thioinosine and draflazine

hENT1 (human equilibrative nucleoside transporter 1) is inhibited by nanomolar concentrations of various structurally distinct coronary vasodilator drugs, including dipyridamole, dilazep, draflazine, soluflazine and NBMPR (nitrobenzylmercaptopurine ribonucleoside). When a library of randomly mutated hENT1 cDNAs was screened using a yeast-based functional complementation assay for resistance to dilazep, a clone containing the W29G mutation was identified. Multiple sequence alignments revealed that this residue was highly conserved. Mutations at Trp29 were generated and tested for adenosine transport activity and inhibitor sensitivity. Trp29 mutations significantly reduced the apparent V(max) and/or increased the apparent K(m) values for adenosine transport. Trp29 mutations increased the IC50 values for hENT1 inhibition by dipyridamole, dilazep, NBMPR, soluflazine and draflazine. NBMPR and soluflazine displayed remarkably similar trends, with large aromatic substitutions at residue 29 resulting in the lowest IC50 values, suggesting that both drugs could interact via ring-stacking interactions with Trp29. The W29T mutant displayed a selective loss of pyrimidine nucleoside transport activity, which contrasts with the previously identified L442I mutant that displayed a selective loss of purine nucleoside transport. W29T, L442I and the double mutant W29T/L442I were characterized kinetically for nucleoside transport activity. A helical wheel projection of TM (transmembrane segment) 1 suggests that Trp29 is positioned close to Met33, implicated previously in nucleoside and inhibitor recognition, and that both residues line the permeant translocation pathway. The data also suggest that Trp29 forms part of, or lies close to, the binding sites for dipyridamole, dilazep, NBMPR, soluflazine and draflazine.

Topics: Adenosine; Biological Transport; Dilazep; Dipyridamole; Enzyme Activation; Equilibrative Nucleoside Transporter 1; Humans; Kinetics; Models, Biological; Mutation; Nucleosides; Piperazines; Protein Binding; Thioinosine; Tryptophan; Vasodilator Agents

Levels of cardiovascular active metabolites, like adenosine, are regulated by nucleoside transporters of endothelial cells. We characterized the nucleoside and nucleobase transport capabilities of primary human cardiac microvascular endothelial cells (hMVECs). hMVECs accumulated 2-[3H]chloroadenosine via the nitrobenzylmercaptopurine riboside-sensitive equilibrative nucleoside transporter 1 (ENT1) at a V(max) of 3.4 +/- 1 pmol.microl(-1).s(-1), with no contribution from the nitrobenzylmercaptopurine riboside-insensitive ENT2. Inhibition of 2-chloroadenosine uptake by ENT1 blockers produced monophasic inhibition curves, which are also compatible with minimal ENT2 expression. The nucleobase [3H]hypoxanthine was accumulated within hMVECs (K(m) = 96 +/- 37 microM; V(max) = 1.6 +/- 0.3 pmol.microl(-1).s(-1)) despite the lack of a known nucleobase transport system. This novel transporter was dipyridamole-insensitive but could be inhibited by adenine (K(i) = 19 +/- 7 microM) and other purine nucleobases, including chemotherapeutic analogs. A variety of other cell types also expressed the nucleobase transporter, including the nucleoside transporter-deficient PK(15) cell line (PK15NTD). Further characterization of [3H]hypoxanthine uptake in the PK15NTD cells showed no dependence on Na(+) or H(+). PK15NTD cells expressing human ENT2 accumulated 4.5-fold more [3H]hypoxanthine in the presence of the ENT2 inhibitor dipyridamole than did PK15NTD cells or hMVECs, suggesting trapping of ENT2-permeable metabolites. Understanding the nucleoside and nucleobase transporter profiles in the vasculature will allow for further study into their roles in pathophysiological conditions such as hypoxia or ischemia.

Topics: 2-Chloroadenosine; Animals; Cell Culture Techniques; Cell Line; Cells, Cultured; Child, Preschool; Coronary Vessels; Dilazep; Dipyridamole; Dogs; Endothelial Cells; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Female; Humans; Hypoxanthine; Kinetics; Microcirculation; Nucleobase Transport Proteins; Piperazines; Protein Binding; Purines; Rats; Swine; Thioinosine; Transfection; Tritium

We studied the binding of [3H]nitrobenzylthioinosine (NBMPR) and the uptake of [3H]formycin B by the es (equilibrative inhibitor-sensitive) nucleoside transporter of Madin Darby Canine Kidney (MDCK) cells. NBMPR inhibited [3H]formycin B uptake with a Ki of 2.7+/-0.6 nM, and [3H]NBMPR had a KD of 1.3+/-0.3 nM for binding to these cells; these values are significantly higher than those obtained in human and mouse cell models. In contrast, other recognized es inhibitors, such as dipyridamole, were significantly more effective as inhibitors of [3H]NBMPR binding and [3H]formycin B uptake by MDCK cells relative to that seen for human cells. We isolated a cDNA encoding the canine es nucleoside transporter (designated cENT1), and assessed its function by stable expression in nucleoside transport deficient PK15NTD cells. The PK15-cENT1 cells displayed inhibitor sensitivities that were comparable to those obtained for the endogenous es nucleoside transporter in MDCK cells. These data indicate that the dog es/ENT1 transporter has distinctive inhibitor binding characteristics, and that these characteristics are a function of the protein structure as opposed to the environment in which it is expressed.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Carrier Proteins; Cell Line; Cloning, Molecular; Dilazep; Dipyridamole; DNA, Complementary; Dogs; Dose-Response Relationship, Drug; Equilibrative Nucleoside Transporter 1; Formycins; Kinetics; Molecular Sequence Data; Piperazines; Protein Binding; Protein Conformation; Radioligand Assay; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Structure-Activity Relationship; Thioinosine; Tritium

1. Microvascular endothelial cells (MVECs) form a barrier between circulating metabolites, such as adenosine, and the surrounding tissue. We hypothesize that MVECs have a high capacity for the accumulation of nucleosides, such that inhibition of the endothelial nucleoside transporters (NT) would profoundly affect the actions of adenosine in the microvasculature. 2. We assessed the binding of [(3)H]nitrobenzylmercaptopurine riboside (NBMPR), a specific probe for the inhibitor-sensitive subtype of equilibrative NT (es), and the uptake of [(3)H]formycin B (FB), by MVECs isolated from rat skeletal muscle. The cellular expression of equilibrative (ENT1, ENT2, ENT3) and concentrative (CNT1, CNT2, CNT3) NT subtypes was also determined using both qualitative and quantitative polymerase chain reaction techniques. 3. In the absence of Na(+), MVECs accumulated [(3)H]FB with a V(max) of 21+/-1 pmol microl(-1) s(-1). This uptake was mediated equally by es (K(m) 260+/-70 microm) and ei (equilibrative inhibitor-insensitive; K(m) 130+/-20 microm) NTs. 4. A minor component of Na(+)-dependent cif (concentrative inhibitor-insensitive FB transporter)/CNT2-mediated [(3)H]FB uptake (V(i) 0.008+/-0.005 pmol microl(-1) s(-1) at 10 microm) was also observed at room temperature upon inhibition of ENTs with dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d]pyrimidine)/NBMPR. 5. MVECs had 122,000 high-affinity (K(d) 0.10 nm) [(3)H]NBMPR binding sites (representing es transporters) per cell. A lower-affinity [(3)H]NBMPR binding component (K(d) 4.8 nm) was also observed that may be related to intracellular es-like proteins. 6. Rat skeletal muscle MVECs express es/ENT1, ei/ENT2, and cif/CNT2 transporters with characteristics typical of rat tissues. This primary cell culture model will enable future studies on factors influencing NT subtype expression, and the consequent effect on adenosine bioactivity, in the microvasculature.

Topics: Animals; Capillaries; Cell Separation; Cells, Cultured; Dilazep; Dipyridamole; DNA Primers; Endothelial Cells; Formycins; Muscle, Skeletal; Nucleoside Transport Proteins; Piperazines; Radioligand Assay; Rats; Reverse Transcriptase Polymerase Chain Reaction; Thioinosine; Vasodilator Agents

The equilibrative nucleoside transporters of mammalian cells play an important role in the regulation of extracellular adenosine concentrations, and inhibition of these transporters potentiates the biological effects of adenosine. Two subtypes of equilibrative transporters have been defined by their differential sensitivities to inhibition by nitrobenzylthioinosine (NBMPR; es/ENT1, sensitive; ei/ENT2, insensitive). In addition, significant species differences have been noted in es/ENT1 transporter affinity for a subset of inhibitors including draflazine and dipyridamole. Draflazine and a series of 15 chemically related compounds were compared for their abilities to: (a) inhibit the binding of [3H]NBMPR to the es/ENT1 transporter in mouse Ehrlich cell and human erythrocyte membranes, and (b) inhibit the es/ENT1 and ei/ENT2 transporter-mediated uptake of [3H]uridine in Ehrlich cells. Compounds within this series represented over a 1000-fold range of affinities for the es/ENT1 and ei/ENT2 transporters with subtype selectivities (ENT1/ENT2) ranging from 370 for R70527 to 0.17 for soluflazine. Five other analogues were identified, in addition to soluflazine, that had significantly higher affinity for the ei/ENT2 transporter compared with es/ENT1. Structure activity analyses of these data identified the requirement of a hydrophobic group connected to a 2-aminocarbonyl piperazine by a 5-carbon chain for high-affinity interactions with es/ENT1. This hydrophobic moiety was not as important for ei/ENT2 affinity and, in contrast to es/ENT1, a shorter alkyl chain enhanced binding to ei/ENT2. These draflazine analogues also varied in their differential affinities for mouse vs. human es/ENT1 transporters, and the degree of species discrimination was strongly dependent on the position of the aminocarbonyl group on the piperazine ring. This information, combined with structural data derived from molecular studies with ENT1 and ENT2 recombinant proteins, should guide further development of subtype-selective inhibitors of the equilibrative nucleoside transporters.

Topics: Animals; Carcinoma, Ehrlich Tumor; Carrier Proteins; Cell Membrane; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Erythrocytes; Humans; In Vitro Techniques; Membrane Proteins; Mice; Piperazines; Radioligand Assay; Species Specificity; Thioinosine; Tumor Cells, Cultured; Uridine

Plasmodium, the aetiologic agent of malaria, cannot synthesize purines de novo, and hence depends upon salvage from the host. Here we describe the molecular cloning and functional expression in Xenopus oocytes of the first purine transporter to be identified in this parasite. This 422-residue protein, which we designate PfENT1, is predicted to contain 11 membrane-spanning segments and is a distantly related member of the widely distributed eukaryotic protein family the equilibrative nucleoside transporters (ENTs). However, it differs profoundly at the sequence and functional levels from its homologous counterparts in the human host. The parasite protein exhibits a broad substrate specificity for natural nucleosides, but transports the purine nucleoside adenosine with a considerably higher apparent affinity (K(m) 0.32+/-0.05 mM) than the pyrimidine nucleoside uridine (K(m) 3.5+/-1.1 mM). It also efficiently transports nucleobases such as adenine (K(m) 0.32+/-0.10 mM) and hypoxanthine (K(m) 0.41+/-0.1 mM), and anti-viral 3'-deoxynucleoside analogues. Moreover, it is not sensitive to classical inhibitors of mammalian ENTs, including NBMPR [6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, or nitrobenzylthioinosine] and the coronary vasoactive drugs, dipyridamole, dilazep and draflazine. These unique properties suggest that PfENT1 might be a viable target for the development of novel anti-malarial drugs.

Topics: Adenine; Amino Acid Sequence; Animals; Antimalarials; Biological Transport; Blotting, Southern; Carrier Proteins; Cations; Cell Membrane; Cloning, Molecular; Dilazep; Dipyridamole; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins; Nucleosides; Phylogeny; Piperazines; Plasmodium falciparum; Platelet Aggregation Inhibitors; Protein Structure, Secondary; Protozoan Proteins; Sequence Homology, Amino Acid; Substrate Specificity; Thioinosine; Time Factors; Uridine; Vasodilator Agents; Xenopus

Inhibitors of adenosine membrane transport cause vasodilation and enhance the plasma adenosine concentration. However, it is unclear why the plasma adenosine concentration rises rather than falls when membrane transport is inhibited. We tested the hypothesis that the cytosolic adenosine concentration exceeds the interstitial concentration under well-oxygenated conditions.. In isolated, isovolumically working guinea pig hearts (n=50), the release rate of adenosine and accumulation of S-adenosylhomocysteine (after 20 minutes of 200 micromol/L homocysteine), a measure of the free cytosolic adenosine concentration, were determined in the absence and presence of specific and powerful blockers of adenosine membrane transport (nitrobenzylthioinosine 1 micromol/L), adenosine deaminase (erythro-9-hydroxy-nonyl-adenine 5 micromol/L), and adenosine kinase (iodotubericidine 10 micromol/L). Data analysis with a distributed multicompartment model revealed a total cardiac adenosine production rate of 2294 pmol. min-1. g-1, of which 8% was produced in the extracellular region. Because of a high rate of intracellular metabolism, however, 70.3% of extracellularly produced adenosine was taken up into cellular regions, an effect that was effectively eliminated by membrane transport block. The resulting approximately 2.8-fold increase of the interstitial adenosine concentration evoked near-maximal coronary dilation.. We rejected the hypothesis that the cytosolic adenosine concentration exceeds the interstitial. Rather, there is significant extracellular production, and the parenchymal cell represents a sink, not a source, for adenosine under well-oxygenated conditions.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Biological Transport; Bradykinin; Coronary Circulation; Cytosol; Depression, Chemical; Dipyridamole; Drug Synergism; Enzyme Inhibitors; Extracellular Space; Guinea Pigs; Heart; Models, Biological; Myocardium; Osmolar Concentration; Oxygen; Oxygen Consumption; Piperazines; Thioinosine; Tubercidin

1. The aim of the present study was to determine the effect of the nucleoside transporter inhibitor, draflazine, on the force of contraction in human myocardium and the affinity of the compound for the nucleoside transporter. Nucleoside transport inhibitors, like draflazine, are of potential importance for cardiopreservation of donor hearts for heart transplantation. 2. Functional experiments were performed in isolated electrically driven (1 Hz, 1.8 mmol l-1 Ca2+) human atrial trabeculae and ventricular papillary muscle strips. The affinity of draflazine for the myocardial nucleoside transporter was studied in isolated membranes from human ventricular myocardium and human erythrocytes in radioligand binding experiments using [3H]-nitrobenzylthioinosine ([3H]-NBTI). Dipyridamole was studied for comparison. 3. In membranes from human myocardium and erythrocytes, [3H]-NTBI labelled 1.18 pmol mg-1 protein and 23.0 pmol mg-1 protein, respectively, nucleoside transporter molecules with a KD value of 0.8 nmol l-1. Draflazine concentration-dependently inhibited binding of [3H]-NBTI to myocardial and erythrocyte membranes with a K(i)-value of 4.5 nmol l-1. The potency as judged from the K(i) values was ten times greater than that of dipyridamole in both myocardial and erythrocyte membranes. 4. Draflazine, at concentrations up to 100 mumol l-1, did not produce negative inotropic effects in atrial and ventricular myocardium. (-)-N6-phenylisopropyladenosine (R-PIA) and carbachol did not reduce force of contraction in ventricular myocardium, but exerted concentration-dependent direct negative inotropic effects in atrial myocardium. 5. The data provide evidence that draflazine specifically binds to the nucleoside transporter of the human heart and erythrocytes with high affinity. The compound does not produce negative inotropic effects at concentrations as high as 100 micromol 1-1.6. Draflazine could be a useful agent for cardio preservation because it does not produce cardio depressant effects. Thus, it may be possible to perfuse explanted hearts directly with this agent without the hazard of cardiodepression.

Topics: Adult; Affinity Labels; Aged; Carbachol; Cardiac Pacing, Artificial; Carrier Proteins; Dipyridamole; Erythrocyte Membrane; Female; Heart; Humans; In Vitro Techniques; Male; Membrane Proteins; Middle Aged; Myocardial Contraction; Myocardium; Nucleoside Transport Proteins; Phenylisopropyladenosine; Piperazines; Radioligand Assay; Thioinosine

In this study, we determined whether R75231, (+/-)-2-(aminocarbonyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis( 4-fluoro- phenyl)pentyl]-1-piperazineacetamide, and its two enantiomers, all nucleoside transport inhibitors, could play a role as anti-aggregatory agents. First, we determined the binding characteristics of [3H]nitrobenzylthioinosine, also a nucleoside transport inhibitor, on intact human erythrocytes. The Kd value was 0.27 +/- 0.04 nM and the Bmax was 23.5 +/- 5.1 pmol/10(9) erythrocytes. Second, we studied the ability of R75231 and its enantiomers R88021 ((-)-R75231, or draflazine) and R88016 ((+)-R75231), to displace [3H]nitrobenzylthioinosine. R75231 had an IC50 value of 2.2 +/- 0.3 nM. R88021 was twice as potent as R75231 and R88016 was approximately 20-fold less potent than R75231. Finally, the ability of these nucleoside transport inhibitors to enhance anti-aggregatory effects of adenosine was examined in whole human blood. Adenosine alone, 10 microM, had no effect on ADP-induced platelet aggregation. However, in the presence of 1 microM R75231, 10 microM of adenosine inhibited the aggregatory response completely. Dose-response curves indicated that the IC50 values of draflazine and R88016 were approximately 0.5 microM and 10 microM, respectively. R75231 and its enantiomers are valuable research tools to assess the role of the nucleoside transporter. Moreover, R75231 and draflazine (R88021) may prove to be useful as anti-aggregatory agents.

Topics: Adenosine; Adenosine Diphosphate; Binding Sites; Biological Transport; Blood Platelets; Erythrocytes; Humans; Piperazines; Platelet Aggregation; Platelet Aggregation Inhibitors; Radioligand Assay; Stereoisomerism; Thioinosine

This study investigated the interaction of the mioflazine derivative R75231 with the nucleoside transport system of rabbit cortical synaptosomes, and assessed the binding of [3H]R75231 to human erythrocyte ghost membranes. R75231 was a potent inhibitor of [3H]nitrobenzylthioinosine binding and [3H]uridine uptake in synaptosomes (Ki < 10 nM). This inhibition was evident even after extensive washing of the synaptosomes, subsequent to exposure to R75231. In addition to its tight binding characteristics, R75231 was shown to be a 'mixed' type inhibitor of [3H]nitrobenzylthioinosine binding (increased KD, decreased Bmax). [3H]R75231 bound with high affinity (KD = 0.4 nM) to erythrocyte membranes with a Bmax of 44 pmol/mg protein, which is comparable to the number of [3H]nitrobenzylthioinosine binding sites in this preparation. Binding of [3H]R75231 to these membranes was reversible, but the rate of dissociation was dependent upon the displacer used. Nitrobenzylthioinosine and dipyridamole each induced a complete dissociation of site-bound [3H]R75231 at rates not significantly different from those observed using a protocol involving a 100-fold dilution with buffer (no displacer). However, R75231 and mioflazine slowed the rate of dissociation of [3H]R75231 and actually caused an initial increase in the amount of site-bound [3H]R75231. Adenosine, on the other hand, enhanced the rate of [3H]R75231 dissociation. These results indicate that R75231 binding to the nucleoside transporter is a complex reaction, which may involve multiple interacting sites demonstrating positive cooperativity.

Topics: Adenosine; Animals; Binding, Competitive; Carrier Proteins; Cerebral Cortex; Erythrocyte Membrane; Half-Life; Humans; In Vitro Techniques; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Piperazines; Rabbits; Synaptosomes; Thioinosine; Uridine

The tritiated analogue of R75231 ((+-)-2-(aminocarbonyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis (4-fluorophenyl)pentyl]-1-piperazineacetamide) has been examined as a new radioligand for (nitrobenzylthioinosine sensitive) nucleoside transport proteins. [3H]R75231 was prepared in two steps from R69064 ((+-)-4-[5,5-bis[4-fluorophenyl)-4-pentenyl]-2-piperazinecarboxamide+ ++ dihydrochloride) with a specific activity of 0.23 TBq/mmol (6.3 Ci/mmol). [3H]R75231 bound in a pseudo-irreversible and saturable manner to a membrane preparation of calf lung tissue. The new radioligand displayed high affinity (KD = 0.32 +/- 0.06 nmol/l at 25 degrees C) and capacity (Bmax = 6.1 +/- 0.3 pmol/mg protein). Specific [3H]R75231 binding could be fully displaced by both structural analogues and reference inhibitors such as dipyridamole, NBI, dilazep and hexobendine, as well as by various nucleosides. The two stereoisomers of R75231, R88016 ((+)-R75231) and R88021 ((-)-R-75231), potently displaced specific [3H]R75231 and [3H]NBI binding, R88021 being 30-fold more active than R88016. Pseudo-Hill coefficients derived from the shape of all the [3H]R75231 displacement curves were approximately unity. In contrast, R75231 and most of its analogues displaced specific [3H]NBI binding with pseudo-Hill coefficients consistently larger than unity under identical experimental conditions. This latter finding is suggestive for the existence of two distinct binding sites for the two radioligands, which may or may not overlap to some extent.

Topics: Affinity Labels; Animals; Cattle; In Vitro Techniques; Lung; Membranes; Piperazines; Radioligand Assay; Stereoisomerism; Thioinosine; Tritium

Topics: Animals; Biological Transport; Dilazep; Dipyridamole; Epinephrine; Erythrocytes; Humans; In Vitro Techniques; Nucleosides; Phosphates; Piperazines; Rabbits; Thioinosine