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.

Nucleoside transporter-guided cytarabine-conjugated liposomes for intracellular methotrexate delivery and cooperative choriocarcinoma therapy.

Gestational trophoblastic tumors seriously endanger child productive needs and the health of women in childbearing age. Nanodrug-based therapy mediated by transporters provides a novel strategy for the treatment of trophoblastic tumors. Focusing on the overexpression of human equilibrative nucleoside transporter 1 (ENT1) on the membrane of choriocarcinoma cells (JEG-3), cytarabine (Cy, a substrate of ENT1)-grafted liposomes (Cy-Lipo) were introduced for the targeted delivery of methotrexate (Cy-Lipo@MTX) for choriocarcinoma therapy in this study. ENT1 has a high affinity for Cy-Lipo and can mediate the endocytosis of the designed nanovehicles into JEG-3 cells. The ENT1 protein maintains its transportation function through circulation and regeneration during endocytosis. Therefore, Cy-Lipo-based formulations showed high tumor accumulation and retention in biodistribution studies. More importantly, the designed DSPE-PEG2k-Cy conjugation exhibited a synergistic therapeutic effect on choriocarcinoma. Finally, Cy-Lipo@MTX exerted an extremely powerful anti-choriocarcinoma effect with fewer side effects. This study suggests that the overexpressed ENT1 on choriocarcinoma cells holds great potential as a high-efficiency target for the rational design of active targeting nanotherapeutics.

Selective Inhibition of Human Equilibrative and Concentrative Nucleoside Transporters by BCR-ABL Kinase Inhibitors: IDENTIFICATION OF KEY hENT1 AMINO ACID RESIDUES FOR INTERACTION WITH BCR-ABL KINASE INHIBITORS.

Human nucleoside transporters (hNTs) mediate cellular influx of anticancer nucleoside drugs, including cytarabine, cladribine, and fludarabine. BCR-ABL tyrosine kinase inhibitors (TKIs) imatinib and dasatinib inhibit fludarabine and cytarabine uptake. We assessed interactions of bosutinib, dasatinib, imatinib, nilotinib, and ponatinib with recombinant hNTs (hENT1, 2; hCNT1, -2, and -3) produced individually in yeast Saccharomyces cerevisiae Nilotinib inhibited hENT1-mediated uridine transport most potently (IC50 value, 0.7 µm) followed by ponatinib > bosutinib > dasatinib > imatinib. Imatinib inhibited hCNT2 with an IC50 value of 2.3 µm Ponatinib inhibited all five hNTs with the greatest effect seen for hENT1 (IC50 value, 9 µm). TKIs inhibited [(3)H]uridine uptake in a competitive manner. Studies in yeast with mutants at two amino acid residues of hENT1 (L442I, L442T, M33A, M33A/L442I) previously shown to be involved in uridine and dipyridamole binding, suggested that BCR-ABL TKIs interacted with Met(33) (TM1) and Leu(442) (TM11) residues of hENT1. In cultured human CEM lymphoblastoid cells, which possess a single hNT type (hENT1), accumulation of [(3)H]cytarabine, [(3)H]cladribine, or [(3)H]fludarabine was reduced by each of the five TKIs, and also caused a reduction in cell surface expression of hENT1 protein. In conclusion, BCR-ABL TKIs variously inhibit five different hNTs, cause a decrease in cell surface hENT1 expression, and decrease uridine accumulation when presented together with uridine or when given before uridine. In experiments with mutant hENT1, we showed for the first time interaction of Met(33) (involved in dipyridamole binding) with BCR-ABL inhibitors and reduced interaction with M33A mutant hENT1.

Functional reconstitution of human equilibrative nucleoside transporter-1 into styrene maleic acid co-polymer lipid particles.

The human equilibrative nucleoside transporter-1 (hENT1) is important for the entry of anti-cancer and anti-viral nucleoside analog therapeutics into the cell, and thus for their efficacy. Understanding of hENT1 structure-function relationship could assist with development of nucleoside analogs with better cellular uptake properties. However, structural and biophysical studies of hENT1 remain challenging as the hydrophobic nature of the protein leads to complete aggregation upon detergent-based membrane isolation. Here we report detergent-free reconstitution of the hENT1 transporter into styrene maleic acid co-polymer lipid particles (SMALPs) that form a native lipid disc. SMALP-purified hENT1, expressed in Sf9 insect cells binds a variety of ligands with a similar affinity as the protein in native membrane, and exhibits increased thermal stability compared to detergent-solubilized hENT1. hENT1-SMALPs purified using FLAG affinity M2 resin yielded ~0.4mg of active and homogenous protein per liter of culture as demonstrated by ligand binding, size-exclusion chromatography and SDS-PAGE analyses. Electrospray ionization mass spectrometry (ESI-MS) analysis showed that each hENT1 lipid disc contains 16 phosphatidylcholine (PC) and 2 phosphatidylethanolamine (PE) lipid molecules. Polyunsaturated lipids are specifically excluded from the hENT1 lipid discs, possibly reflecting a functional requirement for a dynamic lipid environment. Our work demonstrates that human nucleoside transporters can be extracted and purified without removal from their native lipid environment, opening up a wide range of possibilities for their biophysical and structural studies.

Novel regulation of equlibrative nucleoside transporter 1 (ENT1) by receptor-stimulated Ca2+-dependent calmodulin binding.

Equilibrative nucleoside transporters (ENTs) facilitate the flux of nucleosides, such as adenosine, and nucleoside analog (NA) drugs across cell membranes. A correlation between adenosine flux and calcium-dependent signaling has been previously reported; however, the mechanistic basis of these observations is not known. Here we report the identification of the calcium signaling transducer calmodulin (CaM) as an ENT1-interacting protein, via a conserved classic 1-5-10 motif in ENT1. Calcium-dependent human ENT1-CaM protein interactions were confirmed in human cell lines (HEK293, RT4, U-87 MG) using biochemical assays (HEK293) and the functional assays (HEK293, RT4), which confirmed modified nucleoside uptake that occurred in the presence of pharmacological manipulations of calcium levels and CaM function. Nucleoside and NA drug uptake was significantly decreased (∼12% and ∼39%, respectively) by chelating calcium (EGTA, 50 µM; BAPTA-AM, 25 µM), whereas increasing intracellular calcium (thapsigargin, 1.5 µM) led to increased nucleoside uptake (∼26%). Activation of N-methyl-d-aspartate (NMDA) receptors (in U-87 MG) by glutamate (1 mM) and glycine (100 µM) significantly increased nucleoside uptake (∼38%) except in the presence of the NMDA receptor antagonist, MK-801 (50 µM), or CaM antagonist, W7 (50 µM). These data support the existence of a previously unidentified novel receptor-dependent regulatory mechanism, whereby intracellular calcium modulates nucleoside and NA drug uptake via CaM-dependent interaction of ENT1. These findings suggest that ENT1 is regulated via receptor-dependent calcium-linked pathways resulting in an alteration of purine flux, which may modulate purinergic signaling and influence NA drug efficacy.

Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells.

Human equilibrative nucleoside transporter-1 (hENT1) is the major plasma membrane transporter involved in transportation of natural nucleosides as well as nucleoside analog drugs, used in anti-cancer and anti-viral therapies. Despite extensive biochemical and pharmacological studies, little is known about the structure-function relationship of this protein. The major obstacles to purification include a low endogenous expression level, the lack of an efficient expression and purification protocol, and the hydrophobic nature of the protein. Here, we report protein expression, purification and functional characterization of hENT1 from Sf9 insect cells. hENT1 expressed by Sf9 cells is functionally active as demonstrated by saturation binding with a Kd of 1.2±0.2nM and Bmax of 110±5pmol/mg for [(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR). We also demonstrate purification of hENT1 using FLAG antibody affinity resin in lauryl maltose neopentyl glycol detergent with a Kd of 4.3±0.7nM. The yield of hENT1 from Sf9 cells was ∼0.5mg active transporter per liter of culture. The purified protein is functionally active, stable, homogenous and appropriate for further biophysical and structural studies.

Nucleobase transport by human equilibrative nucleoside transporter 1 (hENT1).

The human equilibrative nucleoside transporters hENT1 and hENT2 (each with 456 residues) are 40% identical in amino acid sequence and contain 11 putative transmembrane helices. Both transport purine and pyrimidine nucleosides and are distinguished functionally by a difference in sensitivity to inhibition by nanomolar concentrations of nitrobenzylmercaptopurine ribonucleoside (NBMPR), hENT1 being NBMPR-sensitive. Previously, we used heterologous expression in Xenopus oocytes to demonstrate that recombinant hENT2 and its rat ortholog rENT2 also transport purine and pyrimidine bases, h/rENT2 representing the first identified mammalian nucleobase transporter proteins (Yao, S. Y., Ng, A. M., Vickers, M. F., Sundaram, M., Cass, C. E., Baldwin, S. A., and Young, J. D. (2002) J. Biol. Chem. 277, 24938-24948). The same study also revealed lower, but significant, transport of hypoxanthine by h/rENT1. In the present investigation, we have used the enhanced Xenopus oocyte expression vector pGEMHE to demonstrate that hENT1 additionally transports thymine and adenine and, to a lesser extent, uracil and guanine. Fluxes of hypoxanthine, thymine, and adenine by hENT1 were saturable and inhibited by NBMPR. Ratios of V(max) (pmol/oocyte · min(-1)):K(m) (mm), a measure of transport efficiency, were 86, 177, and 120 for hypoxantine, thymine, and adenine, respectively, compared with 265 for uridine. Hypoxanthine influx was competitively inhibited by uridine, indicating common or overlapping nucleobase and nucleoside permeant binding pockets, and the anticancer nucleobase drugs 5-fluorouracil and 6-mercaptopurine were also transported. Nucleobase transport activity was absent from an engineered cysteine-less version hENT1 (hENT1C-) in which all 10 endogenous cysteine residues were mutated to serine. Site-directed mutagenesis identified Cys-414 in transmembrane helix 10 of hENT1 as the residue conferring nucleobase transport activity to the wild-type transporter.

The Equilibrative Nucleoside Transporter (ENT1) can be phosphorylated at multiple sites by PKC and PKA.

Equilibrative Nucleoside Transporters (SLC29) are a family of proteins that transport nucleosides, nucleobases and nucleoside analogue drugs across cellular membranes. ENT1 is expressed ubiquitously in mammalian tissues and responsible for a significant portion of nucleoside analog drug uptake in humans. Despite the important clinical role of ENT1, many aspects of the regulation of this protein remain unknown. A major outstanding question in this field is the whether ENT1 is phosphorylated directly. To answer this question, we overexpressed tagged human (h) and mouse (m) ENT1, affinity purified protein using the tag, conducted phosphoamino acid analysis and found that m/hENT1 is predominantly phosphorylated at serine residues. The large intracellular loop of ENT1, between transmembrane domains 6 and 7, has been suggested to be a site of regulation by phosphorylation, therefore we generated His/Ubiquitin tagged peptides of this region and used them for in vitro kinase assays to identify target serines. Our data support a role for PKA and PKC in the phosphorylation of ENT1 within the intracellular loop and show that PKA can phosphorylate multiple sites within this loop while PKC specifically targets serines 279 and 286 and threonine 274. These data demonstrate, for the first time, that ENT1 is a phosphoprotein that can be directly phosphorylated at several sites by more than one kinase. The presence of multiple kinase targets within the loop suggests that ENT1 phosphorylation is considerably more complex than previously thought and thus ENT1 may be subject to phosphorylation by multiple pathways.

Analysis of recombinant tagged equilibrative nucleoside transporter 1 (ENT1) expressed in E. coli.

Nucleoside transporters (NTs) are integral membrane proteins necessary for the cellular entry of nucleoside analog drugs used in chemotherapeutic treatment of conditions such as cancer and viral or parasitic infections. NTs are also the targets of certain drugs used in the treatment of various cardiovascular conditions. Because of the importance of NTs in drug uptake, determination of the three-dimensional structure of these proteins, particularly hENT1, has the potential to improve these treatments through structure-based design of more specifically targeted and transported drugs. In this paper, we use NMR spectroscopy to investigate the structure of the large intracellular loop between transmembrane domains 6 and 7 and we also describe a method for the successful overexpression of full-length hENT1 in a bacterial system. Recombinant tandem histidine-affinity (HAT) and 3×FLAG tagged hENT1 was overexpressed in E. coli, affinity purified, and functionally characterized by nitrobenzylthioinosine (NBTI) binding. Anti-3×FLAG immunodetection confirmed the expression of N-HAT-3×FLAG-hENT1, while increased NBTI binding (3.2-fold compared with controls) confirmed the conformational integrity of the recombinant hENT1 within the bacterial inner membrane. Yields of recombinant hENT1 using this approach were ~15 µg/L of bacterial culture and this approach provides a basis for large-scale production of protein for a variety of purposes.

Disrupted plasma membrane localization and loss of function reveal regions of human equilibrative nucleoside transporter 1 involved in structural integrity and activity.

Human Equilibrative Nucleoside Transporter 1 (hENT1) is an integral membrane protein that transports nucleosides and analog drugs across cellular membranes. Very little is known about intracellular processing and localization of hENT1. Here we show that disruption of a highly conserved triplet (PWN) near the N-terminus, or the last eight C-terminal residues (two hydrophobic triplets separated by a positive arginine) result in loss of plasma membrane localization and/or transport function. To understand the role of specific residues within these regions, we studied the localization patterns of N- or C-terminal deletion and/or substitution mutants of GFP-hENT1 using confocal microscopy. Quantification of GFP-hENT1 (mutant and wildtype) protein at the plasma membrane was conducted using nitrobenzylthioinosine (NBTI) binding. Functionality of the GFP-hENT1 mutants was determined by heterologous expression in Xenopus laevis oocytes followed by measurement of uridine uptake. Mutation of the proline within the PWN motif disrupts plasma membrane localization. C-terminal mutations (primarily within the hydrophobic triplets) lead to hENT1 retention within the cell (e.g. in the ER). Some mutants still localize to the plasma membrane but show reduced transport activity. These data suggest that these two regions contribute to the structural integrity and thus correct processing and function of hENT1.

Affinity, binding kinetics and functional characterization of draflazine analogues for human equilibrative nucleoside transporter 1 (SLC29A1).

In the last decade it has been recapitulated that receptor-ligand binding kinetics is a relevant additional parameter in drug discovery to improve in vivo drug efficacy and safety. The equilibrative nucleoside transporter-1 (ENT1, SLC29A1) is an important drug target, as transporter inhibition is a potential treatment of ischemic heart disease, stroke, and cancer. Currently, two non-selective ENT1 inhibitors (dilazep and dipyridamole) are on the market as vasodilators. However, their binding kinetics are unknown; moreover, novel, more effective and selective inhibitors are still needed. Hence, this study focused on the incorporation of binding kinetics for finding new and improved ENT1 inhibitors. We developed a radioligand competition association assay to determine the binding kinetics of ENT1 inhibitors with four chemical scaffolds (including dilazep and dipyridamole). The kinetic parameters were compared to the affinities obtained from a radioligand displacement assay. Three of the scaffolds presented high affinities with relatively fast dissociation kinetics, yielding short to moderate residence times (RTs) at the protein (1-44 min). While compounds from the fourth scaffold, i.e. draflazine analogues, also had high affinity, they displayed significantly longer RTs, with one analogue (4) having a RT of over 10 h. Finally, a label-free assay was used to evaluate the impact of divergent ENT1 inhibitor binding kinetics in a functional assay. It was shown that the potency of compound 4 increased with longer incubation times, which was not observed for draflazine, supporting the importance of long RT for increased target-occupancy and effect. In conclusion, our research shows that high affinity ENT1 inhibitors show a large variation in residence times at this transport protein. As a consequence, incorporation of binding kinetic parameters adds to the design criteria and may thus result in a different lead compound selection. Taken together, this kinetic approach could inspire future drug discovery in the field of ENT1 and membrane transport proteins in general.

IMPROvER: the Integral Membrane Protein Stability Selector.

Identifying stabilising variants of membrane protein targets is often required for structure determination. Our new computational pipeline, the Integral Membrane Protein Stability Selector (IMPROvER) provides a rational approach to variant selection by employing three independent approaches: deep-sequence, model-based and data-driven. In silico tests using known stability data, and in vitro tests using three membrane protein targets with 7, 11 and 16 transmembrane helices provided measures of success. In vitro, individual approaches alone all identified stabilising variants at a rate better than expected by random selection. Low numbers of overlapping predictions between approaches meant a greater success rate was achieved (fourfold better than random) when approaches were combined and selections restricted to the highest ranked sites. The mix of information IMPROvER uses can be extracted for any helical membrane protein. We have developed the first general-purpose tool for selecting stabilising variants of [Formula: see text]-helical membrane proteins, increasing efficiency and reducing workload. IMPROvER can be accessed at http://improver.ddns.net/IMPROvER/ .

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.

Characterization of mENT1Delta11, a novel alternative splice variant of the mouse equilibrative nucleoside transporter 1.

Mammalian cells require specific transport mechanisms for the cellular uptake and release of endogenous nucleosides such as adenosine, and nucleoside analogs used in chemotherapy. We have identified a novel splice variant of the mouse equilibrative nucleoside transporter, mENT1, that results from the exclusion of exon 11 during pre-RNA processing. This variant encodes a truncated protein (mENT1Delta11) missing the last three transmembrane domains of the full-length mENT1. The mENT1Delta11 transcript and protein were found to be differentially distributed among tissues relative to full-length mENT1. PK15-NTD (nucleoside transport deficient) cells were transfected with mENT1 or mENT1Delta11 and assessed for nucleoside transport function. No significant differences were observed between the mENT1 and mENT1Delta11 in terms of transport function or inhibitor binding affinity. PK15-mENT1Delta11 transfected cells bound the ENT1 probe [3H]nitrobenzylthioinosine (NBMPR) with high affinity and mediated the cellular accumulation of both [3H]2-chloroadenosine and [3H]uridine. The only significant differences between the mENT1 variants were that mENT1Delta11 could not be photolabeled with [3H]NBMPR and that mENT1Delta11 was insensitive to the transporter-modifying effects of N-ethylmaleimide. These data suggest that the last three transmembrane domains of mENT1 are not necessary for transport activity, but this region does contain the cysteines responsible for the sensitivity of mENT1 to sulfhydryl reagents, and the residues important for covalent modification of the protein with NBMPR. These results provide important guidelines for future mutagenesis studies aimed at elucidating the tertiary structure of the ENT1 protein and the domains involved in inhibitor binding and substrate translocation.

Synthetic single domain antibodies for the conformational trapping of membrane proteins.

Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.

Functional characterization of human equilibrative nucleoside transporter 1.

Equilibrative nucleoside transporters (ENTs), which facilitate cross-membrane transport of nucleosides and nucleoside-derived drugs, play an important role in the salvage pathways of nucleotide synthesis, cancer chemotherapy, and treatment for virus infections. Functional characterization of ENTs at the molecular level remains technically challenging and hence scant. In this study, we report successful purification and biochemical characterization of human equilibrative nucleoside transporter 1 (hENT1) in vitro. The HEK293F-derived, recombinant hENT1 is homogenous and functionally active in proteoliposome-based counter flow assays. hENT1 transports the substrate adenosine with a Km of 215 ± 34 µmol/L and a Vmax of 578 ± 23.4 nmol mg-1 min-1. Adenosine uptake by hENT1 is competitively inhibited by nitrobenzylmercaptopurine ribonucleoside (NBMPR), nucleosides, deoxynucleosides, and nucleoside-derived anti-cancer and anti-viral drugs. Binding of hENT1 to adenosine, deoxyadenosine, and adenine by isothermal titration calorimetry is in general agreement with results of the competitive inhibition assays. These results validate hENT1 as a bona fide target for potential drug target and serve as a useful basis for future biophysical and structural studies.

Comparative genomic analysis of equilibrative nucleoside transporters suggests conserved protein structure despite limited sequence identity.

Equilibrative nucleoside transporters (ENTs) are a recently characterized and poorly understood group of membrane proteins that are important in the uptake of endogenous nucleosides required for nucleic acid and nucleoside triphosphate synthesis. Despite their central importance in cellular metabolism and nucleoside analog chemotherapy, no human ENT gene has been described and nothing is known about gene structure and function. To gain insight into the ENT gene family, we used experimental and in silico comparative genomic approaches to identify ENT genes in three evolutionarily diverse organisms with completely (or almost completely) sequenced genomes, Homo sapiens, Caenorhabditis elegans and Drosophila melanogaster. We describe the chromosomal location, the predicted ENT gene structure and putative structural topologies of predicted ENT proteins derived from the open reading frames. Despite variations in genomic layout and limited ortholog protein sequence identity (< or =27.45%), predicted topologies of ENT proteins are strikingly similar, suggesting an evolutionary conservation of a prototypic structure. In addition, a similar distribution of protein domains on exons is apparent in all three taxa. These data demonstrate that comparative sequence analyses should be combined with other approaches (such as genomic and proteomic analyses) to fully understand structure, function and evolution of protein families.

Mutation of leucine-92 selectively reduces the apparent affinity of inosine, guanosine, NBMPR [S6-(4-nitrobenzyl)-mercaptopurine riboside] and dilazep for the human equilibrative nucleoside transporter, hENT1.

We developed a yeast-based assay for selection of hENT1 (human equilibrative nucleoside transporter 1) mutants that have altered affinity for hENT1 inhibitors and substrates. In this assay, expression of hENT1 in a yeast strain deficient in adenine biosynthesis (ade2) permits yeast growth on a plate lacking adenine but containing adenosine, a hENT1 substrate. This growth was prevented when inhibitors of hENT1 [e.g. NBMPR [S6-(4-nitrobenzyl)-mercaptopurine riboside], dilazep or dipyridamole] were included in the media. To identify hENT1 mutants resistant to inhibition by these compounds, hENT1 was randomly mutagenized and introduced into this strain. Mutation(s) that allowed growth of yeast cells in the presence of these inhibitors were then identified and characterized. Mutants harbouring amino acid changes at Leu92 exhibited resistance to NBMPR and dilazep but not dipyridamole. The IC50 values of NBMPR and dilazep for [3H]adenosine transport by one of these mutants L92Q (Leu92-->Gln) were approx. 200- and 4-fold greater when compared with the value for the wild-type hENT1, whereas that for dipyridamole remained unchanged. Additionally, when compared with the wild-type transporter, [3H]adenosine transport by L92Q transporter was significantly resistant to inhibition by inosine and guanosine but not by adenosine or pyrimidines. The Km value for inosine transport was approx. 4-fold greater for the L92Q mutant (260+/-16 mM) when compared with the wild-type transporter (65+/-7.8 mM). We have identified for the first time an amino acid residue (Leu92) of hENT1 that, when mutated, selectively alters the affinity of hENT1 to transport the nucleosides inosine and guanosine and its sensitivity to the inhibitors NBMPR and dilazep.

Functional characterization in yeast of genetic variants in the human equilibrative nucleoside transporter, ENT1.

The human equilibrative nucleoside transporter, ENT1, appears to play a critical role in the disposition of nucleosides and nucleoside analogs used clinically as anti-viral and anti-cancer drugs. Recently, we identified variants of ENT1 in an ethnically diverse DNA sample set from 247 individuals, focusing primarily on the coding region. The goal of the present study was to analyse the haplotype structure and functionally characterize the variants of ENT1. We observed that a single haplotype, ENT1*1, accounted for 91.3% of the 494 chromosomes. Functional analysis in Saccharomyces cerevisiae revealed no differences in the kinetics of uptake of nucleosides and nucleoside analogs by the two non-synonymous variant transporters, ENT1-I216T and ENT1-E391K, and the reference ENT1. These results, together with the observation that there are few haplotypes of ENT1, indicate that coding region variants of ENT1 do not contribute to inter-individual differences in response to nucleoside analog drugs.

Inhibition of nucleoside transport by new analogues of 4-nitrobenzylthioinosine: replacement of the ribose moiety by substituted benzyl groups.

4-Nitrobenzylthioinosine (NBTI, 1) is a well-known inhibitor for the nucleoside transport protein ENT1. However, its highly polar nature is unfavorable for oral absorption and/or penetration into the CNS. In the search for compounds with lower polarity than NBTI we replaced its ribose moiety by substituted benzyl groups. Halogen, hydroxyl, (trifluoro)methyl(-oxy), nitro, and amine functionalities were among the substituents at the benzyl group. In general, substitution of the benzyl group resulted in a lower affinity for ENT1. Only 2-hydroxyl substitution showed a higher affinity. Most likely this is the result of hydrogen bonding. Substitution at the 2-position of the benzyl group with aryl groups was also addressed. Compared to parent compound carrying a 2-phenylbenzyl group, all synthesized analogues gave higher affinities. Introduction of fluoro, trifluoromethyl, methoxy, and hydroxyl groups at the phenyl group clearly showed that addition to the 4-position was preferable. Despite the highly different character of a ribose and a benzyl group, Ki values in the low nanomolar range were obtained for the benzyl-substituted derivatives. Compound 35, LUF5919, and compound 60, LUF5929, displayed the highest affinity (Ki = 39 nM for both compounds), having a polar surface area of 101 A2 and 85 A2, respectively.