Transport of antiviral 3′-deoxy-nucleoside drugs by recombinant human and rat equilibrative, nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes

We report identification of a new human nucleoside transporter protein by molecular cloning and functional expression of its cDNA. Previously, we used expression selection in Xenopus oocytes to isolate a cDNA from rat jejunal epithelium encoding the pyrimidine-selective Na+-dependent nucleoside transporter rCNT1 (Q.-Q. Huang, S. Y. M. Yao, M. W. L. Ritzel, A. R. P. Paterson, C. E. Cass, and J. D. Young. J. Biol. Chem. 269: 17757-17760, 1994). cDNAs for a human homologue of rCNT1, designated hCNT1, have been isolated from human kidney by hybridization cloning and reverse transcriptase polymerase chain reaction amplification strategies. hCNT1 was 83% identical to rCNT1 in amino acid sequence and exhibited the transport characteristics of an Na+-dependent nucleoside transporter with selectivity for pyrimidine nucleosides and adenosine when expressed in Xenopus oocytes. Deoxyadenosine, which undergoes net renal secretion, and guanosine were poor permeants. hCNT1 did, however, transport 3'-azido-3'-deoxythymidine. This is the first demonstration that members of the CNT family exist in human cells and provides evidence of their involvement in the renal transport of physiological nucleosides and nucleoside drugs. The hCNT1 gene was mapped to chromosome 15q25-26.

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Molecular biology and regulation of nucleoside and nucleobase transporter proteins in eukaryotes and prokaryotes

Biochemistry and Cell Biology ◽

10.1139/o02-153 ◽

2002 ◽

Vol 80 (5) ◽

pp. 623-638 ◽

Cited By ~ 59

Author(s):

Miguel A Cabrita ◽

Stephen A Baldwin ◽

James D Young ◽

Carol E Cass

Keyword(s):

Molecular Biology ◽

Vital Role ◽

Nucleoside Transporters ◽

Nucleoside Transport ◽

Nucleoside Transporter ◽

Concentration Gradients ◽

Transporter Proteins ◽

Equilibrative Nucleoside Transporters ◽

Nucleoside Drugs ◽

Nucleobase Transport

The molecular cloning of cDNAs encoding nucleoside transporter proteins has greatly advanced understanding of how nucleoside permeants are translocated across cell membranes. The nucleoside transporter proteins identified thus far have been categorized into five distinct superfamilies. Two of these superfamilies, the equilibrative and concentrative nucleoside transporters, have human members and these will be examined in depth in this review. The human equilibrative nucleoside transporters translocate nucleosides and nucleobases bidirectionally down their concentration gradients and are important in the uptake of anticancer and antiviral nucleoside drugs. The human concentrative nucleoside transporters cotranslocate nucleosides and sodium unidirectionally against the nucleoside concentration gradients and play a vital role in certain tissues. The regulation of nucleoside and nucleobase transporters is being studied more intensely now that more tools are available. This review provides an overview of recent advances in the molecular biology and regulation of the nucleoside and nucleobase transporters.Key words: nucleoside transporter, nucleoside transport, nucleobase transporter, nucleobase transport, regulation of nucleoside and nucleobase transport, nucleoside drugs.

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Current Progress on Equilibrative Nucleoside Transporter Function and Inhibitor Design

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555219870123 ◽

2019 ◽

Vol 24 (10) ◽

pp. 953-968 ◽

Cited By ~ 3

Author(s):

Shahid Rehan ◽

Saman Shahid ◽

Tiina A. Salminen ◽

Veli-Pekka Jaakola ◽

Ville O. Paavilainen

Keyword(s):

Small Molecule ◽

Family Members ◽

Nucleoside Analogs ◽

Homology Model ◽

Nucleoside Transporters ◽

Nucleoside Transporter ◽

Surface Receptors ◽

Cellular Import ◽

Nucleoside Drugs ◽

Small Molecule Modulators

Physiological nucleosides are used for the synthesis of DNA, RNA, and ATP in the cell and serve as universal mammalian signaling molecules that regulate physiological processes such as vasodilation and platelet aggregation by engaging with cell surface receptors. The same pathways that allow uptake of physiological nucleosides mediate the cellular import of synthetic nucleoside analogs used against cancer, HIV, and other viral diseases. Physiological nucleosides and nucleoside drugs are imported by two families of nucleoside transporters: the SLC28 concentrative nucleoside transporters (CNTs) and SLC29 equilibrative nucleoside transporters (ENTs). The four human ENT paralogs are expressed in distinct tissues, localize to different subcellular sites, and transport a variety of different molecules. Here we provide an overview of the known structure–function relationships of the ENT family with a focus on ligand binding and transport in the context of a new hENT1 homology model. We provide a generic residue numbering system for the different ENTs to facilitate the interpretation of mutational data produced using different ENT homologs. The discovery of paralog-selective small-molecule modulators is highly relevant for the design of new therapies and for uncovering the functions of poorly characterized ENT family members. Here, we discuss recent developments in the discovery of new paralog-selective small-molecule ENT inhibitors, including new natural product-inspired compounds. Recent progress in the ability to heterologously produce functional ENTs will allow us to gain insight into the structure and functions of different ENT family members as well as the rational discovery of highly selective inhibitors.

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Molecular Cloning and Functional Characterization of Nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) Equilibrative Nucleoside Transporter Proteins (rENT1 and rENT2) from Rat Tissues

Journal of Biological Chemistry ◽

10.1074/jbc.272.45.28423 ◽

1997 ◽

Vol 272 (45) ◽

pp. 28423-28430 ◽

Cited By ~ 160

Author(s):

Sylvia Y. M. Yao ◽

Amy M. L. Ng ◽

William R. Muzyka ◽

Mark Griffiths ◽

Carol E. Cass ◽

...

Keyword(s):

Molecular Cloning ◽

Functional Characterization ◽

Rat Tissues ◽

Nucleoside Transporter ◽

Transporter Proteins ◽

Equilibrative Nucleoside Transporter

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Adenosine transport: Recent advances in the molecular biology of nucleoside transporter proteins

Drug Development Research ◽

10.1002/(sici)1098-2299(199811/12)45:3/4<277::aid-ddr26>3.0.co;2-7 ◽

1998 ◽

Vol 45 (3-4) ◽

pp. 277-287 ◽

Cited By ~ 9

Author(s):

Lori L. Jennings ◽

Carol E. Cass ◽

Mabel W.L. Ritzel ◽

Sylvia Y.M. Yao ◽

James D. Young ◽

...

Keyword(s):

Molecular Biology ◽

Nucleoside Transporter ◽

Transporter Proteins ◽

Recent Advances ◽

Adenosine Transport

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Physiological and Pharmacological Roles of Nucleoside Transporter Proteins

Nucleosides Nucleotides & Nucleic Acids ◽

10.1080/15257770802145819 ◽

2008 ◽

Vol 27 (6-7) ◽

pp. 769-778 ◽

Cited By ~ 32

Author(s):

Míriam Molina-Arcas ◽

Laia Trigueros-Motos ◽

F. Javier Casado ◽

Marçal Pastor-Anglada

Keyword(s):

Nucleoside Transporter ◽

Transporter Proteins

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Identification of Cys140 in helix 4 as an exofacial cysteine residue within the substrate-translocation channel of rat equilibrative nitrobenzylthioinosine (NBMPR)-insensitive nucleoside transporter rENT2

Biochemical Journal ◽

10.1042/bj3530387 ◽

2001 ◽

Vol 353 (2) ◽

pp. 387-393 ◽

Cited By ~ 21

Author(s):

Sylvia Y. M. YAO ◽

Manickavasagam SUNDARAM ◽

Eugene G. CHOMEY ◽

Carol E. CASS ◽

Stephen A. BALDWIN ◽

...

Keyword(s):

Xenopus Oocytes ◽

Cysteine Residue ◽

Integral Membrane Proteins ◽

Structural Domain ◽

Nucleoside Transporter ◽

Vasoactive Drugs ◽

Wild Type ◽

Functional Protein ◽

Equilibrative Nucleoside Transporter ◽

Transmembrane Segments

The human and rat equilibrative nucleoside transporter proteins hENT1, rENT1, hENT2 and rENT2 belong to a family of integral membrane proteins with 11 potential transmembrane segments (TMs), and are distinguished functionally by differences in transport of nucleobases and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and vasoactive drugs. In the present study, we have produced recombinant hENT1, rENT1, hENT2 and rENT2 in Xenopus oocytes and investigated uridine transport following exposure to the impermeant thiol-reactive reagent p-chloromercuriphenyl sulphonate (PCMBS). PCMBS caused reversible inhibition of uridine influx by rENT2, but had no effect on hENT1, hENT2 or rENT1. This difference correlated with the presence in rENT2 of a unique Cys residue (Cys140) in the outer half of TM4 that was absent from the other ENTs. Mutation of Cys140 to Ser produced a functional protein (rENT2/C140S) that was insensitive to inhibition by PCMBS, identifying Cys140 as the exofacial Cys residue in rENT2 responsible for PCMBS inhibition. Uridine protected wild-type rENT2 against PCMBS inhibition, suggesting that Cys140 in TM4 lies within or is closely adjacent to the substrate-translocation channel of the transporter. TM4has been shown previously to be within a structural domain (TMs 3Ő6) responsible for interactions with NBMPR, vasoactive drugs and nucleobases.

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