Equilibrative Nucleoside Transporter Research Articles

Identification of human TRIAC transmembrane transporters.

TRIAC (3,5,3'-triiodothyroacetic acid) is a T3-receptor agonist pharmacologically used in patients to mitigate T3 resistance. It is additionally explored to treat some symptoms of patients with inactivating mutations in the thyroid hormone (TH) transporter MCT8 (SLC16A2). MCT8 is expressed along the blood-brain-barrier, on neurons, astrocytes, and oligodendrocytes. Hence, pathogenic variants in MCT8 limit the access of TH into and their functions within the brain. TRIAC was shown to enter the brain independently of MCT8 and to modulate expression of TH-dependent genes. The aim of the study was to identify transporters that facilitate TRIAC uptake into cells. We performed a whole-genome RNAi screen in HepG2 cells stably expressing a T3-receptor-dependent luciferase reporter gene. Validation of hits from the primary and confirmatory secondary screen involved a counter screen with siRNAs and compared the cellular response to TRIAC to the effect of T3, in order to exclude siRNAs targeting the gene expression machinery. MDCK1 cells were stably transfected with cDNA encoding C-terminally myc-tagged versions of the identified TRIAC-preferring transporters. Several individual clones were selected after immunocytochemical characterization for biochemical characterization of their 125I-TRIAC transport activities. We identified SLC22A9 and SLC29A2 as transporters mediating cellular uptake of TRIAC. SLC22A9 encodes the organic anion transporting polypeptide 7 (OAT7), a sodium-independent organic anion transporter expressed in the plasma membrane in brain, pituitary, liver and other organs. Competition with the SLC22A9/OAT7 substrate estrone-3-sulfate reduced 125I-TRIAC uptake. SLC29A2 encodes the equilibrative nucleoside transporter 2 (ENT2), which is ubiquitously expressed including pituitary and brain. Co-incubation with the SLC29A2/ENT2 inhibitor nitrobenzyl-6-thioinosine reduced 125I-TRIAC uptake. Moreover, ABCD1, an ATP-dependent peroxisomal pump, was identified as a 125I-TRIAC exporter in transfected MDCK1 cells. Knowledge of TRIAC transporter expression patterns, also during brain development, may thus in the future help to interpret observations on TRIAC effects as well as understand why TRIAC may not show a desirable effect on cells or organs not expressing appropriate transporters. The identification of ABCD1 highlights the sensitivity of our established screening assay, but it may not hold significant relevance for patients undergoing TRIAC treatment.

Investigating ZMP Accumulation in C2C12 Myotubes with AICAR and Prodrug-39

5’Adenosine monophosphate (AMP) is a signaling molecule and a known regulator of enzymes in glycolysis, glycogen breakdown, and may play a role in regulating protein synthesis. It is also a known activator of AMP-dependent protein kinase (AMPK), a key energy sensor within the cell. Activation of AMPK has been linked to the regulation of mitochondrial biogenesis, upregulation of BCAA catabolism, and increased skeletal muscle glucose uptake and fatty acid oxidation. AMPK can be activated pharmacologically with 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), an adenosine analogue. AICAR enters the cell through adenosine transporters and undergoes phosphorylation by Adenosine Kinase forming AICAR monophosphate (ZMP). Unfortunately, the poor bioavailability of AICAR limits its use pharmacologically. Prodrug-39 (P39) is comprised of ZMP with a 4-nitrophenol group to allow membrane permeability and subsequent ZMP formation without the need of membrane transporters. We investigated the ZMP accumulation in C2C12 myotubes following exposure to different concentrations of P39 or AICAR. Based on preliminary data, we hypothesized that AICAR treatments would be more effective at producing ZMP over short periods of time and when treatment concentrations were high, but that P39 treatments would result in greater and more consistent ZMP accumulation over longer periods of time when treatment concentrations were low. We first measured the absorbance of 4-nitrophenol (4NP) within the cell media to determine phosphodiesterase cleavage of P39. Nitrophenol absorbance was 3.9 times higher at 1000 μM compared to 100 μM treatment of P39 after 15 minute incubation, and 13.3 times higher at 11 hours. The nitrophenol absorbance per hour increased linearly between 250 μM to 1000 μM of P39. Adenine nucleotide content and ZMP accumulation in treated cells was determined following organic protein extraction and UV-Vis ultra-performance liquid chromatography (UPLC). After 13 hours of treatment with 500 μM AICAR or P39, ZMP accumulation was 14 fold higher following AICAR treatment compared to P39 (59.7±5.0 vs 4.2±0.1 μmol/g protein). Interestingly, ZMP accumulation was not different between 100 μM treatments (5.6±1.3 vs. 5.7±1.5 μmol/g protein). This suggests that phosphodiesterase cleavage of P39 is likely the limiting factor for ZMP accumulation. Interestingly, while ZMP accumulation following 13 hour incubation with 100 μM AICAR or P39 was not different, ATP content was approximately 50% higher in P39 treated cells (33.7±5.8 vs. 51.0±5.8 μmol/g protein). While the measurement of ZMP accumulation at very low treatment concentrations (10 μM) is very diffcult, P39 exposure for 1 to 1.5 hours resulted in a 3 to 8 fold increase in ZMP content compared to AICAR. This data illustrates the potential for P39 as a drug capable of accumulating greater ZMP than AICAR at very low treatment concentrations, however the capacity of phosphodiesterase activity may limit the application of this drug. This work was funded by a grant from Skylark Biosciences. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Can pharmacogenetics impact the therapeutic effect of cytarabine and anthracyclines in adult acute myeloid leukaemia patients? A Serbian experience

Background. Cytarabine-anthracycline based induction chemotherapy remains the standard of care for remission induction among patients with newly diagnosed acute myeloid leukaemia (AML). There are remarkable differences in therapy response among AML patients. This fact could be partly explained by the patients’ genetic variability related to metabolic paths of cytarabine and anthracyclines. The aim of this study is to evaluate the effect of variants in pharmacogenes SLC29A1, DCK, ABCB1, GSTM1 and GSTT1, as well as laboratory and AML-related parameters on clinical outcome in adult AML patients. Methods. A total of 100 AML patients were included in the study. Pharmacogenetic variants SLC29A1 rs9394992, DCK rs12648166, ABCB1 rs2032582 and GSTM1 and GSTT1 gene deletions were detected by methodology based on PCR, fragment analysis and direct sequencing. The methods of descriptive and analytic statistics were used. Survival analysis was done by the Kaplan-Meier method using the Log-Rank test. Results. This is the first study of adult AML pharmacogenetics in Serbian population. Clinical outcomes in our cohort of AML patients were not impacted by analysed variants in SLC29A1, DCK, ABCB1and GSTT1 and GSTM1 genes, independently or in combinations. Achievement of complete remission was identified as an independent prognostic indicator of clinical outcome. Conclusions. The population specific genomic profile has to be considered in pharmacogenetics. Since the data on AML pharmacogenetics in European populations is limited, our results contribute to knowledge in this field and strongly indicate that high-throughput approach has to be applied in order to find particular pharmacogenetic markers of AML in European population.

Effects of nucleoside analogues, lipophilic prodrugs and elaidic acids on core signaling pathways in cancer cells

Objectives Nucleoside analogs such as gemcitabine (GEM; dFdC) and cytarabine (Ara-C) require nucleoside transporters to enter cells, and deficiency in equilibrative nucleoside transporter 1 (ENT1) can lead to resistance to these drugs. To facilitate transport-independent uptake, prodrugs with a fatty acid chain attached to the 5’-position of the ribose group of gemcitabine or cytarabine were developed (CP-4126 and CP-4055, respectively). As antimetabolites can activate cellular survival pathways, we investigated whether the prodrugs or their side-chains had similar or decreased effects. Methods Two cell lines A549 (non-small cell lung cancer) and WiDr (colon cancer cells) were exposed for 2-24hr to IC50 concentrations of GEM, Ara-C, CP-4126, CP4055 and elaidic acid (EA) concentrations corresponding to the CP-4126 and CP-4055 IC50. Cells were harvested and analyzed for proteins in cell survival pathways (p-AKT/AKT, p-ERK/ERK, p-P38/P38, GSK-3β/pGSK-3β) by using Western Blotting. Results All drugs and their derivatives showed time- and cell-line-dependent effects. In A549 cells, GEM, CP-4126 and EA-4126 decreased the p-AKT/AKT ratio at 2 and 24 hr. For the p-ERK/ERK ratio, GEM, EA-4126, Ara-C, CP-4045 and EA-4055 exposure led to an increase after 6 hr in A549 cells. Interestingly, Ara-C, CP-4055 and EA-4055 decreased p-ERK/ERK ratio in WiDr cells after 4 hr. In A549 cells, the p-GSK-3β/GSK-3β ratio decreased after exposure to Ara-C and CP-4055 but in WiDr cells increased after 24 hr. In A549 cells treatment with Ara-C, CP-4055 and EA-4126 decreased the p-P38/P38 after 6 hr. Conclusions The findings suggest that both parent drugs, prodrugs, and the EA chain influence cell survival and signaling pathways.

MSphere of Influence: Deciphering purine auxotrophy in protozoan parasites.

Bruno Martorelli Di Genova works in parasitology, focusing on Toxoplasma gondii metabolism. In this mSphere of Influence article, he reflects on how the articles "Metabolic Reprogramming during Purine Stress in the Protozoan Pathogen Leishmania donovani" and "Yeast-Based High-Throughput Screen Identifies Plasmodium falciparum Equilibrative Nucleoside Transporter 1 Inhibitors That Kill Malaria Parasites" impacted him, informing his research strategies and understanding of metabolic flexibility in Toxoplasma gondii.

Blood-to-Testis Transport of Ribavirin Involves Carrier-Mediated Processes at the Blood–Testis Barrier

Ribavirin, an antiretroviral agent targeting the hepatitis C virus, causes male reproductive toxicity. This study investigated the mechanism of ribavirin transport at the blood–testis barrier (BTB). In vivo mouse integration plot analysis after intravenous administration revealed that the net influx clearance of [3H]ribavirin in the testis was 3.6-fold greater than that of [14C]D-mannitol, a paracellular transport marker, implying transcellular transport of ribavirin across the BTB. Moreover, [3H]ribavirin uptake by TM4 cells, mouse-derived Sertoli cells, was time- and concentration-dependent, with a Km value of 2.49 mM. S-[(4-nitrophenyl)methyl]-6-thioinosine, an inhibitor of Na+-independent equilibrative nucleoside transporters (ENTs), strongly inhibited the [3H]ribavirin uptake by TM4 cells at 100 µM. Compared to the uptake of [3H]adenosine, a typical endogenous nucleoside, [3H]ribavirin uptake was relatively similar to ENT2 transport. [3H]Ribavirin uptake was also observed in mouse ENT2-expressing Xenopus laevis oocytes, and gene silencing via the transfection of ENT2 small interfering RNA significantly reduced the [3H]ribavirin transport into TM4 cells by 13 %. Taken together, these results suggest that ENT2 partially contributes to ribavirin transport at the BTB.

Abstract 734: Inhibition of equilibrative nucleoside transporter 1 relieves intracellular adenosine-mediated immune suppression

Abstract The profound benefit of immune checkpoint blockade for cancer therapy is restricted to limited subsets of patients with specific cancers. Many factors contribute to primary and acquired resistance to immune checkpoint blockade, including local accumulation of immunosuppressive metabolites such as the nucleoside adenosine and downstream adenosine receptor signaling. Pharmacological inhibition of adenosine generation and signaling are active areas of clinical investigation. Here we report a novel mechanism whereby adenosine suppresses anti-cancer immune responses by intracellular accumulation. We show that human T cells readily take up adenosine via equilibrative nucleoside transporter 1 (ENT1), suppressing proliferation and effector function by inhibition of de novo pyrimidine nucleotide synthesis in T cells. ENT1 expression increases upon T cell activation, suggesting uptake of extracellular adenosine is a mechanism to limit T cell responses in high adenosine environments. Quantitative mass spectrometry imaging reveals adenosine concentrations in human tumors up to the range of 100 µM - levels significantly greater than previously demonstrated and consistent with suppression of T cell responses. Deletion of ENT1 leads to potent control of tumor growth in syngeneic mouse models including KPC, a poorly immunogenic model of pancreatic ductal adenocarcinoma, and is associated with increased CD8+ T cell frequency, proliferation and cytokine production within tumors. Furthermore, ENT1 expression was observed by flow cytometry on human tumor-infiltrating CD8+ T cells. We have discovered and characterized EOS301984, a potent ENT1 antagonist that is currently being evaluated in patients with advanced solid tumors. EOS301984 blocks intracellular adenosine transport and restores pyrimidine levels in T cells activated in the presence of adenosine, resulting in enhanced tumor cell killing by memory T cells and increased ex vivo expansion of functional human tumor-infiltrating lymphocytes in adenosine-rich environments. Finally, in a humanized mouse model of triple negative breast cancer resistant to anti-PD-1 blockade (MDA-MB-231), combination of EOS301984 with nivolumab synergistically led to control of tumor growth. Thus, ENT1 inhibition represents a novel approach to augment anti-cancer immune responses through restoring pyrimidine nucleotide synthesis in T cells suppressed by adenosine. Citation Format: Theodore J. Sanders, Christopher S. Nabel, Margreet Brouwer, Annelise L. Hermant, Lucas Chaible, Jean-Philippe Deglasse, Angelique Pabois, Wilfried Cathou, Aurore Smets, Michael Deligny, Joao Marchante, Quentin Dubray, Marie-Claire Lettelier, Chiara Martinoli, Reece Marillier, Olivier De Henau, Yvonne McGrath, Matthew G. Vander Heiden, Erica Houthuys. Inhibition of equilibrative nucleoside transporter 1 relieves intracellular adenosine-mediated immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 734.

Abstract 5801: Targeted delivery of Exatecan to tumors via a novel cell-penetrating, anti-DNA antibody

Abstract The formulation of novel chemotherapeutics capable of precisely targeting and damaging tumor cells, while preserving the integrity of healthy tissues, represents a principal challenge and is recognized as one of the biggest achievements in cancer treatment in the last few decades. Antibody-drug conjugates (ADCs) hold significant potential as an effective cancer treatment, by targeting and delivering cytotoxic drugs exclusively to tumor cells that express distinct antigens associated with malignancy. Due to the necessity for specific surface antigens, therapies with ADCs are limited to specific tumor subtypes. In this study, we show the potential of a lupus-derived, cell-penetrating, anti-DNA antibody for delivering of cytotoxic payload to tumors in vivo. We utilized a fully humanized variant, named as V66, which was chemically linked to the Topoisomerase I inhibitor, Exatecan. The primary mechanism involved in V66 cellular penetration is mediated by the Equilibrative Nucleoside Transporter ENT2. The latter shows elevated expression in a substantial subset of cancer types in comparison to healthy tissues, broadening the range of applications for ADCs in tumor therapy. Using labeled V66, we demonstrated specific tumor delivery in vivo, with minimal impact on healthy tissues. In vitro experiments validated the nuclear localization of V66-Exatecan, where it triggers the activation of the DNA damage repair pathway (DDR). As a consequence of the profound DNA damage, exposure to V66-Exatecan leads to cell death, exhibiting low nanomolar EC50 values across a diverse range of cell lines. Furthermore, V66-Exatecan treatment presents synthetic lethality with BRCA2 deficiency, evidenced by a substantial increase in sensitivity. In vivo efficacy studies conducted in BRCA2 deficient tumors revealed a remarkable effectiveness of V66-Exatecan, leading to almost complete tumor regression and substantial improvement in the probability of survival. In addition to the lack of significant weight changes observed throughout the treatment, further toxicological studies conducted in serum and tissues demonstrated the absence of short- and long-term toxicity, with optimal liver and kidney function and the absence of damage to other tissues. Likewise, bone marrow cellularity remained unaffected. Collectively, these studies indicate that V66-Exatecan achieves targeted treatment of tumor cells, where it induces DNA damage leading to cell death. The in vivo application of V66-Exatecan leads to tumor regression without triggering general toxicity that is otherwise associated with Topoisomerase I inhibitors. This work provides the basis to advance a novel nuclear-penetrating ADC for delivering cytotoxic payloads. Citation Format: Zaira Ianniello, Elias Quijano, Peter M. Glazer. Targeted delivery of Exatecan to tumors via a novel cell-penetrating, anti-DNA antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5801.

Cases with the H syndrome presenting with skin and bone findings.

The H syndrome is an autosomal recessive disease characterized by hyperpigmentation, hypertrichosis and sensorineural hearing loss. A mutation in the coding of the human equilibrative nucleoside transporter 3 (hENT3) within the SLC29A3 gene on chromosome 10q22 leads to the manifestation of this disease. In this report, we present two cases of H syndrome. The first patient exhibits hyperpigmentation, hypogonadism, Type 1 diabetes mellitus, arthritis and osteoporosis. The second patient experiences hyperpigmentation, hypertrichosis, osteopenia and hypogonadism. Our objective is to broaden the clinical spectrum of H syndrome, highlighting the involvement of arthritis, hyperinflammation and low bone mineral density in individuals with this disorder.

Alteration in the number, morphology, function, and metabolism of erythrocytes in high-altitude polycythemia.

Introduction: High-altitude polycythemia (HAPC) is a common chronic high-altitude disease characterized by significantly increased erythrocyte, hemoglobin (Hb), and hematocrit values and decreased arterial oxygen saturation. The mechanisms underlying HAPC development are unclear; we aimed to investigate this in an HAPC rat model. Methods: Twelve Sprague-Dawley rats were divided into control and HAPC groups. The HAPC group was exposed to hypobaric hypoxia. This HAPC model was assessed using routine blood tests and blood gas analyses. Bone marrow, peripheral blood reticulocytes (RETs), and peripheral blood erythrocyte apoptosis were measured using flow cytometry. Erythrocyte osmotic fragility (EOF) tests were conducted. Abnormal erythrocytes were counted using electron microscopy. Plasma-free hemoglobin, 5'-nucleotidase (CD73), adenosine, erythrocyte cytosolic adenosine, sphingosine-1-phosphate (S1P), and 2,3-bisphosphoglycerate (BPG) levels were measured using enzyme-linked immunosorbent assays. Erythrocyte metabolic pathway-related protein [adenosine A2B receptor (ADORA2B), erythrocyte equilibrative nucleoside transporter 1 (eENT1), sphingosine kinase 1 (SPHK1), phospho-SPHK1, bisphosphoglycerate mutase (BPGM), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] levels were assessed by Western blotting. Results: The HAPC rat model was successfully established (Hb > 210g/L). Indices of bone marrow and peripheral blood RET proportions were significantly higher in the HAPC than the control group (p = 0.04 and p < 0.001, respectively). The proportion of peripheral blood erythrocytes in early apoptosis was significantly lower in the HAPC than the control group (p < 0.001). Vesicular erythrocyte and acanthocyte proportions were significantly higher in the HAPC than the control group (p < 0.001 and p = 0.019, respectively). The EOF tests revealed that 50% erythrocyte hemolysis occurred at 4.0-4.5 and 4.5-5.0g/L NaCl in the control and HAPC groups, respectively. Plasma-free hemoglobin, CD73, adenosine, erythrocyte cytosolic adenosine, S1P, and 2,3-BPG levels and ADORA2B, eENT1, phospho-SPHK1, S1P, BPGM, and GAPDH erythrocyte expression levels (all p ≤ 0.02) were significantly higher in the HAPC than the control group. Conclusion: In model rats, an HAPC-related erythrocyte increase was associated with enhanced bone marrow hematopoietic function and reduced erythrocyte apoptosis, whereas numerous abnormal erythrocytes, increased EOF, and reduced hemolysis resistance were associated with erythrocyte metabolism. CD73/adenosine/S1P/2,3-BPG and eENT1/adenosine/BPGM/2,3-BPG metabolic pathways in erythrocytes were activated in HAPC rats, facilitating oxygen release. These findings further reveal the intrinsic HAPC mechanism and forms a basis for future development of preventive and therapeutic strategies for HAPC.

40 Hz light flickering promotes sleep through cortical adenosine signaling

Flickering light stimulation has emerged as a promising non-invasive neuromodulation strategy to alleviate neuropsychiatric disorders. However, the lack of a neurochemical underpinning has hampered its therapeutic development. Here, we demonstrate that light flickering triggered an immediate and sustained increase (up to 3 h after flickering) in extracellular adenosine levels in the primary visual cortex (V1) and other brain regions, as a function of light frequency and intensity, with maximal effects observed at 40 Hz frequency and 4000 lux. We uncovered cortical (glutamatergic and GABAergic) neurons, rather than astrocytes, as the cellular source, the intracellular adenosine generation from AMPK-associated energy metabolism pathways (but not SAM-transmethylation or salvage purine pathways), and adenosine efflux mediated by equilibrative nucleoside transporter-2 (ENT2) as the molecular pathway responsible for extracellular adenosine generation. Importantly, 40 Hz (but not 20 and 80 Hz) light flickering for 30 min enhanced non-rapid eye movement (non-REM) and REM sleep for 2–3 h in mice. This somnogenic effect was abolished by ablation of V1 (but not superior colliculus) neurons and by genetic deletion of the gene encoding ENT2 (but not ENT1), but recaptured by chemogenetic inhibition of V1 neurons and by focal infusion of adenosine into V1 in a dose-dependent manner. Lastly, 40 Hz light flickering for 30 min also promoted sleep in children with insomnia by decreasing sleep onset latency, increasing total sleep time, and reducing waking after sleep onset. Collectively, our findings establish the ENT2-mediated adenosine signaling in V1 as the neurochemical basis for 40 Hz flickering-induced sleep and unravel a novel and non-invasive treatment for insomnia, a condition that affects 20% of the world population.

The prognostic and predictive role of class III β-Tubulin and hENT1 expression in patients with advanced pancreatic ductal adenocarcinoma

BackgroundFOLFIRINOX and gemcitabine-nabpaclitaxel (GnP) are standard first-line treatment regimens for advanced pancreatic ductal adenocarcinoma (PDAC). However, currently, there is a lack of predictive biomarkers to aid in the treatment selection. We aimed to explore the prognostic and predictive value of class III β-Tubulin (TUBB3) and human equilibrative nucleoside transporter 1 (hENT1) expression, which have previously been shown to be associated with taxane and gemcitabine resistance in advanced PDAC. MethodsWe conducted a retrospective analysis of 106 patients with advanced PDAC treated with GnP and/or FOLFIRINOX at our institution. TUBB3 and hENT1 immunohistochemical staining was performed on tumor specimens and subsequently evaluated based on the intensity and percentage of expression. ResultsIn patients who received the GnP regimen, a high combined score (TUBB3low/hENT1high) was associated with a higher DCR and longer PFS compared to those with intermediate (TUBB3high/hENT1high or TUBB3low/hENT1low) and low score (TUBB3high/hENT1low). In the multivariate analysis, a high combined score was an independent predictor of higher DCR (OR:11.96; 95 % CI:2.61–54.82; p = 0.001) and longer PFS (HR:0.33; 95%CI:0.18–0.60; p < 0.001). However, there was no difference in response rates or PFS based on TUBB3 and hENT1 expression among patients receiving the FOLFIRINOX regimen. ConclusionOur findings indicate that tumor TUBB3 and hENT1 expression may predict the efficacy of the GnP regimen, and low TUBB3 and high hENT1 expression (TUBB3low/hENT1high) are associated with a higher DCR and longer PFS in patients treated with GnP. Evaluating TUBB3 and hENT1 jointly can identify the patients most (as well as least) likely to benefit from GnP chemotherapy.

Characterization of equilibrative nucleoside transport of the pancreatic cancer cell line: Panc-1

Characterization of equilibrative nucleoside transport of the pancreatic cancer cell line: Panc-1

Putative purine nucleoside interacting residues in the malaria parasite purine uptake transporter PfENT1 are critical for transporter function.

Malaria remains a major public health threat for billions of people worldwide. Infection with obligate intracellular, unicellular parasites from the genus Plasmodium causes malaria. Plasmodium falciparum causes the deadliest form of human malaria. Plasmodium parasites are purine auxotrophic. They rely on purine import from the host red blood cell cytoplasm via equilibrative nucleoside transporters to supply substrates to the purine salvage pathway. We previously developed a high throughput screening assay to identify inhibitors of the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Screening a small molecule library identified PfENT1 inhibitors that blocked proliferation of P. falciparum parasites in in vitro culture. The goal of the current work was to validate a high-resolution model of PfENT1 predicted by the AlphaFold protein structure prediction program. We superimposed the predicted PfENT1 structure on the human homologue structure, hENT1, and developed a structure-based sequence alignment. We mutated the residues in PfENT1 aligned with and flanking the residues in hENT1 that interact with the purine analog, nitrobenzylthioinosine (NBMPR). Mutation of the PfENT1 residues Q135, D287, and R291 that are predicted to form hydrogen bonds to purine nucleosides eliminated purine and pyrimidine transport function in various yeast-based growth and radiolabeled substrate uptake assays. Mutation of two flanking residues, W53 and S290, also resulted in inactive protein. Mutation of L50 that forms hydrophobic interactions with the purine nucleobase reduced transport function. Based on our results the AlphaFold predicted structure for PfENT1 may be useful in guiding medicinal chemistry efforts to improve the potency of our PfENT1 inhibitors.

Off-Target Effects of P2Y12 Receptor Inhibitors: Focus on Early Myocardial Fibrosis Modulation.

Several studies have demonstrated that, beyond their antithrombotic effects, P2Y12 receptor inhibitors may provide additional off-target effects through different mechanisms. These effects range from the preservation of endothelial barrier function to the modulation of inflammation or stabilization of atherosclerotic plaques, with an impact on different cell types, including endothelial and immune cells. Many P2Y12 inhibitors have been developed, from ticlopidine, the first thienopyridine, to the more potent non-thienopyridine derivatives such as ticagrelor which may promote cardioprotective effects following myocardial infarction (MI) by inhibiting adenosine reuptake through sodium-independent equilibrative nucleoside transporter 1 (ENT1). Adenosine may affect different molecular pathways involved in cardiac fibrosis, such as the Wnt (wingless-type)/beta (β)-catenin signaling. An early pro-fibrotic response of the epicardium and activation of cardiac fibroblasts with the involvement of Wnt1 (wingless-type family member 1)/β-catenin, are critically required for preserving cardiac function after acute ischemic cardiac injury. This review discusses molecular signaling pathways involved in cardiac fibrosis post MI, focusing on the Wnt/β-catenin pathway, and the off-target effect of P2Y12 receptor inhibition. A potential role of ticagrelor was speculated in the early modulation of cardiac fibrosis, thanks to its off-target effect.

Anticonvulsant effect of equilibrative nucleoside transporters 1 inhibitor in a mouse model of Dravet syndrome.

There are limited therapeutic options for patients with Dravet syndrome (DS). The equilibrative nucleoside transporters 1 (ENT1) mediate both the influx and efflux of adenosine across the cell membrane exerted beneficial effects in the treatment of epilepsy. This study aimed to evaluate the anticonvulsant effect of the ENT1 inhibitor in an animal model of DS (Scn1aE1099X/+ mice). J7 (5 mg/kg) treatment was efficacious in elevating seizure threshold in Scn1aE1099X/+ mice after hyperthermia exposure. Moreover, the J7 treatment significantly reduced the frequency of spontaneous excitatory post-synaptic currents (sEPSCs, ~35% reduction) without affecting the amplitude in dentate gyrus (DG) granule cells. Pretreatment with the adenosine A1 receptor (A1R) antagonist, DPCPX, abolished the J7 effects on sEPSCs. These observations suggest that the J7 shows an anticonvulsant effect in hyperthermia-induced seizures in Scn1aE1099X/+ mice. This effect possibly acts on presynaptic A1R-mediated signaling modulation in granule cells.

Transporter modulation of molnupiravir and its metabolite β-D-N4-hydroxycytidine across the blood-brain barrier in a rat

BackgroundThe antiviral drug molnupiravir is an orally bioavailable prodrug of the nucleoside analog β-D-N4-hydroxycytidine (NHC), which is used to treat coronavirus disease 2019 (COVID-19). However, there is very little information on the barrier distribution of molnupiravir. Our hypothesis is that molnupiravir and NHC can penetrate the blood‒brain barrier (BBB) into brain tissue and that nucleoside transporters (equilibrative nucleoside transporters; ENT and concentrative nucleoside transporters; CNT) can modulate this process.MethodsTo investigate the mechanism of molnupiravir transport through the BBB, multiple microdialyses coupled to a validated ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC‒MS/MS) was developed to monitor dialysates, and nitrobenzylthioinosine (NBMPR; an inhibitor of ENT) was administered concomitantly with molnupiravir (100 mg/kg, i.v.) in the male rat.ResultsHere, we show that molnupiravir is rapidly metabolized to NHC in the blood and crossed the BBB in 20 min. Furthermore, when NBMPR is concomitantly administered to inhibit efflux, the concentrations of molnupiravir and NHC in the brain increased significantly.ConclusionsIn summary, molnupiravir rapidly transforms into NHC and crosses the BBB and reaches the brain at approximately 0.3-0.8% of the blood‒brain ratio. The maximum concentration of NHC in the blood and brain is above the average half maximal inhibitory concentration (IC50) of the drug required to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, suggesting a therapeutic effect. The penetration of NHC is modulated by NBMPR. These findings provide constructive information on brain disorders in clinical patients with COVID-19.

Prognostic and predictive value of human equilibrative nucleoside transporter 1 (hENT1) in extrahepatic cholangiocarcinoma: a translational study.

Introduction: Effective (neo) adjuvant chemotherapy for cholangiocarcinoma is lacking due to chemoresistance and the absence of predictive biomarkers. Human equilibrative nucleoside transporter 1 (hENT1) has been described as a potential prognostic and predictive biomarker. In this study, the potential of rabbit-derived (SP120) and murine-derived (10D7G2) antibodies to detect hENT1 expression was compared in tissue samples of patients with extrahepatic cholangiocarcinoma (ECC), and the predictive value of hENT1 was investigated in three ECC cell lines. Methods: Tissues of 71 chemonaïve patients with histological confirmation of ECC were selected and stained with SP120 or 10D7G2 to assess the inter-observer variability for both antibodies and the correlation with overall survival. Concomitantly, gemcitabine sensitivity after hENT1 knockdown was assessed in the ECC cell lines EGI-1, TFK-1, and SK-ChA-1 using sulforhodamine B assays. Results: Scoring immunohistochemistry for hENT1 expression with the use of SP120 antibody resulted in the highest interobserver agreement but did not show a prognostic role of hENT1. However, 10D7G2 showed a prognostic role for hENT1, and a potential predictive role for gemcitabine sensitivity in hENT1 in SK-ChA-1 and TFK-1 cells was found. Discussion: These findings prompt further studies for both preclinical validation of the role of hENT1 and histochemical standardization in cholangiocarcinoma patients treated with gemcitabine-based chemotherapy.

EVALUATION OF COMPONENTS OF THE EXTRACELLULAR PURINERGIC SIGNALING SYSTEM IN HUMAN SEPSIS.

Objective: Extracellular purines such as adenosine triphosphate (ATP), uridine triphosphate (UTP), and uridine diphosphate (UDP) and the ATP degradation product adenosine are biologically active signaling molecules, which accumulate at sites of metabolic stress in sepsis. They have potent immunomodulatory effects by binding to and activating P1 or adenosine and P2 receptors on the surface of leukocytes. Here we assessed the levels of extracellular purines, their receptors, metabolic enzymes, and cellular transporters in leukocytes of septic patients. Methods: Peripheral blood mononuclear cells (PBMCs), neutrophils, and plasma were isolated from blood obtained from septic patients and healthy control subjects. Ribonucleic acid was isolated from cells, and mRNA levels for purinergic receptors, enzymes, and transporters were measured. Adenosine triphosphate, UTP, UDP, and adenosine levels were evaluated in plasma. Results: Adenosine triphosphate levels were lower in septic patients than in healthy individuals, and levels of the other purines were comparable between the two groups. Levels of P1 and P2 receptors did not differ between the two patient groups. mRNA levels of ectonucleoside triphosphate diphosphohydrolase (NTPDase) 1 or CD39 increased, whereas those of NTPDase2, 3, and 8 decreased in PBMCs of septic patients when compared with healthy controls. CD73 mRNA was lower in PBMCs of septic than in healthy individuals. Equilibrative nucleoside transporter (ENT) 1 mRNA concentrations were higher and ENT2, 3, and 4 mRNA concentrations were lower in PBMCs of septic subjects when compared with healthy subjects. Concentrative nucleoside transporter (CNT) 1 mRNA levels were higher in PBMCs of septic versus healthy subjects, whereas the mRNA levels of CNT2, 3, and 4 did not differ. We failed to detect differences in mRNA levels of purinergic receptors, enzymes, and transporters in neutrophils of septic versus healthy subjects. Conclusion: Because CD39 degrades ATP to adenosine monophosphate (AMP), the lower ATP levels in septic individuals may be the result of increased CD39 expression. This increased degradation of ATP did not lead to increased adenosine levels, which may be explained by the decreased expression of CD73, which converts AMP to adenosine. Altogether, our results demonstrate differential regulation of components of the purinergic system in PBMCs during human sepsis.

Loss of function of ENT3 drives histiocytosis and inflammation through TLR-MAPK signaling

AbstractHistiocytoses are inflammatory myeloid neoplasms often driven by somatic activating mutations in mitogen-activated protein kinase (MAPK) cascade genes. H syndrome is an inflammatory genetic disorder caused by germ line loss-of-function mutations in SLC29A3, encoding the lysosomal equilibrative nucleoside transporter 3 (ENT3). Patients with H syndrome are predisposed to develop histiocytosis, yet the mechanism is unclear. Here, through phenotypic, molecular, and functional analysis of primary cells from a cohort of patients with H syndrome, we reveal the molecular pathway leading to histiocytosis and inflammation in this genetic disorder. We show that loss of function of ENT3 activates nucleoside-sensing toll-like receptors (TLR) and downstream MAPK signaling, inducing cytokine secretion and inflammation. Importantly, MEK inhibitor therapy led to resolution of histiocytosis and inflammation in a patient with H syndrome. These results demonstrate a yet-unrecognized link between a defect in a lysosomal transporter and pathological activation of MAPK signaling, establishing a novel pathway leading to histiocytosis and inflammation.