved in the regulation of ENT1, although the effect of its inhibitors on the expression levels of A1R and ENT1 is unclear. Therefore, in the present study, SB203580 was used to inhibit the p38 MAPK signaling pathway in rats, and the expression levels of A1R and ENT1 in the brain tissue of rats with acute LiCl-pilocarpine-induced status epilepticus was detected. SB203580 decreased pathological damage of hippocampal neurons, prolonged seizure latency, reduced the frequency of seizures, and decreased levels of A1R and ENT1 protein in rats.

The PharmacoScan pharmacogenomics platform screens for variation in genes that affect drug absorption, distribution, metabolism, elimination, immune adverse reactions and targets. Among the 1,191 genes tested on the platform, 12 genes are expressed in the red cell membrane: ABCC1, ABCC4, ABCC5, ABCG2, CFTR, SLC16A1, SLC19A1, SLC29A1, ATP7A, CYP4F3, EPHX1 and FLOT1. These genes represent 5 ATP-binding cassette proteins, 3 solute carrier proteins, 1 ATP transport protein and 3 genes associated with drug metabolism and adverse drug reactions. Only ABCG2 and SLC29A1 encode blood group systems, JR and AUG, respectively. We propose red cells as an ex vivo model system to study the effect of heritable variants in genes encoding the transport proteins on the pharmacokinetics of drugs. Altered pharmacodynamics in red cells could also cause adverse reactions, such as haemolysis, hitherto unexplained by other mechanisms.

Identifying stabilising variants of membrane protein targets is often required for structure determination. Our new computational pipeline, the Integral Membrane Protein Stability Selector (IMPROvER) provides a rational approach to variant selection by employing three independent approaches: deep-sequence, model-based and data-driven. In silico tests using known stability data, and in vitro tests using three membrane protein targets with 7, 11 and 16 transmembrane helices provided measures of success. In vitro, individual approaches alone all identified stabilising variants at a rate better than expected by random selection. Low numbers of overlapping predictions between approaches meant a greater success rate was achieved (fourfold better than random) when approaches were combined and selections restricted to the highest ranked sites. The mix of information IMPROvER uses can be extracted for any helical membrane protein. We have developed the first general-purpose tool for selecting stabilising variants of $$\upalpha$$ α -helical membrane proteins, increasing efficiency and reducing workload. IMPROvER can be accessed at http://improver.ddns.net/IMPROvER/.

Background/aim A new class of imidazo[2,1-b][1,3,4]thiadiazole compounds have recently been evaluated as inhibitors of phosphorylation of focal adhesion kinase (FAK) in pancreatic cancer. FAK is overexpressed in mesothelioma and has recently emerged as an interesting target for the treatment of this disease. Materials and methods Ten imidazo[2,1-b][1,3,4]thiadiazole compounds characterized by indole bicycle and a thiophene ring, were evaluated for their cytotoxic activity in two primary cell cultures of peritoneal mesothelioma, MesoII and STO cells. Results Compounds 1a and 1b showed promising antitumor activity with IC50 values in the range of 0.59 to 2.81 μM in both cell lines growing as monolayers or as spheroids. Their antiproliferative and antimigratory activity was associated with inhibition of phospho-FAK, as detected by a specific ELISA assay in STO cells. Interestingly, these compounds potentiated the antiproliferative activity of gemcitabine, and these results might be explained by the increase in the mRNA expression of the key gemcitabine transporter human equilibrative nucleoside transporter-1 (hENT-1). Conclusion These promising results support further studies on new imidazo[2,1-b][1,3,4]thiadiazole compounds as well as on the role of both FAK and hENT-1 modulation in order to develop new drug combinations for peritoneal mesothelioma.

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.

Accurate disease monitoring is essential following transarterial chemoembolization (TACE) in hepatocellular carcinoma (HCC) due to potential for profound adverse event and large variation in survival outcome. Post-treatment changes on conventional imaging can confound determination of residual/recurrent disease, magnifying the clinical challenge. Based on increased expression of thymidylate synthase (TYMS), thymidine kinase-1 (TK-1) and SLC29A1 (Equilibrative nucleoside transporter 1, ENT1) in HCC compared with liver tissue, we conducted a proof of concept study evaluating the efficacy of 18F-fluorothymidine (18F-FLT)-PET to assess response to TACE. As previous PET studies in HCC have been hampered by high background liver signal, we investigated if a temporal-intensity voxel-clustering ("Kinetic Spatial Filtering") (KSF) improved lesion detection. Methods: A tissue microarray (TMA) was built from 36 HCC samples and matched surrounding cirrhotic tissue and was stained for thymidine kinase-1 (TK-1). A prospective study was conducted; eighteen patients with a diagnosis of HCC by American Association for the Study of Liver Diseases criteria (AALSD) who were eligible to treatment with TACE were enrolled. Patients underwent baseline conventional imaging and dynamic 18F-FLT-PET/KSF followed by TACE. Repeat imaging was performed 6-8 weeks post TACE. PET parameters were compared with modified-Response Evaluation in Solid Tumours (mRECIST) enhancement-based criteria. Results: Cancer Genome Atlas analysis revealed increased RNA expression of TYMS, TK-1 and SLC29A1 in HCC. TK-1 protein expression was significantly higher in HCC (p

Gemcitabine (GEM) chemotherapy, as the first-line regimen for pancreatic cancer, tends to induce drug resistance, which ultimately worsens the prognosis of patients with pancreatic cancer. Our previous study indicated a close correlation between pancreatic cancer progression and glucose metabolism, especially at the chemoresistant stage, highlighting the importance of the application of 18F-FDG PET dual-phase imaging in the early detection of pancreatic cancer. We speculate that glycolysis, participates in the development of chemoresistance in pancreatic cancer. In this article, we wanted to determine whether manipulating hENT1 expression in pancreatic cancer cells can reverse GEM chemoresistance and whether glucose transport and glycolysis are involved during this process. We found that hENT1 reversed GEM-induced drug resistance by inhibiting glycolysis and altering glucose transport mediated by HIF-1α in pancreatic cancer. Our findings also suggest that 18F-FDG PET dual-phase imaging after the 4th chemotherapy treatment can accurately identify drug-resistant pancreatic tumors and improve hENT1 reversal therapy. Our findings highlight that the dynamic observation of (retention index) RI changes from the beginning of treatment can also be helpful for evaluating the therapeutic effect.

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.

Clinicopathological parameters derived from initial transurethral resection of bladder tumor (TUR-Bt) have limitations in predicting tumor progression in bladder cancer. Reduced folate carrier 1 (RFC1) and equilibrative nucleoside transporter 1 (ENT1) are solute carrier (SLC) transporters supporting cellular uptake of endogenous bioactive substances and anti-cancer drugs. The aim of this study was to elucidate the role of SLC transporters in bladder cancer and investigate the potential of RFC1 and ENT1 expression as immunohistochemical markers for high-grade malignancy. We compared T-stage with the immunohistochemical expression of RFC1 and ENT1 and other clinicopathological parameters; moreover, we also used multiple logistic regression model to assess relative contributions for T-stage in bladder cancer (n=130). Concurrently, 57 TUR-Bt-derived imprint cytological samples were stained to evaluate the implication of cytological analysis. Elevated expression levels of RFC1 and ENT1 were significantly correlated with higher T-stage (p < .0001) and efficiently predicted tumor progression, compared with other clinicopathological parameters (RFC1, p = .0325; ENT1, p = .0171). Independent variables of optimal model for predicting T-stage were gender, age, histological grade, expression levels of RFC1 and ENT1. Cytological analysis was consistent with immunostained-tissue data. We reveal RFC1 and ENT1 as potential immunohistocytochemical markers for high-grade malignancy in bladder cancer.

Decitabine (DAC), a DNA methylation inhibitor, is transported into cancer cells mainly via equilibrative nucleoside transporter 1 (ENT1) and subsequently phosphorylated by deoxycytidine kinase (dCK). We previously reported that apparent DAC uptake into cells may be described using a simple compartment model with clearance for facilitated diffusion (PS) and subsequent phosphorylation (CLmet). In the present study, time course of apparent intracellular [3H]-DAC uptake was analyzed numerically, and PS and CLmet values were calculated using the compartment model in human colon cancer HCT116 cells. PS at 0.1 μM [3H]-DAC was markedly decreased in the presence of 100 μM irinotecan or etoposide, while CLmet was markedly decreased in the presence of 100 μM cytarabine or gemcitabine. CLmet at 0.1–10 μM [3H]-DAC varied in a concentration-dependent manner and was described by Michaelis-Menten parameters Km,met and Vmax,met. In conclusion, DAC uptake mainly via ENT1 may be described by a bidirectional first-order kinetic parameter, while phosphorylation by dCK may be described by Michaelis-Menten parameters.

Purpose Gemcitabine, the primary drug for the treatment of pancreatobiliary cancer (PBC), requires human equilibrative nucleoside transporter 1 (hENT1) to enter cells. High tumoral hENT1 expression has been linked with improved survival among patients with PBC treated with gemcitabine; however, this finding has been inconsistent, and studies used different expression assays. Methods Databases were reviewed for studies that examined hENT1 and clinical outcome in PBC. Of 307 publications, 34 studies were found that used immunohistochemistry (IHC) with one of eight anti–hENT1 antibody assays. Five studies were excluded for redundancy, and 29 studies underwent detailed review. Results On average, 51% of tumor samples had high hENT1 expression (range, 7% to 92%). Among studies that examined hENT1 expression and overall survival (OS), 58% (15 of 26 studies) showed an association between high tumoral hENT1 and improved OS for gemcitabine-treated patients. Among 10D7G2 antibody studies, 88% (seven of eight studies) demonstrated this association. Studies with other antibodies—in particular, SP120 (two of nine studies)—were less consistent. The ability to detect an association between improved OS and high hENT1 was antibody dependent (χ2 P = .0237). An association between high tumoral hENT1 expression and improved disease-free/progression-free survival (DFS/PFS) was demonstrated in 71% of studies (15 of 21 studies). Pooled hazard ratio (HR) analyses of all antibody studies demonstrated a link between high hENT1 tumor expression and improved OS (HR, 0.674; 95% CI, 0.509 to 0.893; P = .006) and DFS/PFS (HR, 0.740; 95% CI, 0.517 to 0.1.059; P = .10). This signal was stronger among studies that used the 10D7G2 antibody in comparison to those in which another antibody was used, with HRs of 0.488 (95% CI, 0.396 to 0.602; P < .001) and 0.410 (95% CI, 0.280 to 0.599; P < .001), respectively. Conclusion High tumoral hENT1 expression on IHC with 10D7G2 is a strong and reproducible prognostic marker for improved outcome among gemcitabine-treated patients with PBC.

Plasmodium falciparum causes the most severe form of malaria and causes approximately 500 000 deaths per year. P. falciparum parasites resistant to current antimalarial treatments are spreading. Th...

Equilibrative nucleoside transporter 1 (ENT1) transfers nucleosides, such as adenosine, across plasma membranes. We reported previously that mice lacking ENT1 (ENT1−/−) exhibit progressive ectopic calcification of spinal tissues—a phenotype resembling diffuse idiopathic skeletal hyperostosis (DISH) in humans. Our objective was to investigate potential calcification of orofacial tissues in ENT1−/− mice. Heads of wild-type mice and ENT1−/− mice from 3 to 17 months were evaluated using microcomputed tomography (μCT). Some heads were decalcified and processed for histological assessment. Other heads were examined using energy dispersive X-ray spectroscopy and micro X-ray diffraction. Using μCT, ENT1−/− mice showed extensive radiopaque lesions within the mandibular symphysis, the severity of which increased with advancing age. Histologically, at 6 months these ectopic radiopacities were found to correspond to acellular, amorphous, eosinophilic material, with no evidence of inflammatory cells. Because lesions were localised to the symphysis, we identified early pathological changes at 3 months and observed that lesions initiated specifically within the fibrocartilage pad. Energy-dispersive X-ray spectroscopy of ectopic lesions revealed large amounts of calcium and phosphorous in a molar ratio of ~1.59, and X-ray diffraction profiles matched that of calcium-deficient hydroxyapatite. This is the first characterisation of ectopic calcifications within the mandibular symphysis of ENT1−/− mice, indicating a role for ENT1 and adenosine metabolism in regulating calcification of fibrocartilaginous tissues. Moreover, these murine lesions resemble areas of dystrophic calcification in the spinal tissues of humans with DISH. Importantly, ectopic calcifications develop in a reproducible temporal pattern within a well-defined anatomical region and, thus, provide a model for determining the cellular and molecular pathways underlying ectopic calcification in DISH and related disorders.

Striatal adenosine A1receptor (A1R) activation can inhibit dopamine release. A1Rs on other striatal neurons are activated by an adenosine tone that is limited by equilibrative nucleoside transporter 1 (ENT1) that is enriched on astrocytes and is ethanol sensitive. We explored whether dopamine release in nucleus accumbens core is under tonic inhibition by A1Rs, and is regulated by astrocytic ENT1 and ethanol. Inex vivostriatal slices from male and female mice, A1R agonists inhibited dopamine release evoked electrically or optogenetically and detected using fast-scan cyclic voltammetry, most strongly for lower stimulation frequencies and pulse numbers, thereby enhancing the activity-dependent contrast of dopamine release. Conversely, A1R antagonists reduced activity-dependent contrast but enhanced evoked dopamine release levels, even for single optogenetic pulses indicating an underlying tonic inhibition. The ENT1 inhibitor nitrobenzylthioinosine reduced dopamine release and promoted A1R-mediated inhibition, and, conversely, virally mediated astrocytic overexpression of ENT1 enhanced dopamine release and relieved A1R-mediated inhibition. By imaging the genetically encoded fluorescent adenosine sensor [GPCR-activation based (GRAB)-Ado], we identified a striatal extracellular adenosine tone that was elevated by the ENT1 inhibitor and sensitive to gliotoxin fluorocitrate. Finally, we identified that ethanol (50 mm) promoted A1R-mediated inhibition of dopamine release, through diminishing adenosine uptake via ENT1. Together, these data reveal that dopamine output dynamics are gated by a striatal adenosine tone, limiting amplitude but promoting contrast, regulated by ENT1, and promoted by ethanol. These data add to the diverse mechanisms through which ethanol modulates striatal dopamine, and to emerging datasets supporting astrocytic transporters as important regulators of striatal function.SIGNIFICANCE STATEMENTDopamine axons in the mammalian striatum are emerging as strategic sites where neuromodulators can powerfully influence dopamine output in health and disease. We found that ambient levels of the neuromodulator adenosine tonically inhibit dopamine release in nucleus accumbens core via adenosine A1receptors (A1Rs), to a variable level that promotes the contrast in dopamine signals released by different frequencies of activity. We reveal that the equilibrative nucleoside transporter 1 (ENT1) on astrocytes limits this tonic inhibition, and that ethanol promotes it by diminishing adenosine uptake via ENT1. These findings support the hypotheses that A1Rs on dopamine axons inhibit dopamine release and, furthermore, that astrocytes perform important roles in setting the level of striatal dopamine output, in health and disease.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by notorious resistance to current therapies attributed to inherent tumor heterogeneity and highly desmoplastic and immunosuppressive tumor microenvironment (TME). Unique proline isomerase Pin1 regulates multiple cancer pathways, but its role in the TME and cancer immunotherapy is unknown. Here we find that Pin1 is overexpressed both in cancer cells and cancer-associated fibroblasts (CAFs), and correlates with poor survival in PDAC patients. Targeting Pin1 using clinically available drugs induces complete elimination or sustained remissions of aggressive PDAC by synergizing with anti-PD-1 and gemcitabine in diverse model systems. Mechanistically, Pin1 drives the desmoplastic and immunosuppressive TME by acting on CAFs, and induces lysosomal degradation of the PD-1 ligand PD-L1 and the gemcitabine transporter ENT1 in cancer cells, besides activating multiple cancer pathways. Thus, Pin1 inhibition simultaneously blocks multiple cancer pathways, disrupts the desmoplastic and immunosuppressive TME, and upregulates PD-L1 and ENT1, rendering PDAC eradicable by immunochemotherapy.

Gemcitabine or 5-fluorouracil (5-FU) based treatments can be selected for pancreatic cancer. Equilibrative nucleoside transporter 1 (hENT1) predicts adjuvant gemcitabine treatment benefit over 5-FU. Cytidine deaminase (CDA), inside or outside of the cancer cell, will deaminate gemcitabine, altering transporter affinity. ESPAC-3(v2) was a pancreatic cancer trial comparing adjuvant gemcitabine and 5-FU. Tissue microarray sections underwent in situ hybridization and immunohistochemistry. Analysis of both CDA and hENT1 was possible with 277 patients. The transcript did not correlate with protein levels for either marker. High hENT1 protein was prognostic with gemcitabine, median overall survival was 26.0 v 16.8 months (p = 0.006). Low CDA transcript was prognostic regardless of arm, 24.8 v 21.2 months with gemcitabine (p = 0.02) and 26.4 v 14.6 months with 5-FU (p = 0.02). Patients with low hENT1 protein did better with 5-FU, but only if the CDA transcript was low (median survival of 5-FU v gemcitabine, 29.3 v 18.3 months, compared with 14.2 v 14.6 with high CDA). CDA mRNA is an independent prognostic biomarker. When added to hENT1 protein status, it may also provide treatment-specific predictive information and, within the frame of a personalized treatment strategy, guide to either gemcitabine or 5FU for the individual patient.

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