A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes

Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.

Augustine Blood Group System and Equilibrative Nucleoside Transporter 1

Augustine (AUG) is a blood group system comprising four antigens: AUG1, AUG2 (At^a), and AUG4 are of very high frequency; AUG3 is of very low frequency. These antigens are located on ENT1, an equilibrative nucleoside transporter encoded by <i>SLC19A1</i>. AUG antibodies are of clinical relevance in blood transfusion and pregnancy: anti-AUG2 have caused haemolytic transfusion reactions; the only anti-AUG3 was associated with severe haemolytic disease of the fetus and newborn. ENT1 is present in almost all human tissues. It facilitates the transfer of purine and pyrimidine nucleosides and is responsible for the majority of adenosine transport across plasma membranes. Adenosine transport appears to be an important factor in the regulation of bone metabolism. The AUG_null phenotype (AUG:–1,–2,–3,–4) has been found in three siblings, who are homozygous for an inactivating splice-site mutation in <i>SLC29A1</i>. Although ENT1 is very likely to be absent from all cells in these three individuals, they were apparently healthy with normal lifestyles. However, they suffered frequent attacks of pseudogout, a form of arthritis, in various joints with multiple calcifications around their hand joints. Ectopic calcification in the hips, pubic symphysis, and lumbar discs was present in the propositus. The three AUG_null individuals had misshapen red cells with deregulated protein phosphorylation, but no anaemia or shortening of red cell lifespan. Defective in vitro erythropoiesis in the absence of ENT1 was confirmed by shRNA-mediated knockdown of ENT1 during in vitro erythropoiesis of CD34⁺ progenitor cells from individuals with normal ENT1. Nucleoside transporters, such as ENT1, are vital in the uptake of synthetic nucleoside analogue drugs, used in cancer and viral chemotherapy. It is feasible that the efficacy of these drugs would be compromised in patients with the extremely rare AUG_null phenotype.

hENT1 Predicts Benefit from Gemcitabine in Pancreatic Cancer but Only with Low CDA mRNA

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.

RRM1 Expression as a Prognostic Biomarker for Unresectable or Recurrent Biliary Tract Cancer Treated with Gemcitabine plus Cisplatin

The combination of gemcitabine plus cisplatin (GP) is regarded as a first-line treatment for patients with unresectable or recurrent biliary tract cancer (BTC). Several proteins including human equilibrative nucleoside transporter-1 (hENT1), deoxycytidine kinase (DCK), cytidine deaminase (CDA), and ribonucleotide reductase subunit 1 (RRM1) are known to be involved in gemcitabine uptake and metabolism. This study was aimed to identify the predictive and prognostic values of these biomarkers in patients who treated with GP for advanced BTC. Tumor samples were obtained from 34 patients with unresectable or recurrent BTC who were treated with GP between August 2015 and February 2018. Intratumoral expression of hENT1, DCK, CDA and RRM1 was determined by immunohistochemistry and analyzed for association with chemotherapy response, progression-free survival (PFS) and overall survival (OS). Median OS was significantly longer in the RRM1-negative group than in the RRM1-positive (9.9 months vs. 5.9 months, p = 0.037). Multivariate adjustment analyses also demonstrated RRM1 expression as an independent prognostic factor for OS in patients treated with GP chemotherapy. Increased intratumoral expression of RRM1 on immunohistochemical staining may be a biomarker predicting poor survival in patients with GP chemotherapy for advanced BTC. Large-scale well-predefined prospective research is needed to validate the utility of biomarkers in clinical practice.

Contribution of the Adenosine 2A Receptor to Behavioral Effects of Tetrahydrocannabinol, Cannabidiol and PECS-101

The cannabis-derived molecules, ∆9 tetrahydrocannabinol (THC) and cannabidiol (CBD), are both of considerable therapeutic interest for a variety of purposes, including to reduce pain and anxiety and increase sleep. In addition to their other pharmacological targets, both THC and CBD are competitive inhibitors of the equilibrative nucleoside transporter-1 (ENT-1), a primary inactivation mechanism for adenosine, and thereby increase adenosine signaling. The goal of this study was to examine the role of adenosine A2A receptor activation in the effects of intraperitoneally administered THC alone and in combination with CBD or PECS-101, a 4′-fluorinated derivative of CBD, in the cannabinoid tetrad, elevated plus maze (EPM) and marble bury assays. Comparisons between wild-type (WT) and A2AR knock out (A2AR-KO) mice were made. The cataleptic effects of THC were diminished in A2AR-KO; no other THC behaviors were affected by A2AR deletion. CBD (5 mg/kg) potentiated the cataleptic response to THC (5 mg/kg) in WT but not A2AR-KO. Neither CBD nor THC alone affected EPM behavior; their combination produced a significant increase in open/closed arm time in WT but not A2AR-KO. Both THC and CBD reduced the number of marbles buried in A2AR-KO but not WT mice. Like CBD, PECS-101 potentiated the cataleptic response to THC in WT but not A2AR-KO mice. PECS-101 also reduced exploratory behavior in the EPM in both genotypes. These results support the hypothesis that CBD and PECS-101 can potentiate the cataleptic effects of THC in a manner consistent with increased endogenous adenosine signaling.

Equilibrative nucleoside transporter 1 inhibition rescues energy dysfunction and pathology in a model of tauopathy

Tau pathology is instrumental in the gradual loss of neuronal functions and cognitive decline in tauopathies, including Alzheimer’s disease (AD). Earlier reports showed that adenosine metabolism is abnormal in the brain of AD patients while consequences remained ill-defined. Herein, we aimed at investigating whether manipulation of adenosine tone would impact Tau pathology, associated molecular alterations and subsequent neurodegeneration. We demonstrated that treatment with an inhibitor (J4) of equilibrative nucleoside transporter 1 (ENT1) exerted beneficial effects in a mouse model of Tauopathy. Treatment with J4 not only reduced Tau hyperphosphorylation but also rescued memory deficits, mitochondrial dysfunction, synaptic loss, and abnormal expression of immune-related gene signatures. These beneficial effects were particularly ascribed to the ability of J4 to suppress the overactivation of AMPK (an energy reduction sensor), suggesting that normalization of energy dysfunction mitigates neuronal dysfunctions in Tauopathy. Collectively, these data highlight that targeting adenosine metabolism is a novel strategy for tauopathies.

Substrate Dependent Regulation of the Human Equilibrative Nucleoside Transporter 1 (hENT1) in HEK293 Cells

Nucleosides and nucleoside analog drugs enter cells through nucleoside transporters, such as the human equilibrative nucleoside transporter 1 (hENT1). The regulation of nucleoside transporters is poorly understood. In this study, through fluorescence-activated cell sorting (FACS) analyses, confocal microscopy and radio-ligand binding assays, I show a decrease in hENT1 abundance at the plasma membrane (PM) in HEK cells treated in the presence of a bolus amount of cytidine (40μM) for 6 hours. Kinetic and transport assays indicate that the remaining hENT1 population at the PM has a higher Vmax and Km but there is no change in overall substrate uptake compared to untreated cells. I also show that cytidine pre-treatment leads to an increased cytotoxicity from gemcitabine (a nucleoside analog drug). These are the first data that show direct substrate dependent regulation of a nucleoside transporter by a mechanism that may involve increased recycling/internalization of the transporter.