Uridine recognition motifs of human equilibrative nucleoside transporters 1 and 2 produced in Saccharomyces cerevisiae

The sugar moiety of nucleosides has been shown to play a major role in permeant-transporter interaction with human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2). To better understand the structural requirements for interactions with hENT1 and hENT2, a series of uridine analogs with sugar modifications were subjected to an assay that tested their abilities to inhibit [3H]uridine transport mediated by recombinant hENT1 and hENT2 produced in Saccharomyces cerevisiae. hENT1 displayed higher affinity for uridine than hENT2. Both transporters barely tolerated modifications or inversion of configuration at C(3'). The C(2')-OH at uridine was a structural determinant for uridine-hENT1, but not for uridine-hENT2, interactions. Both transporters were sensitive to modifications at C(5') and hENT2 displayed more tolerance to removal of C(5')-OH than hENT1; addition of an O-methyl group at C(5') greatly reduced interaction with either hENT1 or hENT2. The changes in binding energies between transporter proteins and the different uridine analogs suggested that hENT1 formed strong interactions with C(3')-OH and moderate interactions with C(2')-OH and C(5')-OH of uridine, whereas hENT2 formed strong interactions with C(3')-OH, weak interactions with C(5')-OH, and no interaction with C(2')-OH.     

Comparison of the interaction of uridine, cytidine, and other pyrimidine nucleoside analogues with recombinant human equilibrative nucleoside transporter 2 (hENT2) produced in Saccharomyces cerevisiae.

The human equilibrative nucleoside transporters I and 2 (hENT1, hENT2) share 50% amino acid identity and exhibit broad selectivities, accepting purine and pyrimidine nucleosides as permeants. The permeant selectivity of hENT2 is less well understood because of the low abundance of the native transporter in cells amenable to functional analysis. Recent studies of hENT2 produced in recombinant form in functional expression systems have shown that it differs from hENT1 in that it transports nucleobases. To further understand the structural requirements for permeant interaction with hENT2, we compared the relative abilities of uridine, cytidine, and their analogues to inhibit transport of [3H]uridine by recombinant hENT1 and hENT2 produced in yeast. hENT1 and hENT2 tolerated halogen modification at the 5 position of the base and the 2' and 5' positions of the ribose moieties of uridine whereas removal of the hydroxyl group at the 3' position of the ribose moiety of uridine eliminated interaction with both transporters. hENT2 displayed a lower ability, compared with hENT1, to interact with cytidine and cytidine analogues, suggesting a low tolerance for the presence of the amino group at the 4 position of the base.     

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.     

Mutation of residue 33 of human equilibrative nucleoside transporters 1 and 2 alters sensitivity to inhibition of transport by dilazep and dipyridamole.

Human equilibrative nucleoside transporters (hENT) 1 and 2 differ in that hENT1 is inhibited by nanomolar concentrations of dipyridamole and dilazep, whereas hENT2 is 2 and 3 orders of magnitude less sensitive, respectively. When a yeast expression plasmid containing the hENT1 cDNA was randomly mutated and screened by phenotypic complementation in Saccharomyces cerevisiae to identify mutants with reduced sensitivity to dilazep, clones with a point mutation that converted Met33 to Ile (hENT1-M33I) were obtained. Characterization of the mutant protein in S. cerevisiae and Xenopus laevis oocytes revealed that the mutant had less than one-tenth the sensitivity to dilazep and dipyridamole than wild type hENT1, with no change in nitrobenzylmercaptopurine ribonucleoside (NBMPR) sensitivity or apparent uridine affinity. To determine whether the reciprocal mutation in hENT2 (Ile33 to Met) also altered sensitivity to dilazep and dipyridamole, hENT2-I33M was created by site-directed mutagenesis. Although the resulting mutant (hENT2-I33M) displayed >10-fold higher dilazep and dipyridamole sensitivity and >8-fold higher uridine affinity compared with wild type hENT2, it retained insensitivity to NBMPR. These data established that mutation of residue 33 (Met versus Ile) of hENT1 and hENT2 altered the dilazep and dipyridamole sensitivities in both proteins, suggesting that a common region of inhibitor interaction has been identified.     

Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells. Ent2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine

We stably transfected the cloned human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD cells. Although hENT1 and hENT2 are predicted to be 50-kDa proteins, hENT1 runs as 40 kDa and hENT2 migrates as 50 and 47 kDa on SDS-polyacrylamide gel electrophoresis. Peptide N-glycosidase F and endoglycosidase H deglycosylate hENT1 to 37 kDa and hENT2 to 45 kDa. With hENT1 being more sensitive, there is a 7000-fold and 71-fold difference in sensitivity to nitrobenzylthioinosine (NBMPR) (IC(50), 0.4 +/- 0.1 nM versus 2.8 +/- 0.3 microM) and dipyridamole (IC(50), 5.0 +/- 0.9 nM versus 356 +/- 13 nM), respectively. [(3)H]NBMPR binds to ENT1 cells with a high affinity K(d) of 0.377 +/- 0.098 nM, and each ENT1 cell has 34,000 transporters with a turnover number of 46 molecules/s for uridine. Although both transporters are broadly selective, hENT2 is a generally low affinity nucleoside transporter with 2.6-, 2.8-, 7. 7-, and 19.3-fold lower affinity than hENT1 for thymidine, adenosine, cytidine, and guanosine, respectively. In contrast, the affinity of hENT2 for inosine is 4-fold higher than hENT1. The nucleobase hypoxanthine inhibits [(3)H]uridine uptake by hENT2 but has minimal effect on hENT1. Taken together, these results suggest that hENT2 might be important in transporting adenosine and its metabolites (inosine and hypoxanthine) in tissues such as skeletal muscle where ENT2 is predominantly expressed.     

6-Azauridine, a nucleoside with unusual ribose conformation. The molecular and crystal structure

The cytostatic analogue ribo-6-azauridine crystallizes in the orthorhombic space group P2₁2₁2₁ with eight molecules per unit cell of dimensions $\text{a = 20.230, b = 7.709, c = 12.863}\text{A}\text{?}$. A trial structure was obtained by direct methods. Least-squares refinement of co-ordinates and anisotropic thermal parameters based on 1998 reflections measured on a four-circle diffractometer led to a discrepancy index R = 4.0%. Like uridine, 6-azauridine has the anti conformation about the glycosidic bond and a C(3′)-endo sugar pucker. Unlike uridine, it exhibits a close approach of N(6) to C(2′) at only 2.814 and 2.844 Å in the two independent molecules, and a C(5′)(5′) bond that is gauche to C(4′)O(1′) but trans to C(4′)C(3′); this conformation about a C(4′)C(5′) bond has never been observed before for C(3′)-endo puckered riboses in the crystalline state. The crystal structure displays a pseudo-A face centering and very similar conformational parameters for the two independent molecules. Every OH and NH group in the structure serves as a proton donor in a hydrogen bond, including an unusual N(3)—H(3) … O(1′) link. Molecular orbital calculations by the extended Hückel method indicate that from uridine to 6-azauridine the net charge changes sign at ring positions 5 and 6 and disappears at 1.     

Synthesis and biological evaluation of phloridzin analogs as human concentrative nucleoside transporter 3 (hCNT3) inhibitors

Nucleoside transporter inhibitors have potential therapeutic applications as anticancer, antiviral, cardioprotective and neuroprotective agents. Although quite a few potent inhibitors of the equilibrative nucleoside transporters are known, largely missing are the concentrative nucleoside transporter inhibitors. Phloridzin (3, K_i = 16.00 μM) is a known moderate inhibitor of the concentrative nucleoside transporters. We have synthesized and evaluated analogs of phloridzin at the hCNT3 nucleoside transporter. Within the series of synthesized analogs compound 16 (K_i = 2.88 μM), possessing a ribofuranose sugar unit instead of a glucopyranose as present in phloridzin, exhibited the highest binding affinity at the hCNT3 transporter. Phloridzin and compound 16 have also been shown to be selective for the hCNT3 transporter as compared with the hENT1 transporter. Compound 16 can serve as a new lead which after further modifications could yield selective and potent hCNT3 inhibitors.     

PKC regulation of the human equilibrative nucleoside transporter,hENT1

Regulation of nucleoside transporters is poorly understood. We show that acute stimulation of protein kinase C (PKC) causes a rapid increase in S-(4-nitrobenzyl)-6-thioinosine-sensitive (human equilibrative nucleoside transporter 1, hENT1) nucleoside uptake, in human cultured cells, which is not due to increased metabolism and which can be blocked by PKC inhibitors. Use of isoform-specific inhibitors indicates that PKC delta and/or epsilon (but not alpha, beta or gamma) are responsible for the acute effects. Down-regulation of PKC decreases hENT1-dependent uridine uptake. These are the first data to show rapid PKC delta/epsilon-dependent stimulation of hENT1 transport by a mechanism that may involve activation of transporters at the membrane possibly by post-translational modification of the protein.     

Residues 334 and 338 in transmembrane segment 8 of human equilibrative nucleoside transporter 1 are important determinants of inhibitor sensitivity, protein folding, and catalytic turnover

Equilibrative nucleoside transporters (ENTs) are important for the metabolic salvage of nucleosides and the cellular uptake of antineoplastic and antiviral nucleoside analogs. Human equilibrative nucleoside transporter 1 (hENT1) is inhibited by nanomolar concentrations of structurally diverse compounds, including dipyridamole, dilazep, nitrobenzylmercaptopurine ribonucleoside (NBMPR), draflazine, and soluflazine. Random mutagenesis and screening by functional complementation for inhibitor-resistant mutants in yeast revealed mutations at Phe-334 and Asn-338. Both residues are predicted to lie in transmembrane segment 8 (TM 8), which contains residues that are highly conserved in the ENT family. F334Y displayed increased V(max) values that were attributed to increased rates of catalytic turnover, and N338Q and N338C displayed altered membrane distributions that appeared to be because of protein folding defects. Mutations of Phe-334 or Asn-338 impaired interactions with dilazep and dipyridamole, whereas mutations of Asn-338 impaired interactions with draflazine and soluflazine. A helical wheel projection of TM 8 predicted that Phe-334 and Asn-338 lie in close proximity to other highly conserved and/or hydrophilic residues, suggesting that they form part of a structurally important region that influences interactions with inhibitors, protein folding, and rates of conformational change during the transport cycle.     

Identification and mutational analysis of amino acid residues involved in dipyridamole interactions with human and Caenorhabditis elegans equilibrative nucleoside transporters

The equilibrative nucleoside transporters, hENT1 and CeENT1 from humans and Caenorhabditis elegans, respectively, are inhibited by nanomolar concentrations of dipyridamole and share a common 11-transmembrane helix (TM) topology. Random mutagenesis and screening by functional complementation in yeast for clones with reduced sensitivities to dipyridamole yielded mutations at Ile429 in TM 11 of CeENT1 and Met33 in TM 1 of hENT1. Mutational analysis of the corresponding residues of both proteins suggested important roles for these residues in competitive inhibition of hENT1 and CeENT1 by dipyridamole. To verify the roles of these residues in dipyridamole interactions, hENT2, which naturally exhibits low dipyridamole sensitivity, was mutated to contain side chains favorable for high affinity dipyridamole binding (i.e. a Met at the TM 1 and/or an Ile at the TM 11 positions). The single mutants exhibited increased hENT2 sensitivity to inhibition by dipyridamole, and the double mutant was the most sensitive, with an IC50 value that was only 2% of that of wild type. Functional analysis of the TM 1 and 11 mutants of hENT1 and CeENT1 revealed that Ala and Thr in the TM 1 and 11 positions, respectively, impaired uridine and adenosine transport and that Leu442 of hENT1 was involved in permeant selectivity. Mechanistic and structural models of dipyridamole interactions with the TM 1 and 11 residues are proposed. This study demonstrated that the corresponding residues in TMs 1 and 11 of hENT1, hENT2, and CeENT1 are important for dipyridamole interactions and nucleoside transport.     

Renal nucleoside transporters: physiological and clinical implications.

Renal handling of physiological and pharmacological nucleosides is a major determinant of their plasma levels and tissue availabilities. Additionally, the pharmacokinetics and normal tissue toxicities of nucleoside drugs are influenced by their handling in the kidney. Renal reabsorption or secretion of nucleosides is selective and dependent on integral membrane proteins, termed nucleoside transporters (NTs) present in renal epithelia. The 7 known human NTs (hNTs) exhibit varying permeant selectivities and are divided into 2 protein families: the solute carrier (SLC) 29 (SLC29A1, SLC29A2, SLC29A3, SLC29A4) and SLC28 (SLC28A1, SLC28A2, SLC28A3) proteins, otherwise known, respectively, as the human equilibrative NTs (hENTs, hENT1, hENT2, hENT3, hENT4) and human concentrative NTs (hCNTs, hCNT1, hCNT2, hCNT3). The well characterized hENTs (hENT1 and hENT2) are bidirectional facilitative diffusion transporters in plasma membranes; hENT3 and hENT4 are much less well known, although hENT3, found in lysosomal membranes, transports nucleosides and is pH dependent, whereas hENT4-PMAT is a H+-adenosine cotransporter as well as a monoamine-organic cation transporter. The 3 hCNTs are unidirectional secondary active Na+-nucleoside cotransporters. In renal epithelial cells, hCNT1, hCNT2, and hCNT3 at apical membranes, and hENT1 and hENT2 at basolateral membranes, apparently work in concert to mediate reabsorption of nucleosides from lumen to blood, driven by Na+ gradients. Secretion of some physiological nucleosides, therapeutic nucleoside analog drugs, and nucleotide metabolites of therapeutic nucleoside and nucleobase drugs likely occurs through various xenobiotic transporters in renal epithelia, including organic cation transporters, organic anion transporters, multidrug resistance related proteins, and multidrug resistance proteins. Mounting evidence suggests that hENT1 may have a presence at both apical and basolateral membranes of renal epithelia, and thus may participate in both selective secretory and reabsorptive fluxes of nucleosides. In this review, the renal handling of nucleosides is examined with respect to physiological and clinical implications for the regulation of human kidney NTs and adenosine signaling, intracellular nucleoside transport, and nephrotoxicities associated with some nucleoside drugs.     

A single glycine mutation in the equilibrative nucleoside transporter gene, hENT1, alters nucleoside transport activity and sensitivity to nitrobenzylthioinosine

The human equilibrative nucleoside transporter, hENT1, which is sensitive to inhibition by nitrobenzylthioinosine (NBMPR), is expressed in a wide variety of tissues. hENT1 is involved in the uptake of natural nucleosides, including regulation of the physiological effects of extracellular adenosine, and transports nucleoside drugs used in the treatment of cancer and viral diseases. Structure-function studies have revealed that transmembrane domains (TMD) 3 through 6 of hENT1 may be involved in binding of nucleosides. We have hypothesized that amino acid residues within TMD 3-6, which are conserved across equilibrative transporter sequences from several species, may have a critical role in the binding and transport of nucleosides. Therefore, we explored the role of point mutations of two conserved glycine residues, at positions 179 and 184 located in transmembrane domain 5 (TMD 5), using a GFP-tagged hENT1 in a yeast nucleoside transporter assay system. Mutations of glycine 179 to leucine, cysteine, or valine abolished transporter activity without affecting the targeting of the transporter to the plasma membrane, whereas more conservative mutations such as glycine to alanine or serine preserved both targeting to the plasma membrane and transport activity. Similar point mutations at glycine 184 resulted in poor targeting of hENT1 to the plasma membrane and little or no detectable functional activity. Uridine transport by G179A mutant was significantly lower (p < 0.05) and less sensitive (p < 0.05) to inhibition by NBMPR when compared to the wild-type transporter (IC(50) 7.7 +/- 0.8 nM versus 46 +/- 14.6 nM). Based on these data, we conclude that when hENT1 is expressed in yeast, glycine 179 is critical not only to the ability of hENT1 to transport uridine but also as a determinant of hENT1 sensitivity to NBMPR. In contrast, glycine 184 is likely important in targeting the transporter to the plasma membrane. This is the first identification and characterization of a critical amino acid residue of hENT1 that is important in both nucleoside transporter function and sensitivity to inhibition by NBMPR.     

Simultaneous expression of hCNT1-CFP and hENT1-YFP in Madin-Darby canine kidney cells. Localization and vectorial transport studies

To test the hypothesis that human concentrative and equilibrative nucleoside transporters (hCNT1 and hENT1) are present on the apical and basolateral membrane, respectively, we constructed a Madin-Darby canine kidney (MDCK) cell line that simultaneously and stably expresses recombinant hCNT1 and hENT1 gene products tagged with CFP and YFP fluorescent proteins, respectively. Using a confocal microscope, both hCNT1-CFP and hENT1-YFP were found to be distributed uniformly on the plasma membrane of undifferentiated MDCK cells. Upon differentiation of the MDCK cells on Transwell filter inserts, hCNT1-CFP was visualized exclusively on the apical membrane, whereas hENT1-YFP appeared predominantly on the basolateral membrane. As differentiation proceeded, there was an increase in alkaline phosphatase activity, and activity of hENT1 in the apical compartment decreased while hCNT1 activity remained constant. These results suggest that, on differentiation, hENT1 is sorted to the basolateral membrane. This was confirmed when the hCNT1-mediated uptake of [(3)H]uridine from the apical compartment of the differentiated cells was found to be approximately 20-fold higher and that for hENT1 was approximately 4-fold lower than the corresponding uptake from the basal compartment. As observed in vivo, the net transport of [(3)H]adenosine was from the apical to the basal compartment, whereas that for (14)C-deoxyadenosine was from the basal to the apical compartment. In summary, we have shown for the first time that hCNT1 and hENT1 are expressed in polarized MDCK cells on the apical and basolateral membrane, respectively, allowing vectorial transport in both directions depending on the relative activity (ratio of maximal transporter activity to affinity) of each transporter for their substrates.     

Interaction of wheat protein synthesis elongation factor 1 with GTP analogs

Wheat protein synthesis elongation factor 1 was tested for binding to GTP analogs, including structures resembling “caps” that are present at the 5′-termini of most eukaryotic mRNAs. The interaction was assayed by determining the capacity of the analogs to inhibit the binding of [³H]GTP to elongation factor 1. Significant interaction of elongation factor 1 with G(5′)ppp(5′)G, G(5′)pppp(5′)G, and G(5′)ppp(5′)A was observed. Methylation of a ribose 2′-hydroxyl had very little effect, but methylation of the 7 position of guanosine greatly diminished the affinity of elongation factor 1 for these compounds. m⁷G(5′)ppp(5′)Cm, m⁷G(5′)ppp(5′)Um, and m⁷G(5′)ppp(5′)Am gave no detectable binding with EF1.     

Role of Human Nucleoside Transporters in the Uptake and Cytotoxicity of Azacitidine and Decitabine

The nucleoside analogs 5-azacytidine (azacitidine) and 5-aza-2′-deoxycytidine (decitabine) are active against acute myeloid leukemia and myelodysplastic syndromes. Cellular transport across membranes is crucial for uptake of these highly polar hydrophilic molecules. We assessed the ability of azacitidine, decitabine, and, for comparison, gemcitabine, to interact with human nucleoside transporters (hNTs) in Saccharomyces cerevisiae cells (hENT1/2, hCNT1/2/3) or Xenopus laevis oocytes (hENT3/4). All three drugs inhibited hCNT1/3 potently (K _i values, 3–26 μM), hENT1/2 and hCNT2 weakly (K _i values, 0.5–3.1 mM), and hENT3/4 poorly if at all. Rates of transport of [³H]gemcitabine, [¹⁴C]azacitidine, and [³H]decitabine observed in Xenopus oocytes expressing individual recombinant hNTs differed substantially. Cytotoxicity of azacitidine and decitabine was assessed in hNT-expressing or hNT-deficient cultured human cell lines in the absence or presence of transport inhibitors where available. The rank order of cytotoxic sensitivities (IC ₅₀ values, μM) conferred by hNTs were hCNT1 (0.1) > hENT1 (0.3) ? hCNT2 (8.3), hENT2 (9.0) for azacitidine and hENT1 (0.3) > hCNT1 (0.8) ? hENT2, hCNT2 (>100) for decitabine. Protection against cytotoxicity was observed for both drugs in the presence of inhibitors of nucleoside transport, thus suggesting the importance of hNTs in manifestation of toxicity. In summary, all seven hNTs transported azacitidine, with hCNT3 showing the highest rates, whereas hENT1 and hENT2 showed modest transport and hCNT1 and hCNT3 poor transport of decitabine. Our results show for the first time that azacitidine and decitabine exhibit different human nucleoside transportability profiles and their cytotoxicities are dependent on the presence of hNTs, which could serve as potential biomarkers of clinical response.     

The life cycle of human equilibrative nucleoside transporter 1: from ER export to degradation

Nucleoside transporters (NTs) play an essential role in the transport of nucleosides across cellular membranes. Equilibrative NTs (ENTs) allow facilitated diffusion of nucleosides and the prototypic ENT, hENT1, is primarily localized to the plasma membrane (PM). hENT1 is responsible for the uptake of nucleoside analog drugs used in treating viral infections and cancer, but despite its clinical importance, virtually nothing is known about the dynamics of the hENT1 life cycle including trafficking to the PM, endocytosis and degradation. Therefore, we followed the life cycle of tagged hENT1 (GFP- or FLAG-) transiently transfected into mammalian cells to gain insight into the sequence of events, timing and underlying mechanisms regulating the hENT1 life cycle. Protein translocation to the PM was examined using fixed and live cell confocal microscopy while endocytosis and degradation were analyzed by cell surface biotinylation and [³⁵S] pulse chase analysis respectively. We determined that tagged hENT1 is trafficked to the PM in association with microtubules and incorporated in the plasma membrane where it subsequently undergoes clathrin-mediated endocytosis and recycling. Finally, internalized protein is degraded via the lysosomal pathway and observations suggest the complete life cycle of tagged hENT1 within these cells is approximately 14 hours.     

Apple Sucrose Transporter SUT1 and Sorbitol Transporter SOT6 Interact with Cytochrome b5 to Regulate Their Affinity for Substrate Sugars

Sugar transporters are central machineries to mediate cross-membrane transport of sugars into the cells, and sugar availability may serve as a signal to regulate the sugar transporters. However, the mechanisms of sugar transport regulation by signal sugar availability remain unclear in plant and animal cells. Here, we report that a sucrose transporter, MdSUT1, and a sorbitol transporter, MdSOT6, both localized to plasma membrane, were identified from apple (Malus domestica) fruit. Using a combination of the split-ubiquitin yeast two-hybrid, immunocoprecipitation, and bimolecular fluorescence complementation assays, the two distinct sugar transporters were shown to interact physically with an apple endoplasmic reticulum-anchored cytochrome b5 MdCYB5 in vitro and in vivo. In the yeast systems, the two different interaction complexes function to up-regulate the affinity of the sugar transporters, allowing cells to adapt to sugar starvation. An Arabidopsis (Arabidopsis thaliana) homolog of MdCYB5, AtCYB5-A, also interacts with the two sugar transporters and functions similarly. The point mutations leucine-73 → proline in MdSUT1 and leucine-117 → proline in MdSOT6, disrupting the bimolecular interactions but without significantly affecting the transporter activities, abolish the stimulating effects of the sugar transporter-cytochrome b5 complex on the affinity of the sugar transporters. However, the yeast (Saccharomyces cerevisiae) cytochrome b5 ScCYB5, an additional interacting partner of the two plant sugar transporters, has no function in the regulation of the sugar transporters, indicating that the observed biological functions in the yeast systems are specific to plant cytochrome b5s. These findings suggest a novel mechanism by which the plant cells tailor sugar uptake to the surrounding sugar availability.     

Novel C2-purine position analogs of nitrobenzylmercaptopurine riboside as human equilibrative nucleoside transporter 1 inhibitors

Nucleoside transporter inhibitors have potential therapeutic applications as anticancer, antiviral, cardioprotective, and neuroprotective agents. S⁶-(4-nitrobenzyl)mercaptopurine riboside (NBMPR) is a prototype inhibitor of the human equilibrative nucleoside transporter (hENT1), and is a high affinity ligand with a K_d of 0.1–1.0 nM. We have synthesized and flow cytometrically evaluated the binding affinity of a series of novel C²-purine position substituted analogs of NBMPR at the hENT1. The aim of this research was to understand the substituent requirements at the C²-purine position of NBMPR. Structure–activity relationships (SAR) indicate that increasing the steric bulk at the C²-purine position of NBMPR led to a decrease in binding affinity of these ligands at the hENT1. New high affinity inhibitors were identified, with the best compound, 2-fluoro-4-nitrobenzyl mercaptopurine riboside (7), exhibiting a K_i of 2.1 nM. This information, when coupled with the information obtained from other structure–activity relationship studies should prove useful in efforts aimed at modeling the NMBPR and analogs pharmacophore of hENT1 inhibitors.