UNASSIGNED: Levetiracetam (LEV) has been found to have an antihyperalgesic effect via acting on the adenosine system. However, the effects of LEV on the modulation of the adenosine system in the brain have not been elucidated in the prevention of seizures and epilepsy. The present study aimed to explore the possible LEV mechanisms of action in the adenosine signaling systems in an animal model of epilepsy.
UNASSIGNED: A docking study was initially performed to determine the possible interaction of LEV with adenosine A1 receptors (A1Rs) and equilibrative nucleoside transporters-1 (ENT1). The experimental study was divided into an acute seizure test (32 mice distributed into 4 groups) and a chronic kindling model study (40 mice distributed into 5 groups), followed by gene expression analysis and immunohistochemistry. The kindling model lasted 26 days and took 13 subconvulsive doses of pentylenetetrazole (PTZ) to completely kindle the mice in the PTZ control group. Gene expression changes in the A1Rs, potassium inwardly-rectifying channel 3.2 (Kir3.2), and ENT1 in the brain tissue samples of the mice following treatment with LEV were analyzed using reverse transcription-quantitative polymerase chain reaction, and immunohistochemistry was performed for the A1R protein expression.
UNASSIGNED: Docking studies predicted a significant interaction of LEV with A1Rs and ENT1 proteins. Results from the acute testing revealed that caffeine (100 mg/kg) and 8-cyclopentyl-1,3-dipropylxanthine (25 mg/kg) significantly reversed the antiseizure effects of LEV by reversing the percent protection and shortening the onset of the first myoclonic jerk (FMJ) and generalized clonic seizures (GCSs). In the PTZ-induced kindling, LEV demonstrated an increased gene expression of A1Rs and Kir3.2 in the brain. LEV also significantly reduced the gene expression of ENT1. Furthermore, the immunohistochemical analysis showed that LEV increased the protein expression of A1Rs in the brain.
UNASSIGNED: Based on these results, it can be concluded that LEV modulates epileptogenesis by acting on the adenosine pathway in the central nervous 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.

BACKGROUND: Genetic polymorphisms of molecules are known to cause individual differences in the therapeutic efficacy of anticancer drugs. However, to date, germline mutations (but not somatic mutations) for anticancer drugs have not been adequately studied. The objective of this study was to investigate the association between germline polymorphisms of gemcitabine metabolic and transporter genes with carbohydrate antigen 19-9 (CA 19-9) response (decrease ≥50% from the pretreatment level at 8 weeks) and overall survival (OS) in patients with metastatic pancreatic cancer who receive gemcitabine-based chemotherapy.
METHODS: This multicenter, prospective, observational study enrolled patients with metastatic pancreatic cancer patients who were receiving gemcitabine monotherapy or gemcitabine plus nanoparticle albumin-bound paclitaxel combination chemotherapy. Thirteen polymorphisms that may be involved in gemcitabine responsiveness were genotyped, and univariate and multivariate logistic regression analyses were used to determine the association of these genotypes with CA 19-9 response and OS. The significance level was set at 5%.
RESULTS: In total, 180 patients from 11 hospitals in Japan were registered, and 159 patients whose CA 19-9 response could be assessed were included in the final analysis. Patients who had a CA 19-9 response had significantly longer OS (372 vs. 241 days; p = .007). RRM1 2464A>G and RRM2 175T>G polymorphisms suggested a weak association with CA 19-9 response and OS, but it was not statistically significant. COX-2 -765G>C polymorphism did not significantly correlate with CA 19-9 response but was significantly associated with OS (hazard ratio, 2.031; p = .019).
CONCLUSIONS: Genetic polymorphisms from the pharmacokinetics of gemcitabine did not indicate a significant association with efficacy, but COX-2 polymorphisms involved in tumor cell proliferation might affect OS.

BACKGROUND: FOLFIRINOX 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.
METHODS: We 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.
RESULTS: In 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.
CONCLUSIONS: Our 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.

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.

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Evaluation of kinetic parameters of drug-target binding, kon, koff, and residence time (RT), in addition to the traditional in vitro parameter of affinity is receiving increasing attention in the early stages of drug discovery. Target binding kinetics emerges as a meaningful concept for the evaluation of a ligand\'s duration of action and more generally drug efficacy and safety. We report the biological evaluation of a novel series of spirobenzo-oxazinepiperidinone derivatives as inhibitors of the human equilibrative nucleoside transporter 1 (hENT1, SLC29A1). The compounds were evaluated in radioligand binding experiments, i.e., displacement, competition association, and washout assays, to evaluate their affinity and binding kinetic parameters. We also linked these pharmacological parameters to the compounds\' chemical characteristics, and learned that separate moieties of the molecules governed target affinity and binding kinetics. Among the 29 compounds tested, 28 stood out with high affinity and a long residence time of 87 min. These findings reveal the importance of supplementing affinity data with binding kinetics at transport proteins such as hENT1.

NDec is a novel combination of oral decitabine and tetrahydrouridine that is currently under clinical development for the treatment of sickle cell disease (SCD). Here, we investigate the potential for the tetrahydrouridine component of NDec to act as an inhibitor or substrate of key concentrative nucleoside transporters (CNT1-3) and equilibrative nucleoside transporters (ENT1-2). Nucleoside transporter inhibition and tetrahydrouridine accumulation assays were performed using Madin-Darby canine kidney strain II (MDCKII) cells overexpressing human CNT1, CNT2, CNT3, ENT1, and ENT2 transporters. Results showed that tetrahydrouridine did not influence CNT- or ENT-mediated uridine/adenosine accumulation in MDCKII cells at the concentrations tested (25 and 250 µM). Accumulation of tetrahydrouridine in MDCKII cells was initially shown to be mediated by CNT3 and ENT2. However, while time- and concentration-dependence experiments showed active accumulation of tetrahydrouridine in CNT3-expressing cells, allowing for estimation of Km (3,140 µM) and Vmax (1,600 pmol/mg protein/min), accumulation of tetrahydrouridine was not observed in ENT2-expressing cells. Potent CNT3 inhibitors are a class of drugs not generally prescribed to patients with SCD, except in certain specific circumstances. These data suggest that NDec can be administered safely with drugs that act as substrates and inhibitors of the nucleoside transporters included in this study.

Previous studies implicate extracellular adenosine signaling in attenuating myocardial ischemia and reperfusion injury (IRI). This extracellular adenosine signaling is terminated by its uptake into cells by equilibrative nucleoside transporters (ENTs). Thus, we hypothesized that targeting ENTs would function to increase cardiac adenosine signaling and concomitant cardioprotection against IRI. Mice were exposed to myocardial ischemia and reperfusion injury. Myocardial injury was attenuated in mice treated with the nonspecific ENT inhibitor dipyridamole. A comparison of mice with global Ent1 or Ent2 deletion showed cardioprotection only in Ent1-/- mice. Moreover, studies with tissue-specific Ent deletion revealed that mice with myocyte-specific Ent1 deletion (Ent1loxP/loxP Myosin Cre+ mice) experienced smaller infarct sizes. Measurements of cardiac adenosine levels demonstrated that postischemic elevations of adenosine persisted during reperfusion after targeting ENTs. Finally, studies in mice with global or myeloid-specific deletion of the Adora2b adenosine receptor (Adora2bloxP/loxP LysM Cre+ mice) implied that Adora2b signaling on myeloid-inflammatory cells in cardioprotection provided by ENT inhibition. These studies reveal a previously unrecognized role for myocyte-specific ENT1 in cardioprotection by enhancing myeloid-dependent Adora2b signaling during reperfusion. Extension of these findings implicates adenosine transporter inhibitors in cardioprotection against ischemia and reperfusion injury.

Nucleoside transporters (NTs) play an important role in the metabolism of nucleoside substances and the efficacy of nucleoside drugs. Its spatial information related to biofunctions at the single-molecule level remains unclear, owing to the limitation of the existing labeling methods and traditional imaging methods. Therefore, we synthesize the inhibitor-based fluorescent probe SAENTA-Cy5 and apply direct stochastic optical reconstruction microscopy (dSTORM) to conduct refined observation of human equilibrative nucleoside transporter 1 (hENT1), the most important and famous member of NTs. We first demonstrate the labeling specificity and superiority of SAENTA-Cy5 to the antibody probe. Then, we found different assembly patterns of hENT1 on the apical and basal membranes, which are further investigated to be caused by varying associations of membrane carbohydrates, membrane classical functional domains (lipid rafts), and associated membrane proteins (EpCAM). Our work provides an efficient method for labeling hENT1, which contributes to realize fine observation of NTs. The findings on the assembly features and potential assembly mechanism of hENT1 promote a better understanding of its biofunction, which facilitates further investigations on how NTs work in the metabolism of nucleoside and nucleoside analogues.