Equilibrative Nucleoside Transporter Research Articles

Differential participation of CaMKII/ROCK and NOS pathways in the cholinergic inhibitory drive operated by nicotinic α7 receptors in perisynaptic Schwann cells

Nicotinic α7 receptors (α7 nAChRs) present in perisynaptic Schwann cells (PSCs) control acetylcholine (ACh) spillover from the neuromuscular synapse by transiently increasing intracellular Ca2+, which fosters adenosine release via type 1 equilibrative nucleoside transporters (ENT1) and retrograde activation of presynaptic A1 inhibitory receptors. The putative Ca2+-dependent pathways downstream α7 nAChRs involved in the sensing inhibitory drive operated by PSCs is unknown. Herein, we used phrenic nerve-hemidiaphragm preparations from Wistar rats. Time-lapse video-microscopy was instrumental to assess nerve-evoked (50-Hz bursts) transmitter exocytosis and intracellular NO oscillations in nerve terminals and PSCs loaded with FM4-64 and DAF-FM diacetate fluorescent dyes, respectively. Selective activation of α7 nAChRs with PNU 282987 reduced transmitter exocytosis (FM4-64 dye unloading) during 50-Hz bursts. Inhibition of calmodulin activity (with W-7), Ca2+/calmodulin-dependent protein kinase II (CaMKII; with KN-62) and Rho-kinase (ROCK; with H1152) all prevented the release inhibitory effect of PNU 282987. The α7 nAChR agonist transiently increased NO inside PSCs; the same occurred during phrenic nerve stimulation with 50-Hz bursts in the presence of the cholinesterase inhibitor, neostigmine. The nitric oxide synthase (NOS) inhibitor, L-NOARG, but not with the guanylylcyclase (GC) inhibitor, ODQ, prevented inhibition of transmitter exocytosis by PNU 282987. Inhibition of adenosine kinase with ABT 702 favors the intracellular accumulation and translocation of the nucleoside to the synaptic cleft, thus overcoming prevention of the PNU 282987 effect caused by H1152, but not by L-NOARG. In conclusion, the α7nAChR-mediated cholinergic inhibitory drive operated by PSCs involves two distinct Ca2+-dependent intracellular pathways: a CaMKII/ROCK cascade along with a GC-independent NO pathway with divergent end-effects concerning ADK inhibition.

Adenosine-Dependent Arousal Induced by Astrocytes in a Brainstem Circuit.

Astrocytes play a crucial role in regulating sleep-wake behavior. However, how astrocytes govern a specific sleep-arousal circuit remains unknown. Here, the authors show that parafacial zone (PZ) astrocytes responded to sleep-wake cycles with state-differential Ca2+ activity, peaking during transitions from sleep to wakefulness. Using chemogenetic and optogenetic approaches, they find that activating PZ astrocytes elicited and sustained wakefulness by prolonging arousal episodes while impeding transitions from wakefulness to non-rapid eye movement (NREM) sleep. Activation of PZ astrocytes specially induced the elevation of extracellular adenosine through the ATP hydrolysis pathway but not equilibrative nucleoside transporter (ENT) mediated transportation. Strikingly, the rise in adenosine levels induced arousal by activating A1 receptors, suggesting a distinct role for adenosine in the PZ beyond its conventional sleep homeostasis modulation observed in the basal forebrain (BF) and cortex. Moreover, at the circuit level, PZ astrocyte activation induced arousal by suppressing the GABA release from the PZGABA neurons, which promote NREM sleep and project to the parabrachial nucleus (PB). Thus, their study unveils a distinctive arousal-promoting effect of astrocytes within the PZ through extracellular adenosine and elucidates the underlying mechanism at the neural circuit level.

Adenosine Metabolism Pathway Alterations in Frontal Cortical Neurons in Schizophrenia.

Schizophrenia is a neuropsychiatric illness characterized by altered neurotransmission, in which adenosine, a modulator of glutamate and dopamine, plays a critical role that is relatively unexplored in the human brain. In the present study, postmortem human brain tissue from the anterior cingulate cortex (ACC) of individuals with schizophrenia (n = 20) and sex- and age-matched control subjects without psychiatric illness (n = 20) was obtained from the Bronx-Mount Sinai NIH Brain and Tissue Repository. Enriched populations of ACC pyramidal neurons were isolated using laser microdissection (LMD). The mRNA expression levels of six key adenosine pathway components-adenosine kinase (ADK), equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2), ectonucleoside triphosphate diphosphohydrolases 1 and 3 (ENTPD1 and ENTPD3), and ecto-5'-nucleotidase (NT5E)-were quantified using real-time PCR (qPCR) in neurons from these individuals. No significant mRNA expression differences were observed between the schizophrenia and control groups (p > 0.05). However, a significant sex difference was found in ADK mRNA expression, with higher levels in male compared with female subjects (Mann-Whitney U = 86; p < 0.05), a finding significantly driven by disease (t(17) = 3.289; p < 0.05). Correlation analyses also demonstrated significant associations (n = 12) between the expression of several adenosine pathway components (p < 0.05). In our dementia severity analysis, ENTPD1 mRNA expression was significantly higher in males in the "mild" clinical dementia rating (CDR) bin compared with males in the "none" CDR bin (F(2, 13) = 5.212; p < 0.05). Lastly, antipsychotic analysis revealed no significant impact on the expression of adenosine pathway components between medicated and non-medicated schizophrenia subjects (p > 0.05). The observed sex-specific variations and inter-component correlations highlight the value of investigating sex differences in disease and contribute to the molecular basis of schizophrenia's pathology.

Development of a Novel HEK293 Cell Model Lacking SLC29A1 to Study the Pharmacology of Endogenous SLC29A2-Encoded Equilibrative Nucleoside Transporter Subtype 2.

Equilibrative nucleoside transporters (ENTs) mediate the transmembrane flux of endogenous nucleosides and nucleoside analogs used clinically. The predominant subtype, ENT1, has been well characterized. However, the other subtype, ENT2, has been less well characterized in its native milieu due to its relatively low expression and the confounding influence of coexpressed ENT1. We created a cell model where ENT1 was removed from human embryonic kidney (HEK293) cells using CRISPR/cas9 [ENT1 knockout (KO) cells]; this cell line has ENT2 as the only functional purine transporter. Transporter function was assessed through measurement of [3H]2-chloroadenosine uptake. ENT1 protein was quantified based on the binding of [3H]nitrobenzylthioinosine, and ENT1/ENT2 protein was detected by immunoblotting. Changes in expression of relevant transporters and enzymes involved in purine metabolism were examined by quantitative polymerase chain reaction. Wild-type HEK293 cells and ENT1KO cells had a similar expression of SLC29A2/ENT2 transcript/protein and ENT2-mediated [3H]2-chloroadenosine transport activity (Vmax values of 1.02 ± 0.06 and 1.50 ± 0.22 pmol/μl/s, respectively). Of the endogenous nucleosides/nucleobases tested, adenosine had the highest affinity (Ki) for ENT2 (2.6 μM), while hypoxanthine was the only nucleobase with a submillimolar affinity (320 μM). A range of nucleoside/nucleobase analogs were also tested for their affinity for ENT2 in this model, with affinities (Ki) ranging from 8.6 μM for ticagrelor to 2,300 μM for 6-mercaptopurine. Our data suggest that the removal of endogenous ENT1 from these cells does not change the expression or function of ENT2. This cell line should prove useful for the analysis of novel drugs acting via ENT2 and to study ENT2 regulation. SIGNIFICANCE STATEMENT: We have created a cell line whereby endogenous ENT2 can be studied in detail in the absence of the confounding influence of ENT1. Loss of ENT1 has no impact on the expression and function of ENT2. This novel cell line will provide an ideal model for studying drug interactions with ENT2 as well as the cellular regulation of ENT2 expression and function.

Rheumatological manifestations of H syndrome.

H syndrome (HS) is a rare autosomal recessive genodermatosis characterised by cutaneous hyperpigmentation, hypertrichosis, sclerodermatous thickening, and multisystemic involvement. It results from mutations in the SLC29A3 gene encoding the human equilibrative nucleoside transporter 3, leading to impaired histiocyte apoptosis and unchecked proliferation. We report the case of a 24-year-old Moroccan male who had a history of insulin-dependent diabetes mellitus. He developed hyperpigmented skin patches with hypertrichosis and induration. Musculoskeletal findings included bilateral hallux valgus, pes planus, reducible flexion contractures of the proximal interphalangeal joints, and restricted ankle dorsiflexion. Additional findings consist of lymphadenopathy, hepatomegaly, hypogonadism, and ophthalmic manifestations. Investigations showed elevated sedimentation rate, anaemia, and osteopaenia. Ankle ultrasound revealed calcaneal enthesopathy and subcutaneous infiltration. In reporting this case, we aim to highlight the significant rheumatological involvement that can arise in patients with H syndrome and explore potential treatment options to improve the musculoskeletal findings.

Pharmacological inhibition of ENT1 enhances the impact of specific dietary fats on energy metabolism gene expression

Medium chain fatty acids are commonly consumed as part of diets for endurance sports and as medical treatment in ketogenic diets where these diets regulate energy metabolism and increase adenosine levels. However, the role of the equilibrative nucleoside transporter 1 (ENT1), which is responsible for adenosine transport across membranes in this process, is not well understood. Here, we investigate ENT1 activity in controlling the effects of two dietary medium chain fatty acids (decanoic and octanoic acid), employing the tractable model system Dictyostelium. We show that genetic ablation of three ENT1 orthologues unexpectedly improves cell proliferation specifically following decanoic acid treatment. This effect is not caused by increased adenosine levels triggered by both fatty acids in the presence of ENT1 activity. Instead, we show that decanoic acid increases expression of energy-related genes relevant for fatty acid β-oxidation, and that pharmacological inhibition of ENT1 activity leads to an enhanced effect of decanoic acid to increase expression of tricarboxylicacid cycle and oxidative phosphorylation components. Importantly, similar transcriptional changes have been shown in the rat hippocampus during ketogenic diet treatment. We validated these changes by showing enhanced mitochondria load and reduced lipid droplets. Thus, our data show that ENT1 regulates the medium chain fatty acid-induced increase in cellular adenosine levels and the decanoic acid-induced expression of important metabolic enzymes in energy provision, identifying a key role for ENT1 proteins in metabolic effects of medium chain fatty acids.

Trypanocide Use and Molecular Characterization of Trypanosomes Resistant to Diminazene Aceturate in Cattle in Northern Côte D'Ivoire.

The resistance of trypanosomes to the doses of trypanocide administered by farmers to their animals acts as a real brake on efforts to control to combat African trypanosomiasis. Thus, in-depth knowledge of the use of these different molecules and their resistance profiles will be necessary to establish an integrated strategy to combat African trypanosomiasis. To achieve these objectives, a participatory survey among farmers and a resistance diagnosis of trypanosome strains identified in three regions of northern Côte d'Ivoire (Bagoué, Poro and Tchologo) was carried out using the PCR-RFLP technique, followed by sequencing of genes of interest. This study made it possible to identify three molecules that are commonly used by 85% (63/74) of farmers. In descending order of use, we identified Isometamidium chloride (43%), Diminazene aceturate (28%) and Homidium bromide (14%). Three species of trypanosomes, Trypanosoma congolense, Trypanosoma. theileri and Trypanosoma vivax, were identified in farms, and only one strain had the adenosine transporter gene (Trypanosoma congolense), but this strain was sensitive to the Diminazene aceturate molecule. Comparison of the sequence of this trypanosome strain showed that it is different to the Kenyan strain diagnosed as resistant to the Diminazene aceturate molecule. This study shows that a variety of trypanocides are used by farmers, and that the resistance profile of the strains to the Diminazene aceturate molecule could not be observed. However, it is important to further investigate the other molecules encountered in Côte d'Ivoire.

Extracellular adenosine oppositely regulates the purinome machinery in glioblastoma and mesenchymal stem cells.

Glioblastoma (GB) is a lethal brain tumor that rapidly adapts to the dynamic changes of the tumor microenvironment (TME). Mesenchymal stem/stromal cells (MSCs) are one of the stromal components of the TME playing multiple roles in tumor progression. GB progression is prompted by the immunosuppressive microenvironment characterized by high concentrations of the nucleoside adenosine (ADO). ADO acts as a signaling molecule through adenosine receptors (ARs) but also as a genetic and metabolic regulator. Herein, the effects of high extracellular ADO concentrations were investigated in a human glioblastoma cellular model (U343MG) and MSCs. The modulation of the purinome machinery, i.e., the ADO production (CD39, CD73, and adenosine kinase [ADK]), transport (equilibrative nucleoside transporters 1 (ENT1) and 2 (ENT2)), and degradation (adenosine deaminase [ADA]) were investigated in both cell lines to evaluate if ADO could affect its cell management in a positive or negative feed-back loop. Results evidenced a different behavior of GB and MSC cells upon exposure to high extracellular ADO levels: U343MG were less sensitive to the ADO concentration and only a slight increase in ADK and ENT1 was evidenced. Conversely, in MSCs, the high extracellular ADO levels reduced the ADK, ENT1, and ENT2 expression, which further sustained the increase of extracellular ADO. Of note, MSCs primed with the GB-conditioned medium or co-cultured with U343MG cells were not affected by the increase of extracellular ADO. These results evidenced how long exposure to ADO could produce different effects on cancer cells with respect to MSCs, revealing a negative feedback loop that can support the GB immunosuppressive microenvironment. These results improve the knowledge of the ADO role in the maintenance of TME, which should be considered in the development of therapeutic strategies targeting adenosine pathways as well as cell-based strategies using MSCs.

Deep Brain Stimulation Inhibits Epileptic Seizures via Increase of Adenosine Release and Inhibition of ENT1, CD39, and CD73 Expression.

Deep brain stimulation (DBS) of the anterior nucleus of the thalamus is an efficacious treatment option for patients with refractory epilepsy. Our previous study demonstrates that adenosine is a potential target of DBS for the treatment of epilepsy. Equilibrative nucleoside transporters-1 (ENT1) and ectonucleotidases (CD39, CD73) function as regulators of extracellular adenosine in the brain. It is unclear whether ENT1, CD39, and CD73 are involved in the mechanism of DBS for epilepsy. A total of 48 SD male rats were divided into four groups: control (naïve rats), Pilo (pilocarpine induced rats with epilepsy), DBS (rats with epilepsy treated with DBS for 8weeks), and sham. In the present study, video electroencephalogram monitoring, Morris water maze assays, in vivo measurements of adenosine using fiber photometry, histochemistry, and western blot were performed on the hippocampus. DBS markedly attenuated spontaneous recurrent seizures (SRSs) and enhanced spatial learning in rats with epilepsy, assessed through video-EEG and water maze assays. Fibred photometry measurements of an adenosine sensor revealed dynamic increase in extracellular adenosine during DBS. The expressions of ENT1, CD39, and CD73 in Pilo group and sham group increased compared with the control group, while the expressions of ENT1, CD39, and CD73 in DBS group decreased compared to that of Pilo group and sham group. The findings indicate that DBS reduces the number of SRSs and improves spatial memory in rats with epilepsy with concomitant decrease of ENT1, CD39, and CD73 expressions. Adenosine-modulating enzymes might be the potential targets of DBS for the treatment of epilepsy.

Lessons from the physiological role of guanosine in neurodegeneration and cancer: Toward a multimodal mechanism of action?

Neurodegenerative diseases and brain tumours represent important health challenges due to their severe nature and debilitating consequences that require substantial medical care. Interestingly, these conditions share common physiological characteristics, namely increased glutamate, and adenosine transmission, which are often associated with cellular dysregulation and damage. Guanosine, an endogenous nucleoside, is safe and exerts neuroprotective effects in preclinical models of excitotoxicity, along with cytotoxic effects on tumour cells. However, the lack of well-defined mechanisms of action for guanosine hinders a comprehensive understanding of its physiological effects. In fact, the absence of specific receptors for guanosine impedes the development of structure-activity research programs to develop guanosine derivatives for therapeutic purposes. Alternatively, given its apparent interaction with the adenosinergic system, it is plausible that guanosine exerts its neuroprotective and anti-tumorigenic effects by modulating adenosine transmission through undisclosed mechanisms involving adenosine receptors, transporters, and purinergic metabolism. Here, several potential molecular mechanisms behind the protective actions of guanosine will be discussed. First, we explore its potential interaction with adenosine receptors (A1R and A2AR), including the A1R-A2AR heteromer. In addition, we consider the impact of guanosine on extracellular adenosine levels and the role of guanine-based purine-converting enzymes. Collectively, the diverse cellular functions of guanosine as neuroprotective and antiproliferative agent suggest a multimodal and complementary mechanism of action.

Identification of Human TRIAC Transmembrane Transporters.

Background: 3,5,3'-Triiodothyroacetic acid (TRIAC) 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 monocarboxylate transporter 8 (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. Methods: 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. Results: We identified SLC22A9 and SLC29A2 as transporters mediating cellular uptake of TRIAC. SLC22A9 encodes the organic anion transporter 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. Coincubation 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. Conclusions: 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.

Equilibrative nucleoside transporter 3 supports microglial functions and protects against the progression of Huntington’s disease in the mouse model

Huntington’s disease (HD) is a hereditary neurodegenerative disorder characterized by involuntary movements, cognitive deficits, and psychiatric symptoms. Currently, there is no cure, and only limited treatments are available to manage the symptoms and to slow down the disease’s progression. The molecular and cellular mechanisms of HD’s pathogenesis are complex, involving immune cell activation, altered protein turnover, and disturbance in brain energy homeostasis. Microglia have been known to play a dual role in HD, contributing to neurodegeneration through inflammation but also enacting neuroprotective effects by clearing mHTT aggregates. However, little is known about the contribution of microglial metabolism to HD progression. This study explores the impact of a microglial metabolite transporter, equilibrative nucleoside transporter 3 (ENT3), in HD. Known as a lysosomal membrane transporter protein, ENT3 is highly enriched in microglia, with its expression correlated with HD severity. Using the R6/2 ENT3−/− mouse model, we found that the deletion of ENT3 increases microglia numbers yet worsens HD progression, leading to mHTT accumulation, cell death, and disturbed energy metabolism. These results suggest that the delicate balance between microglial metabolism and function is crucial for maintaining brain homeostasis and that ENT3 has a protective role in ameliorating neurodegenerative processes.

Exploring novel dilazep derivatives as hENT1 inhibitors and potentially covalent molecular tools.

The human equilibrative nucleoside transporter 1 (SLC29A1, hENT1) is a solute carrier that modulates the passive transport of nucleosides and nucleobases, such as adenosine. This nucleoside regulates various physiological processes, such as vasodilation and -constriction, neurotransmission and immune defense. Marketed drugs such as dilazep and dipyridamole have proven useful in cardiovascular afflictions, but the application of hENT1 inhibitors can be beneficial in a number of other diseases. In this study, 39 derivatives of dilazep's close analogue ST7092 were designed, synthesized and subsequently assessed using [3H]NBTI displacement assays and molecular docking. Different substitution patterns of the trimethoxy benzoates of ST7092 reduced interactions within the binding pocket, resulting in diminished hENT1 affinity. Conversely, [3H]NBTI displacement by potentially covalent compounds 14b, 14c, and 14d resulted in high affinities (Ki values between 1.1 and 17.5 nM) for the transporter, primarily by the ability of accommodating the inhibitors in various ways in the binding pocket. However, any indication of covalent binding with amino acid residue C439 remained absent, conceivably as a result of decreased nucleophilic residue reactivity. In conclusion, this research introduces novel dilazep derivatives that are active as hENT1 inhibitors, along with the first high affinity dilazep derivatives equipped with an electrophilic warhead. These findings will aid the rational and structure-based development of novel hENT1 inhibitors and pharmacological tools to study hENT1's function, binding mechanisms, and its relevance in (patho)physiological conditions.

A review of the Augustine blood group system.

Augustine is a newly identified blood group system comprising four antigens, one of which is the high-frequency antigen Ata in the original "series". Four antigens are located on a multipass membrane glycoprotein equilibrative nucleoside transporter 1 (ENT1), and equilibrative nucleoside transporter is encoded by SLC29A1. In 2016, the International Society of Blood Transfusion (ISBT) recognised Augustine as a blood group system and numbered it as 036. The glycoprotein ENT1 transports nucleotides into cells to participate in the synthesis of DNA and RNA, and this is an important link for chemotherapeutic glycosides to enter tumour cells. Augustine antibodies are clinically relevant in blood transfusion and pregnancy.

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.

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.

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.

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.