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.

Vortex dynamics in NbTi films at high frequency and high DC magnetic fields.

We report on the characterization of NbTi films at [Formula: see text] 11 GHz and in DC magnetic fields up to 4 T, performed by means of the coplanar waveguide resonator technique, providing quantitative information about the penetration depth, the complex impedance, and the vortex-motion-induced complex resistivity. This kind of characterization is essential for the development of radiofrequency cavity technology. To access the vortex-pinning parameters, the complex impedance was analyzed within the formalism of the Campbell penetration depth. Measurements in this frequency range allowed us to determine the complete set of vortex-pinning parameters and the flux flow resistivity, both analyzed and discussed in the framework of high-frequency vortex dynamics models. The analysis also benefits from the comparison with results obtained by a dielectric-loaded resonator technique on similar samples and by other ancillary structural and electromagnetic characterization techniques that provide us with a comprehensive picture of the material. It turns out that the normalized flux flow resistivity follows remarkably well the trend predicted by the time dependent Ginzburg-Landau theory, while the pinning constant exhibits a decreasing trend with the field which points to a collective pinning regime.

Diminishing hERG inhibitory activity of aminopiperidine-naphthyridine linked NBTI antibacterials by structural and physicochemical optimizations.

Novel bacterial topoisomerase inhibitors (NBTIs) are an important new class of antibacterials targeting bacterial type II topoisomerases (DNA gyrase and topoisomerase IV). Notwithstanding their potent antibacterial activity, they suffer from a detrimental class-related hERG blockage. In this study, we designed and synthesized an optimized library of NBTIs comprising different linker moieties that exhibit reduced hERG inhibition and retain inhibitory potencies on DNA gyrase and topoisomerase IV of Staphylococcus aureus and Escherichia coli, respectively, as well as potent antibacterial activities. Substitution of the linker's tertiary amine with polar groups outcome in diminished hERG inhibition. Compound 17 expresses nanomolar enzyme inhibitory potency and antibacterial activity against both Gram-positive and Gram-negative bacteria as well as reduced hERG inhibition relative to our previously published NBTI analogs. Here, we point to some important NBTI's structural features that influence their hERG inhibitory activity.

Structure activity relationship of N-1 substituted 1,5-naphthyrid-2-one analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-9).

Novel bacterial topoisomerase inhibitors (NBTIs) are the newest members of gyrase inhibitor broad-spectrum antibacterial agents, represented by the most advanced member, gepotidacin, a 4-amino-piperidine linked NBTI, which is undergoing phase III clinical trials for treatment of urinary tract infections (UTI). We have extensively reported studies on oxabicyclooctane linked NBTIs, including AM-8722. The present study summarizes structure activity relationship (SAR) of AM-8722 leading to identification of 7-fluoro-1-cyanomethyl-1,5-naphthyridin-2-one based NBTI (16, AM-8888) with improved potency and spectrum (MIC values of 0.016-4 µg/mL), with Pseudomonas aeruginosa being the least sensitive strain (MIC 4 µg/mL).

Isolation of Thioinosine and Butenolides from a Terrestrial Actinomycetes sp. GSCW-51 and Their in Silico Studies for Potential against SARS-CoV-2.

In our continuous screening for bioactive microbial natural products, the culture extracts of a terrestrial Actinomycetes sp. GSCW-51 yielded two new metabolites, i. e., 5-hydroxymethyl-3-(1-hydroxy-6-methyl-7-oxooctyl)dihydrofuran-2(3H)-one (1), 5-hydroxymethyl-3-(1,7-dihydroxy-6-methyloctyl)dihydrofuran-2(3H)-one (2), and two known compounds; 5'-methylthioinosine (3), and 5'-methylthioinosine sulfoxide (4), which are isolated first time from any natural source, along with four known compounds (5-8). The structures of the new compounds were deduced by HR-ESI-MS, 1D and 2D NMR data, and in comparison with related compounds from the literature. Additionally, owing to the current COVID-19 pandemic situation, we also computationally explored the therapeutic potential of our isolated compounds against SARS-CoV-2. Compound 4 showed the best binding energies of -6.2 and -6.6 kcal/mol for Mpro and spike proteins, respectively. The intermolecular interactions were also studied using 2-D and 3-D imagery, which also supported the binding energies as well as put several insights under the spotlight. Furthermore, Lipinski's rule of 5 was used to predict the drug likeness of compounds 1-4, which indicated all compounds obey Lipinski's rule of 5. The study of bioavailability radars of the compounds 1-4 also confirmed their drug likeness properties where all the five crucial drug likeness parameters are in color area, which is safe to be used as drugs. Our isolation and computational findings highly encourage the scientific community to do further in vitro and in vivo studies of compounds 1-4.

A Mycobacterium tuberculosis NBTI DNA Gyrase Inhibitor Is Active against Mycobacterium abscessus.

Fluoroquinolones-the only clinically used DNA gyrase inhibitors-are effective against tuberculosis (TB) but are in limited clinical use for nontuberculous mycobacteria (NTM) lung infections due to intrinsic drug resistance. We sought to test alternative DNA gyrase inhibitors for anti-NTM activity. Mycobacterium tuberculosis gyrase inhibitors (MGIs), a subclass of novel bacterial topoisomerase inhibitors (NBTIs), were recently shown to be active against the tubercle bacillus. Here, we show that the MGI EC/11716 not only has potent anti-tubercular activity but is active against M. abscessus and M. avium in vitro. Focusing on M. abscessus, which causes the most difficult to cure NTM disease, we show that EC/11716 is bactericidal, active against drug-tolerant biofilms, and efficacious in a murine model of M. abscessus lung infection. Based on resistant mutant selection experiments, we report a low frequency of resistance to EC/11716 and confirm DNA gyrase as its target. Our findings demonstrate the potential of NBTIs as anti-M. abscessus and possibly broad-spectrum anti-mycobacterial agents.

NBTI attenuates neuroinflammation and apoptosis partly by ENT1/NLRP3/Bcl2 pathway after subarachnoid hemorrhage in rats.

OBJECTIVES: Neuroinflammation and apoptosis are two key factors contributing to early brain injury (EBI) after subarachnoid hemorrhage (SAH) and are strongly associated with a poor prognosis. Recently, equilibrative nucleoside transporter 1 (ENT1) was emerged to accelerate the severity of inflammation and cell apoptosis in several nervous system diseases, including cerebral ischemia, neurodegeneration and epilepsy. However, no study has yet elaborated the expression levels and effects of ENT1 in EBI after SAH. METHODS: Sprague-Dawley rats were subjected to SAH by endovascular perforation. Nitrobenzylthioinosine (NBTI) was intranasally administered at 0.5 h after SAH. The protein expression levels of ENT1, NLRP3, Bcl2, Bax, ACS, Caspase-1, IL-1 were detected by western blot. The modified Garcia score and beam balance score were employed to evaluate the neurologic function of rats following SAH. In addition, hematoxylin-eosin, fluoro-jade C and TdT-mediated dUTP nick-end labeling staining were then used to evaluate brain tissue damage and neuronal apoptosis. RESULTS: Analysis indicated that endogenous levels of ENT1 were significantly upregulated at 24-hour post-SAH, accompanied by NLRP3 inflammasome activation and Bcl2 decline. The administration of NBTI, an inhibitor of ENT1, at a dose of 15 mg/kg, ameliorated neurologic deficits and morphologic lesions at both 24 and 72 h after SAH. Moreover, ENT1 inhibition efficiently mitigated neuronal degeneration and cell apoptosis. In addition, NBTI at 15 mg/kg observably increased Bcl2 content and decreased Bax level. Furthermore, suppression of ENT1 notably reduced the expression levels of NLRP3, apoptosis associated speck like protein containing CARD, caspase-1 and IL-1ß. CONCLUSIONS: NBTI relieved SAH-induced EBI partly through ENT1/NLRP3/Bcl2 pathway.

Testicular disposition of clofarabine in rats is dependent on equilibrative nucleoside transporters.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5-year survival rate is high, some patients respond poorly to chemotherapy or have recurrence in locations such as the testis. The blood-testis barrier (BTB) can prevent complete eradication by limiting chemotherapeutic access and lead to testicular relapse unless a chemotherapeutic is a substrate of drug transporters present at this barrier. Equilibrative nucleoside transporter (ENT) 1 and ENT2 facilitate the movement of substrates across the BTB. Clofarabine is a nucleoside analog used to treat relapsed or refractory ALL. This study investigated the role of ENTs in the testicular disposition of clofarabine. Pharmacological inhibition of the ENTs by 6-nitrobenzylthioinosine (NBMPR) was used to determine ENT contribution to clofarabine transport in primary rat Sertoli cells, in human Sertoli cells, and across the rat BTB. The presence of NBMPR decreased clofarabine uptake by 40% in primary rat Sertoli cells (p = .0329) and by 53% in a human Sertoli cell line (p = .0899). Rats treated with 10 mg/kg intraperitoneal (IP) injection of the NBMPR prodrug, 6-nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), or vehicle, followed by an intravenous (IV) bolus 10 mg/kg dose of clofarabine, showed a trend toward a lower testis concentration of clofarabine than vehicle (1.81 ± 0.59 vs. 2.65 ± 0.92 ng/mg tissue; p = .1160). This suggests that ENTs could be important for clofarabine disposition. Clofarabine may be capable of crossing the human BTB, and its potential use as a first-line treatment to avoid testicular relapse should be considered.

Multiple Computational Approaches for Predicting Drug Interactions with Human Equilibrative Nucleoside Transporter 1.

Equilibrativenucleoside transporters (ENTs) participate in the pharmacokinetics and disposition of nucleoside analog drugs. Understanding drug interactions with the ENTs may inform and facilitate the development of new drugs, including chemotherapeutics and antivirals that require access to sanctuary sites such as the male genital tract. This study created three-dimensional pharmacophores for ENT1 and ENT2 substrates and inhibitors using Kt and IC50 data curated from the literature. Substrate pharmacophores for ENT1 and ENT2 are distinct, with partial overlap of hydrogen bond donors, whereas the inhibitor pharmacophores predominantly feature hydrogen bond acceptors. Mizoribine and ribavirin mapped to the ENT1 substrate pharmacophore and proved to be substrates of the ENTs. The presence of the ENT-specific inhibitor 6-S-[(4-nitrophenyl)methyl]-6-thioinosine (NBMPR) decreased mizoribine accumulation in ENT1 and ENT2 cells (ENT1, ∼70% decrease, P = 0.0046; ENT2, ∼50% decrease, P = 0.0012). NBMPR also decreased ribavirin accumulation in ENT1 and ENT2 cells (ENT1: ∼50% decrease, P = 0.0498; ENT2: ∼30% decrease, P = 0.0125). Darunavir mapped to the ENT1 inhibitor pharmacophore and NBMPR did not significantly influence darunavir accumulation in either ENT1 or ENT2 cells (ENT1: P = 0.28; ENT2: P = 0.53), indicating that darunavir's interaction with the ENTs is limited to inhibition. These computational and in vitro models can inform compound selection in the drug discovery and development process, thereby reducing time and expense of identification and optimization of ENT-interacting compounds. SIGNIFICANCE STATEMENT: This study developed computational models of human equilibrative nucleoside transporters (ENTs) to predict drug interactions and validated these models with two compounds in vitro. Identification and prediction of ENT1 and ENT2 substrates allows for the determination of drugs that can penetrate tissues expressing these transporters.

Predicting Drug Interactions with Human Equilibrative Nucleoside Transporters 1 and 2 Using Functional Knockout Cell Lines and Bayesian Modeling.

Equilibrative nucleoside transporters (ENTs) 1 and 2 facilitate nucleoside transport across the blood-testis barrier (BTB). Improving drug entry into the testes with drugs that use endogenous transport pathways may lead to more effective treatments for diseases within the reproductive tract. In this study, CRISPR/CRISPR-associated protein 9 was used to generate HeLa cell lines in which ENT expression was limited to ENT1 or ENT2. We characterized uridine transport in these cell lines and generated Bayesian models to predict interactions with the ENTs. Quantification of [3H]uridine uptake in the presence of the ENT-specific inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBMPR) demonstrated functional loss of each transporter. Nine nucleoside reverse-transcriptase inhibitors and 37 nucleoside/heterocycle analogs were evaluated to identify ENT interactions. Twenty-one compounds inhibited uridine uptake and abacavir, nevirapine, ticagrelor, and uridine triacetate had different IC50 values for ENT1 and ENT2. Total accumulation of four identified inhibitors was measured with and without NBMPR to determine whether there was ENT-mediated transport. Clofarabine and cladribine were ENT1 and ENT2 substrates, whereas nevirapine and lexibulin were ENT1 and ENT2 nontransported inhibitors. Bayesian models generated using Assay Central machine learning software yielded reasonably high internal validation performance (receiver operator characteristic > 0.7). ENT1 IC50-based models were generated from ChEMBL; subvalidations using this training data set correctly predicted 58% of inhibitors when analyzing activity by percent uptake and 63% when using estimated-IC50 values. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can thereby circumvent the BTB through this transepithelial transport pathway in Sertoli cells. SIGNIFICANCE STATEMENT: This study is the first to predict drug interactions with equilibrative nucleoside transporter (ENT) 1 and ENT2 using Bayesian modeling. Novel CRISPR/CRISPR-associated protein 9 functional knockouts of ENT1 and ENT2 in HeLa S3 cells were generated and characterized. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can circumvent the blood-testis barrier through this transepithelial transport pathway in Sertoli cells.

S-(4-nitrobenzyl)-6-thioinosine: Tritium labelling at high specific activity.

The one-step tritiation of S-(4-nitrobenzyl)-6-thioinosine is described with characterization of the product by tritium NMR as well as mass spectrometry. The storage, stability, and repurification of [benzyl methylene-3 H]S-(4-nitrobenzyl)-6-thioinosine are also discussed.

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.

PURPOSE: S-(4-Nitrobenzyl)-6-thioinosine (NBMPR) is routinely used at concentrations of 0.10 µM and 0.10 mM to specifically inhibit transport of nucleosides mediated by equilibrative nucleoside transporters 1 (ENT1) and 2 (ENT2), respectively. We recently showed that NBMPR (0.10 mM) 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). METHODS: 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. RESULTS: NBMPR inhibited Hoechst 33342 accumulation in MDCKII-ABCG2 cells (IC50 = 53 µM) but not in MDCKII-ABCB1 and MDCKII-parental cells. NBMPR (0.10 mM) 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. CONCLUSION: NBMPR at a concentration of 0.10 mM 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.

Blockade of equilibrative nucleoside transporter 1/2 protects against Pseudomonas aeruginosa-induced acute lung injury and NLRP3 inflammasome activation.

Pseudomonas aeruginosa infections are increasingly multidrug resistant and cause healthcare-associated pneumonia, a major risk factor for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Adenosine is a signaling nucleoside with potential opposing effects; adenosine can either protect against acute lung injury via adenosine receptors or cause lung injury via adenosine receptors or equilibrative nucleoside transporter (ENT)-dependent intracellular adenosine uptake. We hypothesized that blockade of intracellular adenosine uptake by inhibition of ENT1/2 would increase adenosine receptor signaling and protect against P. aeruginosa-induced acute lung injury. We observed that P. aeruginosa (strain: PA103) infection induced acute lung injury in C57BL/6 mice in a dose- and time-dependent manner. Using ENT1/2 pharmacological inhibitor, nitrobenzylthioinosine (NBTI), and ENT1-null mice, we demonstrated that ENT blockade elevated lung adenosine levels and significantly attenuated P. aeruginosa-induced acute lung injury, as assessed by lung wet-to-dry weight ratio, BAL protein levels, BAL inflammatory cell counts, pro-inflammatory cytokines, and pulmonary function (total lung volume, static lung compliance, tissue damping, and tissue elastance). Using both agonists and antagonists directed against adenosine receptors A2AR and A2BR, we further demonstrated that ENT1/2 blockade protected against P. aeruginosa -induced acute lung injury via activation of A2AR and A2BR. Additionally, ENT1/2 chemical inhibition and ENT1 knockout prevented P. aeruginosa-induced lung NLRP3 inflammasome activation. Finally, inhibition of inflammasome prevented P. aeruginosa-induced acute lung injury. Our results suggest that targeting ENT1/2 and NLRP3 inflammasome may be novel strategies for prevention and treatment of P. aeruginosa-induced pneumonia and subsequent ARDS.

Role of equilibrative nucleoside transporter 1 (ENT1) in the disposition of cytarabine in mice.

Cytarabine (Ara-C) is a nucleoside analog used in the treatment of acute myeloid leukemia (AML). Despite the many years of clinical use, the identity of the transporter(s) involved in the disposition of Ara-C remains poorly studied. Previous work demonstrated that concurrent administration of Ara-C with nitrobenzylmercaptopurine ribonucleoside (NBMPR) causes an increase in Ara-C plasma levels, suggesting involvement of one or more nucleoside transporters. Here, we confirmed the presence of an NMBPR-mediated interaction with Ara-C resulting in a 2.5-fold increased exposure. The interaction was unrelated to altered blood cell distribution, and subsequent studies indicated that the disposition of Ara-C was unaffected in mice with a deficiency of postulated candidate transporters, including ENT1, OCTN1, OATP1B2, and MATE1. These studies indicate the involvement of an unknown NBMPR-sensitive Ara-C transporter that impacts the pharmacokinetic properties of this clinically important agent.

Structures of human ENT1 in complex with adenosine reuptake inhibitors.

The human equilibrative nucleoside transporter 1 (hENT1), a member of the SLC29 family, plays crucial roles in adenosine signaling, cellular uptake of nucleoside for DNA and RNA synthesis, and nucleoside-derived anticancer and antiviral drug transport in humans. Because of its central role in adenosine signaling, it is the target of adenosine reuptake inhibitors (AdoRI), several of which are used clinically. Despite its importance in human physiology and pharmacology, the molecular basis of hENT1-mediated adenosine transport and its inhibition by AdoRIs are limited, owing to the absence of structural information on hENT1. Here, we present crystal structures of hENT1 in complex with two chemically distinct AdoRIs: dilazep and S-(4-nitrobenzyl)-6-thioinosine (NBMPR). Combined with mutagenesis study, our structural analyses elucidate two distinct inhibitory mechanisms exhibited on hENT1 and provide insight into adenosine recognition and transport. Our studies provide a platform for improved pharmacological intervention of adenosine and nucleoside analog drug transport by hENT1.

Roles of organic anion transporter 2 and equilibrative nucleoside transporter 1 in hepatic disposition and antiviral activity of entecavir during non-pregnancy and pregnancy.

BACKGROUND AND PURPOSE: Entecavir (ETV), a first-line antiviral drug against hepatitis B virus (HBV), has the possibility to be used to prevent mother-to-child transmission. The aim of present study was to clarify the mechanism of ETV uptake into hepatocytes and evaluate the alteration of ETV's hepatic distribution during pregnancy. EXPERIMENTAL APPROACH: The roles of equilibrative nucleotide transporter (ENT) 1 and organic anion transporter (OAT) 2 in ETV accumulation and anti-HBV efficacy were studied in human ENT1 or OAT2 overexpressed cell models and HepG2.2.15 cells, respectively; meanwhile, the liver-to-plasma ETV concentration ratios in non-pregnant and pregnant mice were measured to evaluate the effect of pregnancy on ETV hepatic distribution. KEY RESULTS: ETV was shown to be a substrate of ENT1 and OAT2. An ENT1 inhibitor significantly decreased the efficacy of ETV in HepG2.2.15 cells, while overexpression of OAT2 increased susceptibility of HBV to ETV. The liver-to-plasma ETV concentration ratios in pregnant mice were sharply reduced; whereas, the absolute concentration of ETV in the liver did not obviously alter in pregnancy. Although oestradiol and progesterone showed a concentration-dependent inhibition on ETV accumulation both in hepatic cell lines and in primary human hepatocytes, a physiologically relevant concentration of oestradiol and progesterone did not affect antiviral activity of ETV. CONCLUSIONS AND IMPLICATIONS: OAT2 and ENT1 are the main transporters involved in the hepatic uptake and anti-HBV efficacy of ETV. The concentration of ETV in the liver was not obviously altered during pregnancy, which indicates that dosage adjustment in pregnancy is not necessary.

Inhibitions of human parainfluenza virus type 2 replication by ribavirin and mycophenolate mofetil are restored by guanosine and S-(4-nitrobenzyl)-6-thioinosine.

The antiviral activities of a nucleoside analog antiviral drug (ribavirin) and a non-nucleoside drug (mycophenolate mofetil) against human parainfluenza virus type 2 (hPIV-2) were investigated, and the restoration of the inhibition by guanosine and S-(4-nitrobenzyl)-6-thioinosine (NBTI: equilibrative nucleoside transporter 1 inhibitor) were also investigated. Ribavirin (RBV) and mycophenolate mofetil (MMF) inhibited cell fusion induced by hPIV-2. Both RBV and MMF considerably reduced the number of viruses released from the cells. Virus genome synthesis was inhibited by RBV and MMF as determined by polymerase chain reaction (PCR) and real time PCR. mRNA syntheses were also reduced. An indirect immunofluorescence study showed that RBV and MMF largely inhibited viral protein syntheses. Using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP), it was found that virus entry into the cells and multinucleated giant cell formation were almost completely blocked by RBV and MMF. RBV and MMF did not disrupt actin microfilaments or microtubules. Both guanosine and NBTI completely or partially reversed the inhibition by RBV and MMF in the viral replication, syntheses of genome RNA, mRNA and protein, and multinucleated giant cell formation. NBTI caused a little damage in actin microfilaments, but had no effect on microtubules. Both RBV and MMF inhibited the replication of hPIV-2, mainly by inhibiting viral genome RNA, mRNA and protein syntheses. The inhibition was almost completely recovered by guanosine. These results indicate that the major mechanism of the inhibition is the depletion of intracellular GTP pools.

FSCPX, a Chemical Widely Used as an Irreversible A1 Adenosine Receptor Antagonist, Modifies the Effect of NBTI, a Nucleoside Transport Inhibitor, by Reducing the Interstitial Adenosine Level in the Guinea Pig Atrium.

Based on in silico results, recently we have assumed that FSCPX, an irreversible A1 adenosine receptor antagonist, inhibits the action of NBTI that is apparent on E/c curves of adenosine receptor agonists. As a mechanism for this unexpected effect, we hypothesized that FSCPX might modify the equilibrative and NBTI-sensitive nucleoside transporter (ENT1) in a way that allows ENT1 to transport adenosine but impedes NBTI to inhibit this transport. This assumption implies that our method developed to estimate receptor reserve for agonists with short half-life such as adenosine, in its original form, overestimates the receptor reserve. In this study, therefore, our goals were to experimentally test our assumption on this effect of FSCPX, to improve our receptor reserve-estimating method and then to compare the original and improved forms of this method. Thus, we improved our method and assessed the receptor reserve for the direct negative inotropic effect of adenosine with both forms of this method in guinea pig atria. We have found that FSCPX inhibits the effects of NBTI that are mediated by increasing the interstitial concentration of adenosine of endogenous (but not exogenous) origin. As a mechanism for this action of FSCPX, inhibition of enzymes participating in the interstitial adenosine production can be hypothesized, while modification of ENT1 can be excluded. Furthermore, we have shown that, in comparison with the improved form, the original version of our method overestimates receptor reserve but only to a small extent. Nevertheless, use of the improved form is recommended in the future.

Role of cysteine 416 in N-ethylmaleimide sensitivity of human equilibrative nucleoside transporter 1 (hENT1).

Human equilibrative nucleoside transporter 1 (hENT1), the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for cellular uptake of physiologic nucleosides and many antineoplastic and antiviral nucleoside drugs. hENT1, which is potently inhibited by nitrobenzylthioinosine (NBMPR), possesses 11 transmembrane helical domains with an intracellular N-terminus and an extracellular C-terminus. As a protein with 10 endogenous cysteine residues, it is sensitive to inhibition by the membrane permeable sulfhydryl-reactive reagent N-ethylmaleimide (NEM) but is unaffected by the membrane impermeable sulfhydryl-reactive reagent p-chloromercuriphenyl sulfonate. To identify the residue(s) involved in NEM inhibition, we created a cysteine-less version of hENT1 (hENT1C-), with all 10 endogenous cysteine residues mutated to serine, and showed that it displays wild-type uridine transport and NBMPR-binding characteristics when produced in the Xenopus oocyte heterologous expression system, indicating that endogenous cysteine residues are not essential for hENT1 function. We then tested NEM sensitivity of recombinant wild-type hENT1, hENT1 mutants C1S to C10S (single cysteine residues replaced by serine), hENT1C- (all cysteine residues replaced by serine), and hENT1C- mutants S1C to S10C (single serine residues converted back to cysteine). Mutants C9S (C416S/hENT1) and S9C (S416C/hENT1C-) were insensitive and sensitive, respectively, to inhibition by NEM, identifying Cys416 as the endofacial cysteine residue in hENT1 responsible for NEM inhibition. Kinetic experiments suggested that NEM modification of Cys416, which is located at the inner extremity of TM10, results in the inhibition of hENT1 uridine transport and NBMPR binding by constraining the protein in its inward-facing conformation.