RESUMO
5'-S-(2-aminoethyl)-6-N-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), 5'-S-(2-acetamidoethyl)-6-N-[(4-substituted)benzyl]-5'-thioadenosine analogues, 5'-S-[2-(6-aminohexanamido)]ethyl-6-N-(4-nitrobenzyl)-5'-thioadenosine (SAHENTA), and related compounds were synthesized by S(N)Ar displacement of fluoride from 6-fluoropurine intermediates with 4-(substituted)benzylamines. Conjugation of the pendant amino groups of SAENTA and SAHENTA with fluorescein-5-yl isothiocyanate (FITC) gave fluorescent probes that bound at nanomolar concentrations specifically to human equilibrative nucleoside transporter 1 (hENT1) produced in recombinant form in model expression systems and in native form in cancer cell lines. Transporter binding effects were studied and the ability of the probes to predict the potential antitumor efficacy of 2'-deoxy-2',2'-difluorocytidine (gemcitabine) was demonstrated.
Assuntos
Adenosina/análogos & derivados , Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/síntese química , Corantes Fluorescentes/síntese química , Tionucleosídeos/síntese química , Adenosina/síntese química , Adenosina/química , Animais , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/química , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Humanos , Camundongos , Oócitos/metabolismo , Ligação Proteica , Estereoisomerismo , Relação Estrutura-Atividade , Tioinosina/análogos & derivados , Tioinosina/farmacologia , Tionucleosídeos/química , Xenopus , Leveduras/efeitos dos fármacos , Leveduras/metabolismo , GencitabinaRESUMO
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.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/genética , Mutação , Nucleosídeos/metabolismo , Triptofano/genética , Vasodilatadores/farmacologia , Adenosina/metabolismo , Transporte Biológico/efeitos dos fármacos , Dilazep/farmacologia , Dipiridamol/farmacologia , Ativação Enzimática/efeitos dos fármacos , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Humanos , Cinética , Modelos Biológicos , Piperazinas/farmacologia , Ligação Proteica , Tioinosina/análogos & derivados , Tioinosina/farmacologia , Triptofano/metabolismoRESUMO
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.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Compostos Heterocíclicos/farmacologia , Conformação Proteica , Dobramento de Proteína , Adenosina/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Dilazep/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/química , Transportador Equilibrativo 1 de Nucleosídeo/genética , Teste de Complementação Genética , Humanos , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutagênese , Mutação , Nucleosídeos/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência de Aminoácidos , Tioinosina/farmacologiaRESUMO
4'-Thio-beta-D-arabinofuranosyl cytosine (TaraC) is in phase I development for treatment of cancer. In human equilibrative nucleoside transporter (hENT) 1-containing CEM cells, initial rates of uptake (10 microM; picomoles per microliter of cell water per second) of [3H]TaraC and [3H]1-beta-D-arabinofuranosyl cytosine (araC) were low (0.007 +/- 003 and 0.034 +/- 0.003, respectively) compared with that of [3H]uridine (0.317 +/- 0.048), a highactivity hENT1 permeant. In hENT1- and hENT2-containing HeLa cells, initial rates of uptake (10 microM; picomoles per cell per second) of [3H]TaraC, [3H]araC, and [3H]deoxycytidine were low (0.30 +/- 0.003, 0.42 +/- 0.03, and 0.51 +/- 0.11, respectively) and mediated primarily by hENT1 (approximately 74, approximately 65, and approximately 61%, respectively). In HeLa cells with recombinant human concentrative nucleoside transporter (hCNT) 1 or hCNT3 and pharmacologically blocked hENT1 and hENT2, transport of 10 microM[3H]TaraC and [3H]araC was not detected. The apparent affinities of recombinant transporters (produced in yeast) for a panel of cytosine-containing nucleosides yielded results that were consistent with the observed low-permeant activities of TaraC and araC for hENT1/2 and negligible permeant activities for hCNT1/2/3. During prolonged drug exposures of CEM cells with hENT1 activity, araC was more cytotoxic than TaraC, whereas coexposures with nitrobenzylthioinosine (to pharmacologically block hENT1) yielded identical cytotoxicities for araC and TaraC. The introduction by gene transfer of hENT2 and hCNT1 activities, respectively, into nucleoside transport-defective CEM cells increased sensitivity to both drugs moderately and slightly. These results demonstrated that nucleoside transport capacity (primarily via hENT1, to a lesser extent by hENT2 and possibly by hCNT1) is a determinant of pharmacological activity of both drugs.
Assuntos
Arabinonucleosídeos/farmacocinética , Citarabina/farmacocinética , Proteínas de Transporte de Nucleosídeos/fisiologia , Animais , Arabinonucleosídeos/metabolismo , Arabinonucleosídeos/farmacologia , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citarabina/metabolismo , Citarabina/farmacologia , Citidina/análogos & derivados , Citidina/farmacologia , Relação Dose-Resposta a Droga , Transportador Equilibrativo 2 de Nucleosídeo/genética , Transportador Equilibrativo 2 de Nucleosídeo/fisiologia , Feminino , Células HeLa , Humanos , Potenciais da Membrana/fisiologia , Proteínas de Transporte de Nucleosídeos/genética , Oócitos/metabolismo , Oócitos/fisiologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tioinosina/análogos & derivados , Tioinosina/farmacologia , Transfecção , Trítio , Uridina/farmacocinética , Xenopus laevisRESUMO
2-Chloro-9-(2'-deoxy-2'-fluoro-beta-d-arabinofuranosyl)adenine (Cl-F-ara-A, clofarabine), a purine nucleoside analog with structural similarity to 2-chloro-2'-deoxyadenosine (Cl-dAdo, cladribine) and 9-beta-d-arabinofuranosyl-2-fluoroadenine (F-ara-A, fludarabine), has activity in adult and pediatric leukemias. Mediated transport of the purine nucleoside analogs is believed to occur through the action of two structurally unrelated protein families, the equilibrative nucleoside transporters (ENTs) and the concentrative nucleoside transporters (CNTs). The current work assessed the transportability of Cl-F-ara-A, Cl-dAdo, and F-ara-A in cultured human leukemic CEM cells that were either nucleoside transport-defective or possessed individual human nucleoside transporter types and in Xenopus laevis oocytes and Saccharomyces cerevisiae yeast that produced individual recombinant human nucleoside transporter types. Cells producing hENT1 or hCNT3 exhibited the highest uptake of Cl-F-ara-A, whereas nucleoside transport-deficient cells and cells producing hCNT1 lacked uptake altogether. When Cl-F-ara-A transport rates by hENT1 were compared with those of Cl-dAdo and F-ara-A, Cl-dAdo had the highest efficiency of transport, although Cl-F-ara-A showed the greatest accumulation during 5-min exposures. In cytotoxicity studies with the CEM lines, Cl-F-ara-A was more cytotoxic to cells producing hENT1 than to the nucleoside transport-deficient cells. The efficiency of Cl-F-ara-A transport by oocytes with recombinant transporters was hCNT3 > hENT2 > hENT1 > hCNT2; no transport was observed with hCNT1. Affinity studies with recombinant transporters produced in yeast showed that hENT1, hENT2, and hCNT3 all had higher affinities for Cl-F-ara-A than for either Cl-dAdo or F-ara-A. These results suggest that the nature and activity of the plasma membrane proteins capable of inward transport of nucleosides are important determinants of Cl-F-ara-A activity in human cells.
Assuntos
Antineoplásicos/metabolismo , Arabinonucleosídeos/metabolismo , Cladribina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Vidarabina/análogos & derivados , Nucleotídeos de Adenina , Animais , Antineoplásicos/farmacologia , Arabinonucleosídeos/farmacologia , Transporte Biológico , Linhagem Celular , Cladribina/farmacologia , Clofarabina , Humanos , Proteínas Recombinantes/metabolismo , Vidarabina/metabolismo , Vidarabina/farmacologia , Xenopus laevisRESUMO
hCNT3 (human concentrative nucleoside transporter 3) is a nucleoside-sodium symporter that transports a broad range of naturally occurring purine and pyrimidine nucleosides as well as anticancer nucleoside drugs. To understand its uridine binding and translocation mechanisms, a cysteine-less version of hCNT3 was constructed and used for cysteine-accessibility and permeant-protection assays. Cysteine-less hCNT3, with 14 endogenous cysteine residues changed to serine, displayed wild-type properties in a yeast expression system, indicating that endogenous cysteine residues are not essential for hCNT3-mediated nucleoside transport. A series of cysteine-substitution mutants spanning predicted TMs (transmembrane domains) 11-13 was constructed and tested for accessibility to thiol-specific reagents. Mutants M496C, G498C, F563C, A594C, G598C and A606C had no detectable transport activity, indicating that a cysteine substitution at each of these positions was not tolerated. Two functional mutants in putative TM 11 (L480C and S487C) and four in putative TM 12 (N565C, T557C, G567C and I571C) were partially inhibited by MTS (methanethiosulphonate) reagent and high concentrations of uridine protected against inhibition, indicating that TMs 11 and 12 may form part of the nucleoside translocation pathway. The lack of accessibility of MTS reagents to TM 13 mutants suggests that TM 13 is not exposed to the nucleoside translocation pathway. Furthermore, G567C, N565C and I571C mutants were only sensitive to MTSEA (MTS-ethylammonium), a membranepermeant thiol reagent, indicating that these residues may be accessible from the cytoplasmic side of the membrane, providing evidence in support of the predicted orientation of TM 12 in the current putative topology model of hCNT3.
Assuntos
Membrana Celular/metabolismo , Cisteína/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Cisteína/química , Cisteína/genética , Expressão Gênica , Humanos , Cinética , Proteínas de Membrana Transportadoras/genética , Mutação , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Uridina/farmacologiaRESUMO
Human concentrative nucleoside transporters 1, 2, and 3 (hCNT1, hCNT2, and hCNT3) exhibit different functional characteristics, and a better understanding of their permeant selectivities is critical for development of nucleoside analog drugs with optimal pharmacokinetic properties. In this study, the sensitivity of a high-throughput yeast expression system used previously for hCNT1 and hCNT3 was improved and used to characterize determinants for interaction of uridine (Urd) with hCNT2. The observed changes of binding energy between hCNT2 and different Urd analogs suggested that it interacts with C3'-OH, C5'-OH, and N3-H of Urd. The C2' and C5 regions of Urd played minor but significant roles for Urd-hCNT2 binding, possibly through Van der Waals interactions. Because the yeast assay only provided information about potential transportability, the permeant selectivities of recombinant hCNT1, hCNT2, and hCNT3 produced in Xenopus laevis oocytes were investigated using a two-electrode voltage clamp assay. hCNT1-mediated transport was sensitive to modifications of the N3, C3', and C5' positions of Urd. hCNT2 showed some tolerance for transporting Urd analogs with C2' or C5 modifications, little tolerance for N3 modifications, and no tolerance for any modifications at C3' or C5' of Urd. Although hCNT3 was sensitive to C3' modifications, it transported a broad range of variously substituted Urd analogs. The transportability profiles identified in this study, which reflected the binding profiles well, should prove useful in the development of anticancer and antiviral therapies with nucleoside drugs that are permeants of members of the hCNT protein family.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Uridina/metabolismo , Sequência de Bases , Primers do DNA , Humanos , Moduladores de Transporte de Membrana , Proteínas de Membrana Transportadoras/antagonistas & inibidores , Ligação Proteica , Proteínas Recombinantes/metabolismoRESUMO
INTRODUCTION: Human concentrative nucleoside transporter 3, hCNT3 (SLC28A3), which mediates transport of purine and pyrimidine nucleosides and a variety of antiviral and anticancer nucleoside drugs, was investigated to determine if there are single nucleotide polymorphisms in the coding regions of the hCNT3 gene. METHODS AND RESULTS: Ninety-six DNA samples from Caucasians (Coriell Panel) were sequenced and sixteen variants in exons and flanking intronic regions were identified, of which five were coding variants; three of these were non-synonymous (S5N, L131F, Y513F) and were further investigated for functional alterations of the resulting recombinant proteins in Saccharomyces cerevisiae and Xenopus laevis oocytes. In yeast, immunostaining and fluorescence quantitation of the reference (wild-type) and variant CNT3 proteins showed similar levels of expression. Kinetic studies were undertaken in yeast with a high through-put semi-automated assay process; reference hCNT3 exhibited Km values of 1.7+/-0.3, 3.6+/-1.3, 2.2+/-0.7, and 2.1+/-0.6 muM and Vmax values of 1402+/-286, 1310+/-113, 1020+/-44, and 1740+/-114 pmol/mg/min, respectively, for uridine, cytidine, adenosine and inosine. Similar Km and Vmax values were obtained for the three variant proteins assayed in yeast under identical conditions. All of the characterized hCNT3 variants produced in oocytes retained sodium and proton dependence of uridine transport based on measurements of radioisotope flux and two-electrode voltage-clamp studies. CONCLUSION: These results suggested a high degree of conservation of function for hCNT3 in the Caucasian population.