Nucleoside Transport Inhibitor Research Articles

Abstract PO-034: Targeting metabolic plasticity and flexibility via SIRT3 inhibition to restrain nucleotides availability in DLBCLs

Abstract Cancer cells need metabolic adaptations to survive in hostile environment such as low nutrient and drug treatment conditions. DLBCLs (Diffuse large B cell lymphoma) originate from a metabolic competitive/challenging environment of germinal center (GC) when B cells undergo positive selections against antigens. However, it is largely unknown whether and how metabolic flexibility (the ability to use different nutrients) and plasticity (the ability to process the same nutrient differently) contribute to DLBCLs. Our previous study identified that SIRT3, the only mitochondrial protein deacetylase, is critical to survival and proliferation of DLBCLs by promoting glutaminolysis. Our mitochondrial localized SIRT3 inhibitor, YC8-02, has pan-toxicity to all DLBCL cells tested regardless of different subtypes with somatic mutations. Here, we observed glucose as an alternative nutrient source to compensate in the TCA cycle in SIRT3 deficient cells. DLBCLs exhibited resistance to SIRT3 inhibition in high glucose condition, while SIRT3 inhibited DLBCLs highly depend on glucose metabolism. The combination of YC8-02 with glucose metabolism inhibitors induces synergistic killing to DLBCLs. As glucose is the main carbon source of cells, it is unknown how DLBCLs manage the flexibility of using glucose for different purposes. Using in vivo metabolic imaging (MALDI), we observed that, in contrast to normal GC B cells, lymphomas specifically use the glucose for de novo nucleotide synthesis. SIRT3 inhibition decreased nucleotide levels in DLBCL cells in vitro and in vivo. The metabolic flux tracing showed that DLBCL cells requires SIRT3 activity to activate a glucose/serine/one-carbon metabolism pathway for de novo nucleotide biosynthesis, and Inhibition of SIRT3 decreased the glucose and serine being used for nucleotides in DLBCLs. Serine as another potent source for nucleotides can rescue DLBCLs from SIRT3 inhibition. Furthermore, we validated that overexpression of enzymes, like MTHFD2, in one carbon metabolism pathway can rescue the proliferation in SIRT3 deficient cells. High nucleotides levels in medium also help DLBCLs survival in YC8-02 treatments. To overcome the metabolic flexibility on environmental nucleotides, we used inhibitors of nucleoside transporters (ENT1/2), dipyridamole and draflazine, which can synergize with YC8-02 to kill DLBCLs in vitro and eradicate systemic lymphomas in vivo. In summary, our results illustrate that DLBCLs with active SIRT3 exhibit metabolic plasticity by repurposing glucose to nucleotide biosynthesis, while lymphoma cells can also reply on metabolic flexibility to manage nutrient usage and gain extracellular nucleotides for survival under SIRT3 inhibition. Our combination treatment SIRT3 and ENT inhibitors can be a promising therapy for DLBCLs by restraining their metabolic plasticity and flexibility. Citation Format: Meng Li, Noel R Park, Chris Chin, Coraline Mlynarczyk, Hening Lin, Shawn Davidson, Ari M Melnick. Targeting metabolic plasticity and flexibility via SIRT3 inhibition to restrain nucleotides availability in DLBCLs [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-034.

Suppression of Ehrlich ascites tumor cell proliferation via G1 arrest induced by dietary nucleic acid-derived nucleosides.

The nucleic acids found in food play a crucial role in maintaining various bodily functions. This study investigated the potential anticancer effects of dietary nucleic acids, an area that is still not fully understood. By utilizing an in vivo mouse model and an in vitro cell model, we discovered an anti-proliferative impact of RNA in both systems. DNA exhibited anti-proliferative effects in the mouse model, while this phenomenon wasn't observed in the in vitro cell model using Ehrlich ascites tumor (EAT) cells. Conversely, DNA hydrolysate demonstrated distinct anti-proliferative effects in EAT cells, suggesting that nucleotides or nucleosides generated during nucleic acid digestion act as active constituents. Furthermore, we examined various nucleosides and two sodium-independent equilibrative nucleoside transporter inhibitors (ENTs), identifying guanosine and 2'-deoxyguanosine as pivotal in the anti-proliferative effect. We also found that the anti-proliferation activity with both nucleosides was suppressed by the treatment of dipyridamole, a non-selective inhibitor for ENT1 and ENT2, but not nitrobenzylthioinosine, a low inhibitor for ENT2. The uptake of these compounds into cells is likely facilitated by ENT2. These nucleotides impeded the progression of cancer cells from the G1 phase to the S phase in the cell cycle. Another significant finding is the increased expression of CCAAT/enhancer-binding protein (C/EBPβ) induced by guanosine and 2'-deoxyguanosine. Furthermore, immunostaining revealed that C/EBPβ diffuses into the nucleus, indicating its presence. This suggests that guanosine or 2-deoxyguanosine induces G1 arrest in cancer cells via the activation of C/EBPβ. Encouraged by these promising results, guanosine and 2'-deoxyguanosine show potential applications in cancer prevention.

Kinetic profiling of novel spirobenzo-oxazinepiperidinone derivatives as equilibrative nucleoside transporter 1 inhibitors

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.

In Vitro Interaction of Tetrahydrouridine with Key Human Nucleoside Transporters

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.

The Effect of Adenosine Signaling on Memory Impairment Induced by Pentylenetetrazole in Zebrafish.

Epilepsy is characterized by the manifestation of spontaneous and recurrent seizures. The high prevalence of comorbidities associated with epilepsy, such as cognitive dysfunction, affects the patients quality oflife. Adenosine signaling modulation might be an effective alternative to control seizures and epilepsy-associated comorbidities. This study aimed to verify the role of adenosine modulation on the seizure development and cognitive impairment induced by pentylenetetrazole (PTZ) in zebrafish. At first, animals were submitted to a training session in the inhibitory avoidance test and, after 10min, they received an intraperitoneal injection of valproate, adenosine A1 receptor agonist cyclopentyladenosine (CPA), adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), adenosine A2A receptor antagonist ZM 241385, adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nony1)-adenine hydrochloride (EHNA) or the nucleoside transporter inhibitor dipyridamole. Thirty min after the intraperitoneal injection, the animals were exposed to 7.5mM PTZ for 10min, where they were evaluated for latency to reach the seizure stages (I, II, and III). Finally, 24h after the training session, the animals were submitted to the inhibitory avoidance test to verify their cognitive performance during the test session. Valproate, CPA, and EHNA showed antiseizure effects and prevented the memory impairment induced by PTZ exposure. DPCPX, ZM 241385, and dipyridamole pretreatments caused no changes in seizure development; however, these drugs prevented memory impairment without altering locomotion. Our results reinforce the antiseizure effects of adenosine signaling and support the idea that the involvement of adenosine in memory processes may be a target for preventive strategies against cognitive impairment associated with epilepsy.

Inhibition of uterine contractility by guanine-based purines in non-pregnant rats.

Growing evidence pointed out that guanine-based purines are able to modulate smooth muscle contractile activity of blood vessels and gastrointestinal tract. Since, so far, possible guanine-based purine modulation of uterine musculature is unknown, the aim of the present study was to investigate in vitro, using organ bath technique, guanosine and guanine effects on spontaneous uterine contraction, and uterine contraction induced by K+-depolarization and oxytocin in a non-pregnant rat. Guanosine, but not guanine, reduced the amplitude of spontaneous contraction of the uterine muscle in a dose-dependent manner. The inhibitory response was antagonized by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a membrane nucleoside transporter inhibitor, but persisted in the presence of theophylline, a nonselective adenosine receptor antagonist, or propanolol, β1/β2 adrenoreceptor antagonist or blockers of a nitrergic pathway. In addition, potassium channel blockers did not influence guanosine-induced effects. Guanosine was able to inhibit the external calcium (Ca2+) influx-induced contraction, but it did not affect the contraction induced by high-KCl solution, indicating that guanosine does not interact with L-type voltage-gated calcium channel. Guanosine prevented/reduced uterine contractions induced by oxytocin, even in the absence of external calcium. In conclusion, guanosine is able to reduce both spontaneous and oxytocin-induced contractions of rat myometrium, likely subsequently to its intracellular intake. The blockade of extracellular Ca2+ influx and reduction of Ca2+ release from the intracellular store are the mechanisms involved in the guanosine-induced tocolytic effects.

Modulation of adenosine signaling reverses 3-nitropropionic acid-induced bradykinesia and memory impairment in adult zebrafish

Huntington's disease (HD) is a neurodegenerative disorder, characterized by motor dysfunction, psychiatric disturbance, and cognitive decline. In the early stage of HD, occurs a decrease in dopamine D2 receptors and adenosine A2A receptors (A2AR), while in the late stage also occurs a decrease in dopamine D1 receptors and adenosine A1 receptors (A1R). Adenosine exhibits neuromodulatory and neuroprotective effects in the brain and is involved in motor control and memory function. 3-Nitropropionic acid (3-NPA), a toxin derived from plants and fungi, may reproduce HD behavioral phenotypes and biochemical characteristics. This study investigated the effects of acute exposure to CPA (A1R agonist), CGS 21680 (A2AR agonist), caffeine (non-selective of A1R and A2AR antagonist), ZM 241385 (A2AR antagonist), DPCPX (A1R antagonist), dipyridamole (inhibitor of nucleoside transporters) and EHNA (inhibitor of adenosine deaminase) in an HD pharmacological model induced by 3-NPA in adult zebrafish. CPA, CGS 21680, caffeine, ZM 241385, DPCPX, dipyridamole, and EHNA were acutely administered via i.p. in zebrafish after 3-NPA (at dose 60 mg/kg) chronic treatment. Caffeine and ZM 241385 reversed the bradykinesia induced by 3-NPA, while CGS 21680 potentiated the bradykinesia caused by 3-NPA. Moreover, CPA, caffeine, ZM 241385, DPCPX, dipyridamole, and EHNA reversed the 3-NPA-induced memory impairment. Together, these data support the hypothesis that A2AR antagonists have an essential role in modulating locomotor function, whereas the activation of A1R and blockade of A2AR and A1R and modulation of adenosine levels may reduce the memory impairment, which could be a potential pharmacological strategy against late-stage symptoms HD.

Metformin Protects Against Sunitinib-induced Cardiotoxicity: Investigating the Role of AMPK.

Sunitinib is associated with cardiotoxicity through inhibition of AMP-protein kinase (AMPK) signaling. By contrast, the common antidiabetic agent metformin has demonstrated cardioprotection through indirect AMPK activation. In this study, we investigate the effects of metformin during sunitinib-induced cytotoxicity. Left ventricular developed pressure, coronary flow, heart rate, and infarct size were measured in Langendorff-perfused rat hearts treated with 1 µM sunitinib ±50 µM metformin ±1 µM human equilibrative nucleoside transporter inhibitor S-(4-Nitrobenzyl)-6-thioinosine (NBTI). Western blot analysis was performed for p-AMPKα levels. Primary isolated cardiac myocytes from the left ventricular tissue were used to measure live cell population levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess adjunctive treatment of and metformin in human hepatoma G2 and promyelocytic leukemia (HL-60) cells treated with 0.1-100 µM sunitinib ±50 µM metformin. In the perfused hearts, coadministration of metformin attenuated the sunitinib-induced changes to left ventricular developed pressure, infarct size, and cardiac myocyte population. Western blot analysis revealed a significant decrease in p-AMPKα during sunitinib treatment, which was attenuated after coadministration with metformin. All metformin-induced effects were attenuated, and NBTI was coadministered. The MTT assay demonstrated an increase in the EC50 value during coadministration of metformin with sunitinib compared with sunitinib monotherapy in hepatoma G2 and HL-60 cell lines, demonstrating the impact and complexity of metformin coadministration and the possible role of AMPK signaling. This study highlights the novel cardioprotective properties of metformin and AMPK activation during sunitinib-induced cardiotoxicity when administered together in the Langendorff heart model.

Cladribine as a Potential Object of Nucleoside Transporter-Based Drug Interactions.

Cladribine is a nucleoside analog that is phosphorylated in its target cells (B and T-lymphocytes) to its active triphosphate form (2-chlorodeoxyadenosine triphosphate). Cladribine tablets 10 mg (Mavenclad®), administered for up to 10 days per year in 2 consecutive years (3.5-mg/kg cumulative dose over 2 years), are used to treat patients with relapsing multiple sclerosis. Cladribine has been shown to be a substrate of various nucleoside transporters (NTs). Intestinal absorption and distribution of cladribine throughout the body appear to be essentially mediated by equilibrative NTs (ENTs) and concentrative NTs (CNTs), specifically by ENT1, ENT2, ENT4, CNT2 (low affinity), and CNT3. Other efficient transporters of cladribine are the ABC efflux transporters, specifically breast cancer resistance protein, which likely modulates the oral absorption and renal excretion of cladribine. A key transporter for the intracellular uptake of cladribine into B and T-lymphocytes is ENT1 with ancillary contributions of ENT2 and CNT2. Transporter-based drug interactions affecting absorption and target cellular uptake of a prodrug such as cladribine are likely to reduce systemic bioavailability and target cell exposure, thereby possibly hampering clinical efficacy. In order to manage optimized therapy, i.e., to ensure uncompromised target cell uptake to preserve the full therapeutic potential of cladribine, it is important that clinicians are aware of the existence of NT-inhibiting medicinal products, various lifestyle drugs, and food components. This article reviews the existing knowledge on inhibitors of NT, which may alter cladribine absorption, distribution, and uptake into target cells, thereby summarizing the existing knowledge on optimized methods of administration and concomitant drugs that should be avoided during cladribine treatment.

A Body of Circumstantial Evidence for the Irreversible Ectonucleotidase Inhibitory Action of FSCPX, an Agent Known as a Selective Irreversible A1 Adenosine Receptor Antagonist So Far.

In previous studies using isolated, paced guinea pig left atria, we observed that FSCPX, known as a selective A1 adenosine receptor antagonist, paradoxically increased the direct negative inotropic response to A1 adenosine receptor agonists (determined using concentration/effect (E/c) curves) if NBTI, a nucleoside transport inhibitor, was present. Based on mathematical modeling, we hypothesized that FSCPX blunted the cardiac interstitial adenosine accumulation in response to nucleoside transport blockade, probably by inhibiting CD39 and/or CD73, which are the two main enzymes of the interstitial adenosine production in the heart. The goal of the present study was to test this hypothesis. In vitro CD39 and CD73 inhibitor assays were carried out; furthermore, E/c curves were constructed in isolated, paced rat and guinea pig left atria using adenosine, CHA and CPA (two A1 adenosine receptor agonists), FSCPX, NBTI and NBMPR (two nucleoside transport inhibitors), and PSB-12379 (a CD73 inhibitor), measuring the contractile force. We found that FSCPX did not show any inhibitory effect during the in vitro enzyme assays. However, we successfully reproduced the paradox effect of FSCPX in the rat model, mimicked the “paradox” effect of FSCPX with PSB-12379, and demonstrated the lipophilia of FSCPX, which could explain the negative outcome of inhibitor assays with CD39 and CD73 dissolved in a water-based solution. Taken together, these three pieces of indirect evidence are strong enough to indicate that FSCPX possesses an additional action besides the A1 adenosine receptor antagonism, which action may be the inhibition of an ectonucleotidase. Incidentally, we found that POM-1 inhibited CD73, in addition to CD39.

Review of Transporter Substrate, Inhibitor, and Inducer Characteristics of Cladribine.

Cladribine is a nucleoside analog that is phosphorylated in its target cells (B- and T-lymphocytes) to its active adenosine triphosphate form (2-chlorodeoxyadenosine triphosphate). Cladribine tablets 10 mg (Mavenclad®) administered for up to 10 days per year in 2 consecutive years (3.5-mg/kg cumulative dose over 2 years) are used to treat patients with relapsing multiple sclerosis. The ATP-binding cassette, solute carrier, and nucleoside transporter substrate, inhibitor, and inducer characteristics of cladribine are reviewed in this article. Available evidence suggests that the distribution of cladribine across biological membranes is facilitated by a number of uptake and efflux transporters. Among the key ATP-binding cassette efflux transporters, only breast cancer resistance protein has been shown to be an efficient transporter of cladribine, while P-glycoprotein does not transport cladribine well. Intestinal absorption, distribution throughout the body, and intracellular uptake of cladribine appear to be exclusively mediated by equilibrative and concentrative nucleoside transporters, specifically by ENT1, ENT2, ENT4, CNT2 (low affinity), and CNT3. Renal excretion of cladribine appears to be most likely driven by breast cancer resistance protein, ENT1, and P-glycoprotein. The latter may play a role despite its poor cladribine transport efficiency in view of the renal abundance of P-glycoprotein. There is no evidence that solute carrier uptake transporters such as organic anion transporting polypeptides, organic anion transporters, and organic cation transporters are involved in the transport of cladribine. Available in vitro studies examining the inhibitor characteristics of cladribine for a total of 13 major ATP-binding cassette, solute carrier, and CNT transporters indicate that in vivo inhibition of any of these transporters by cladribine is unlikely.

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.

A Randomized, Placebo-Controlled Crossover Study with Dipyridamole for Restless Legs Syndrome.

ABSTRACTBackgroundNew pharmacological targets are needed for restless legs syndrome. Preclinical data suggest that a hypoadenosinergic state plays an important pathogenetic role.ObjectiveThe objective of this study was to determine whether inhibitors of equilibrative nucleoside transporters, for example, dipyridamole, could provide effective symptomatic treatment.MethodsA 2‐week double‐blind, placebo‐controlled crossover study assessed the efficacy of dipyridamole (possible up‐titration to 300 mg) in untreated patients with idiopathic restless legs syndrome. Multiple suggested immobilization tests and polysomnography were performed after each treatment phase. Severity was assessed weekly using the International Restless Legs Rating Scale, Clinical Global Impression, and the Medical Outcomes Study Sleep scale. The primary end point was therapeutic response.ResultsTwenty‐eight of 29 patients recruited were included. International Restless Legs Rating Scale scores improved from a mean ± standard deviation of 24.1 ± 3.1 at baseline to 11.1 ± 2.3 at the end of week 2, versus 23.7 ± 3.4 to 18.7 ± 3.2 under placebo (P < 0.001). Clinical Global Impression, Medical Outcomes Study Sleep, and Multiple Suggested Immobilization Test scores all improved (P < 0.001). The mean effective dose of dipyridamole was 217.8 ± 33.1 mg/d. Sleep variables improved. The mean periodic leg movement index at the end of treatment with dipyridamole was 8.2 ± 3.5 versus. 28.1 ± 6.7 under placebo. Side effects (dipyridamole vs placebo) included abdominal distension (18% vs. 7%), dizziness (10.7% vs 7.1%), diarrhea, and asthenia (each 7.1% vs 3.6%).ConclusionsDipyridamole has significant therapeutic effects on both sensory and motor symptoms of restless legs syndrome and on sleep. Our findings confirm the efficacy of dipyridamole in restless legs syndrome predicted from preclinical studies and support a key role of adenosine in restless legs syndrome. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

Investigating the Role of Guanosine on Human Neuroblastoma Cell Differentiation and the Underlying Molecular Mechanisms

Neuroblastoma arises from neural crest cell precursors failing to complete the process of differentiation. Thus, agents helping tumor cells to differentiate into normal cells can represent a valid therapeutic strategy. Here, we evaluated whether guanosine (GUO), a natural purine nucleoside, which is able to induce differentiation of many cell types, may cause the differentiation of human neuroblastoma SH-SY5Y cells and the molecular mechanisms involved. We found that GUO, added to the cell culture medium, promoted neuron-like cell differentiation in a time- and concentration-dependent manner. This effect was mainly due to an extracellular GUO action since nucleoside transporter inhibitors reduced but not abolished it. Importantly, GUO-mediated neuron-like cell differentiation was independent of adenosine receptor activation as it was not altered by the blockade of these receptors. Noteworthy, the neuritogenic activity of GUO was not affected by blocking the phosphoinositide 3-kinase pathway, while it was reduced by inhibitors of protein kinase C or soluble guanylate cyclase. Furthermore, the inhibitor of the enzyme heme oxygenase-1 but not that of nitric oxide synthase reduced GUO-induced neurite outgrowth. Interestingly, we found that GUO was largely metabolized into guanine by the purine nucleoside phosphorylase (PNP) enzyme released from cells. Taken together, our results suggest that GUO, promoting neuroblastoma cell differentiation, may represent a potential therapeutic agent; however, due to its spontaneous extracellular metabolism, the role played by the GUO-PNP-guanine system needs to be further investigated.

The Dihydroorotate Dehydrogenase Inhibitor Brequinar Is Synergistic with ENT1/2 Inhibitors.

The dihydroorotate dehydrogenase (DHODH) inhibitor brequinar failed all clinical trials for solid tumors. To investigate mechanisms to increase brequinar's efficacy, we employed a combination strategy to simultaneously inhibit the nucleotide salvage pathways. Brequinar is synergistic with the equilibrative nucleoside transporter (ENT) inhibitor dipyridamole, but not the concentrative nucleoside transporter inhibitor phlorizin. This synergy carries over to ENT1/2 inhibition, but not ENT4. Our previously described brequinar analogue 41 was also synergistic with dipyridamole as were the FDA-approved DHODH inhibitors leflunomide and teriflunomide but the latter required much higher concentrations than brequinar. Therefore, a combination of brequinar and ENT inhibitors presents a potential anti-cancer strategy in select tumors.

Transport of ribavirin in human myelogenous leukemia cell line K562.

The anticancer effect of ribavirin, a purine nucleoside analogue, has been studied using cultured cancer cells such as the human myelogenous leukemia cell line K562. In order to exert its pharmacological effect, ribavirin has to enter cancer cells. However, there is little information concerning the transport mechanism of ribavirin into K562 cells. In this study, therefore, we examined the uptake mechanism of ribavirin in K562 cells. The uptake of ribavirin in K562 cells was time- and temperature-dependent, and was saturable with a Km value of 1.5 mM. Ribavirin uptake was inhibited by nucleosides such as adenosine and uridine, and by inhibitors of equilibrative nucleoside transporter 1 (ENT1) such as S-(4-nitrobenzyl)-6-thioinosine and dipyridamole in a concentration-dependent manner. In addition, the expression of ENT1 mRNA in K562 cells was confirmed by real-time PCR. On the other hand, Na+-dependence of ribavirin uptake was not observed, suggesting the involvement of ENT1, but not Na+-dependent concentrative nucleoside transporters, in ribavirin uptake in K562 cells. Treatment of K562 cells with sodium butyrate induced erythroid differentiation, but ribavirin uptake activity and sensitivity of the uptake to various inhibitors were not different between native and differentiated K562 cells. These results suggest that ribavirin uptake into K562 cells is mainly mediated by ENT1, which may have a pivotal role in anticancer effect of ribavirin.

Nucleoside transporters are critical to the uptake and antioxidant activity of 7,8-dihydroneopterin in monocytic cells

7,8-Dihydroneopterin protects cells intracellularly from oxidative stress-induced death, but its mode of transport across the cell membrane is unknown. Nucleosides, such as guanosine, are transported via nucleoside transporters of the equilibrative and concentrative forms. Therefore, the objective of this study was to identify which membrane transporters are responsible for 7,8-dihydroneopterin transport in cells and whether this is necessary for protection against oxidative stress. Monocytic cell lines U937, THP-1 and human monocytes were incubated with varying concentrations of 7,8-dihydroneopterin with or without nucleoside transporter inhibitors nitrobenzylthioinosine (NBMPR; ENT1), dipyridamole (DP; ENT1 and ENT2) or indomethacin (INDO; CNT). Only DP inhibited 7,8-dihydroneopterin uptake in U937 cells, while NBMPR and DP inhibited 7,8-dihydroneopterin uptake in THP-1 cells. All three inhibitors limited 7,8-dihydroneopterin uptake in human monocytes at short time points only. When the cells were incubated with 10 mM of the peroxyl radical generator 2,2'-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) a 50–80% loss of cell viability was measured. 7,8-dihydroneopterin protected all cell lines against AAPH-induced cell death, which was prevented with DP in U937 cells, NBMPR in THP-1 cells and a combination of all three nucleoside inhibitors in human monocytes. These data indicate 7,8-dihydroneopterin is transported across the cell membrane of monocytic cells via equilibrative and concentrative nucleoside transporters in a cell lineage-dependent manner. The data also indicate protection from peroxyl radical-generated cell death with 7,8-dihydroneopterin is intracellular and facilitated through nucleoside transporters in monocytic cells.

Apoptotic and anti-proliferative effect of guanosine and guanosine derivatives in HuT-78 T lymphoma cells

The effects of 100 μM of 3′,5′-cGMP, cAMP, cCMP, and cUMP as well as of the corresponding membrane-permeant acetoxymethyl esters on anti-CD3-antibody (OKT3)-induced IL-2 production of HuT-78 cutaneous T cell lymphoma (Sézary lymphoma) cells were analyzed. Only 3′,5′-cGMP significantly reduced IL-2 production. Flow cytometric analysis of apoptotic (propidium iodide/annexin V staining) and anti-proliferative (CFSE staining) effects revealed that 3′,5′-cGMP concentrations > 50 μM strongly inhibited proliferation and promoted apoptosis of HuT-78 cells (cultured in the presence of αCD3 antibody). Similar effects were observed for the positional isomer 2′,3′-cGMP and for 2′,-GMP, 3′-GMP, 5′-GMP, and guanosine. By contrast, guanosine and guanosine-derived nucleotides had no cytotoxic effect on peripheral blood mononuclear cells (PBMCs) or acute lymphocytic leukemia (ALL) xenograft cells. The anti-proliferative and apoptotic effects of guanosine and guanosine-derived compounds on HuT-78 cells were completely eliminated by the nucleoside transport inhibitor NBMPR (S-(4-Nitrobenzyl)-6-thioinosine). By contrast, the ecto-phosphodiesterase inhibitor DPSPX (1,3-dipropyl-8-sulfophenylxanthine) and the CD73 ecto-5′-nucleotidase inhibitor AMP-CP (adenosine 5′-(α,β-methylene)diphosphate) were not protective. We hypothesize that HuT-78 cells metabolize guanosine-derived nucleotides to guanosine by yet unknown mechanisms. Guanosine then enters the cells by an NBMPR-sensitive nucleoside transporter and exerts cytotoxic effects. This transporter may be ENT1 because NBMPR counteracted guanosine cytotoxicity in HuT-78 cells with nanomolar efficacy (IC50 of 25–30 nM). Future studies should further clarify the mechanism of the observed effects and address the question, whether guanosine or guanosine-derived nucleotides may serve as adjuvants in the therapy of cancers that express appropriate nucleoside transporters and are sensitive to established nucleoside-derived cytostatic drugs.

S-(4-Nitrobenzyl)-6-thioinosine (NBMPR) is Not a Selective Inhibitor of Equilibrative Nucleoside Transporters but Also Blocks Efflux Activity of Breast Cancer Resistance Protein.

S-(4-Nitrobenzyl)-6-thioinosine (NBMPR) is routinely used at concentrations of 0.10μM and 0.10mM to specifically inhibit transport of nucleosides mediated by equilibrative nucleoside transporters 1 (ENT1) and 2 (ENT2), respectively. We recently showed that NBMPR (0.10mM) might also inhibit placental active efflux of [3H]zidovudine and [3H]tenofovir disoproxil fumarate. Here we test the hypothesis that NBMPR abolishes the activity of P-glycoprotein (ABCB1) and/or breast cancer resistance protein (ABCG2). We performed accumulation assays with Hoechst 33342 (a model dual substrate of ABCB1 and ABCG2) and bi-directional transport studies with the ABCG2 substrate [3H]glyburide in transduced MDCKII cells, accumulation studies in choriocarcinoma-derived BeWo cells, and in situ dual perfusions of rat term placenta with glyburide. NBMPR inhibited Hoechst 33342 accumulation in MDCKII-ABCG2 cells (IC50 = 53μM) but not in MDCKII-ABCB1 and MDCKII-parental cells. NBMPR (0.10mM) also inhibited bi-directional [3H]glyburide transport across monolayers of MDCKII-ABCG2 cells and blocked ABCG2-mediated [3H]glyburide efflux by rat term placenta in situ. NBMPR at a concentration of 0.10mM abolishes ABCG2 activity. Researchers using NBMPR to evaluate the effect of ENTs on pharmacokinetics must therefore interpret their results carefully if studying compounds that are substrates of both ENTs and ABCG2.

Mutual role of ecto-5'-nucleotidase/CD73 and concentrative nucleoside transporter 3 in the intestinal uptake of dAMP.

2'-Deoxyadenosine 5'-monophosphate (dAMP), a deoxyribonucleotide found in DNA, affects intestinal cell growth. The molecular mechanisms underlying gastrointestinal absorption of foreign DNA ingested along with food has hardly been investigated. The aim of this study was to investigate the mechanism underlying intestinal absorption of dAMP. The uptake of [3H]dAMP by Caco-2 cells was Na+- and pH-dependent and was inhibited by various nucleosides. In contrast, nitrobenzylthioinosine (NMBPR), an equilibrative nucleoside transporter inhibitor, showed little inhibitory effects on [3H]dAMP uptake. Additionally, human concentrative nucleoside transporter (CNT) 3, transiently expressed in COS-7 cells, mediated the uptake of [3H]dAMP. A kinetic study revealed that the Km value of CNT3-mediated uptake of dAMP (59.6 μM) was close to that of 2'-deoxyadenosine (dAdo) (56.3 μM), whereas the dAMP Vmax (15.6 pmol·mg protein–1min–1) was 500-fold lesser than the dAdo Vmax (7782 pmol·mg protein–1min–1). Further, [3H]dAMP uptake was greater in COS-7 cells expressing ecto-5'-nucleotidase/CD73 with CNT3 than in those expressing CNT3 alone. These data suggest that, although dAMP is a substrate of CNT3, it is dephosphorylated to dAdo by CD73 and is efficiently absorbed as dAdo from the intestinal lumen.