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Drug Metab Dispos
2018 May 01;465:600-609. doi: 10.1124/dmd.117.079400.
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Identification of Structural and Molecular Features Involved in the Transport of 3'-Deoxy-Nucleoside Analogs by Human Equilibrative Nucleoside Transporter 3.
Rahman MF
,
Raj R
,
Govindarajan R
.
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Combination antiretroviral drug treatments depend on 3'-deoxy-nucleoside analogs such as 3'-azido-3'-deoxythymidine (AZT) and 2'3'-dideoxyinosine (DDI). Despite being effective in inhibiting human immunodeficiency virus replication, these drugs produce a range of toxicities, including myopathy, pancreatitis, neuropathy, and lactic acidosis, that are generally considered as sequelae to mitochondrial damage. Although cell surface-localized nucleoside transporters, such as human equilibrative nucleoside transporter 2 (hENT2) and human concentrative nucleoside transporter 1 (hCNT1), are known to increase the carrier-mediated uptake of 3'-deoxy-nucleoside analogs into cells, another ubiquitously expressed intracellular nucleoside transporter (namely, hENT3) has been implicated in the mitochondrial transport of 3'-deoxy-nucleoside analogs. Using site-directed mutagenesis, generation of chimeric hENTs, and 3H-permeant flux measurements in mutant/chimeric RNA-injected Xenopus oocytes, here we identified the molecular determinants of hENT3 that dictate membrane translocation of 3'-deoxy-nucleoside analogs. Our findings demonstrated that whereas hENT1 had no significant transport activity toward 3'-deoxy-nucleoside analogs, hENT3 was capable of transporting 3'-deoxy-nucleoside analogs similar to hENT2. Transport analyses of hENT3-hENT1 chimeric constructs demonstrated that the N-terminal half of hENT3 is primarily responsible for the hENT3-3'-deoxy-nucleoside analog interaction. In addition, mutagenic studies identified that 225D and 231L in the N-terminal half of hENT3 partially contribute to the ability of hENT3 to transport AZT and DDI. The identification of the transporter segment and amino acid residues that are important in hENT3 transport of 3'-deoxy-nucleoside analogs may present a possible mechanism for overcoming the adverse toxicities associated with 3'-deoxy-nucleoside analog treatment and may guide rational development of novel nucleoside analogs.
Fig. 1. Determination of the differential 3′-deoxy-nucleoside analog (AZT and DDI) transport by hENTs. (A) Transport activities of 3H-AZT (20 µM) were measured and plotted at pH 7.4 of oocytes at 25°C 24 hours after injection of H2O, hENT1, or hENT2 transcripts. (B) Transport activities of 3H-AZT (20 µM) at pH 5.5 after injection of oocytes with H2O or ∆36hENT3 transcripts. (C) Transport activities of 3H-DDI (20 µM) were measured and plotted at pH 7.4 of oocytes at 37°C 24 hours after injection of H2O, hENT1, or hENT2 transcripts. (D) Transport activities of 3H-DDI at pH 5.5, with H2O or ∆36hENT3 transcript injection. ****P < 0.0001 (one-way analysis of variance/t test with hENTs compared with H2O). Bars represent the average ± S.E. (n = 8–12 oocytes).
Fig. 2. Generation and transport characterization of chimeras of hENT3 and hENT1. (A) Diagrammatic representation of the hENT3 and hENT1 chimeras constructed by replacement of the C-terminal hENT3 region by corresponding regions of hENT1. N-terminal segments of hENT3 and C-terminal segments of hENT1 were joined with increasing increments of approximately 40 amino acids from ∆36hENT3. The constructs were named as in the following example: 80(3+1) is 80 amino acids starting from the N terminus of hENT3 and the remaining length of the protein from hENT1. (B) Uptake of 3H-adenosine (20 µM) into oocytes was measured and plotted at 25°C 24 hours after injection of hENT1, ∆36ENT3, or chimeric transcripts. (C and D) Uptake of 3H-adenosine (20 µM) into oocytes at 25°C 24 hours after injection of hENT1, ∆36ENT3, or chimeric transcripts in the absence and presence of NBMPR at pH 5.5 (C) and pH 7.4 (D), respectively. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-way analysis of variance). Data represent the average ± S.E.M. (n = 8–12 oocytes).
Fig. 3. Identification of the region(s) of hENT3 responsible for 3′-deoxy-nucleoside analog transport by hENT1 and hENT3 chimeras. (A) Uptake of 3H-adenosine (20 µM) into oocytes was measured and plotted at 25°C 24 hours after injection of hENT1, ∆36ENT3, or chimeric transcripts. (B) Uptake of 3H-AZT (20 µM) into oocytes at 25°C 24 hours after injection of hENT1, ∆36ENT3, or chimeric transcripts. (C) Uptake of 3H-DDI (20 µM) into oocytes at 25°C 24 hours after injection of hENT1, ∆36ENT3, and chimeric transcripts. *P < 0.05; **P < 0.01; ****P < 0.0001 (one-way analysis of variance). Data represent the average ± S.E.M. (n = 8–12 oocytes).
Fig. 4. Determination of relative dominance of the intracellular targeting signal(s) of N-terminal 36 amino acids of hENT3 and the C-terminal half of hENT1 in deciding cell surface transport activity. (A) Diagrammatic representation of the 267(3+1) chimera with or without the N-terminal 36 amino acids from hENT3. (B) Uptake of 3H-adenosine (20 µM) into oocytes was measured and plotted at 25°C 24 hours after injection of hENT1, ∆36ENT3, or chimeric transcripts. (C) Uptake of 3H-AZT (20 µM) into oocytes at 25°C 24 hours after injection of hENT1, ∆36ENT3, or chimeric transcripts. (D) Uptake of 3H-DDI (20 µM) into oocytes at 25°C 24 hours after injection of hENT1, ∆36ENT3, or chimeric transcripts. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-way analysis of variance). Data represent the average ± S.E.M. (n = 8–12 oocytes).
Fig. 5. Identification of residues dictating the selectivity of hENT3 in transporting 3′-deoxy-nucleoside analogs. (A) Sequence alignment of hENT1, hENT2, and hENT3: the residues that are common in hENT2 and hENT3 but not in hENT1 are indicated in the boxes. (B–D) Transport activities of 3H-adenosine (20 µM; B), 3H-AZT (20 µM; C), and 3H-DDI (20 µM; D) of H2O, ∆36hENT3, and mutant transcript-injected oocytes. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-way analysis of variance of mutants compared with ∆36hENT3). For (C) and (D), statistical analysis was performed for mutants retaining adenosine transport activity. Data represent the average ± S.E.M. (n = 8–12 oocytes).
Adams,
Preparation and anti-HIV activity of N-3-substituted thymidine nucleoside analogs.
1997, Pubmed
Adams,
Preparation and anti-HIV activity of N-3-substituted thymidine nucleoside analogs.
1997,
Pubmed
Agarwal,
Synthesis and evaluation of thymidine kinase 1-targeting carboranyl pyrimidine nucleoside analogs for boron neutron capture therapy of cancer.
2015,
Pubmed
Aguirrebengoa,
[Nucleoside analogs inhibiting reverse transcriptase: use in adult patient with HIV infection].
1996,
Pubmed
Albrecht,
3'-deoxy-3'-(dihydroxyphosphinylmethyl)nucleosides. Isosteric phosphonate analogs of nucleoside 3'-phosphates.
1970,
Pubmed
Ambrose,
The human immunodeficiency virus type 1 nonnucleoside reverse transcriptase inhibitor resistance mutation I132M confers hypersensitivity to nucleoside analogs.
2009,
Pubmed
Anderson,
Recent developments in the clinical pharmacology of anti-HIV nucleoside analogs.
2008,
Pubmed
Angusti,
Design, synthesis and anti flaviviridae activity of N(6)-, 5',3'-O- and 5',2'-O-substituted adenine nucleoside analogs.
2008,
Pubmed
Aseervatham,
The Role of Flexible Loops in Folding, Trafficking and Activity of Equilibrative Nucleoside Transporters.
2015,
Pubmed
,
Xenbase
Baldwin,
The equilibrative nucleoside transporter family, SLC29.
2004,
Pubmed
Baldwin,
Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes.
2005,
Pubmed
,
Xenbase
Bavoux,
[Antiretroviral drugs and pregnancy: apropos of an alert regarding mitochondrial pathology and nucleoside analogs].
2000,
Pubmed
Billioud,
The main hepatitis B virus (HBV) mutants resistant to nucleoside analogs are susceptible in vitro to non-nucleoside inhibitors of HBV replication.
2011,
Pubmed
Burchenal,
Experimental and clinical studies on nucleoside analogs as antitumor agents.
1975,
Pubmed
Campeau,
Whole-exome sequencing identifies mutations in the nucleoside transporter gene SLC29A3 in dysosteosclerosis, a form of osteopetrosis.
2012,
Pubmed
Cao,
Efficacy and safety of nucleoside analogs on blocking father-to-infant vertical transmission of hepatitis B virus.
2015,
Pubmed
Chan,
Antiviral nucleoside toxicity in canine bone marrow progenitor cells and its relationship to drug permeation.
1992,
Pubmed
Cliffe,
SLC29A3 gene is mutated in pigmented hypertrichosis with insulin-dependent diabetes mellitus syndrome and interacts with the insulin signaling pathway.
2009,
Pubmed
Colacino,
Effect of fialuridine on replication of mitochondrial DNA in CEM cells and in human hepatoblastoma cells in culture.
1994,
Pubmed
Colacino,
Mechanisms for the anti-hepatitis B virus activity and mitochondrial toxicity of fialuridine (FIAU).
1996,
Pubmed
Damaraju,
Interaction of fused-pyrimidine nucleoside analogs with human concentrative nucleoside transporters: High-affinity inhibitors of human concentrative nucleoside transporter 1.
2011,
Pubmed
,
Xenbase
Ehteshami,
Nucleotide Substrate Specificity of Anti-Hepatitis C Virus Nucleoside Analogs for Human Mitochondrial RNA Polymerase.
2017,
Pubmed
Endres,
Residues Met89 and Ser160 in the human equilibrative nucleoside transporter 1 affect its affinity for adenosine, guanosine, S6-(4-nitrobenzyl)-mercaptopurine riboside, and dipyridamole.
2005,
Pubmed
Galmarini,
Nucleoside analogues: mechanisms of drug resistance and reversal strategies.
2001,
Pubmed
Gordon,
Severe toxicity of fialuridine (FIAU).
1996,
Pubmed
Govindarajan,
Facilitated mitochondrial import of antiviral and anticancer nucleoside drugs by human equilibrative nucleoside transporter-3.
2009,
Pubmed
,
Xenbase
Gray,
The concentrative nucleoside transporter family, SLC28.
2004,
Pubmed
Hirschi,
Visualizing multistep elevator-like transitions of a nucleoside transporter.
2017,
Pubmed
Honkoop,
Mitochondrial injury. Lessons from the fialuridine trial.
1997,
Pubmed
Hsu,
Equilibrative nucleoside transporter 3 deficiency perturbs lysosome function and macrophage homeostasis.
2012,
Pubmed
Johnson,
Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4 Å.
2012,
Pubmed
Kang,
Human equilibrative nucleoside transporter-3 (hENT3) spectrum disorder mutations impair nucleoside transport, protein localization, and stability.
2010,
Pubmed
Kleiner,
Histopathologic changes associated with fialuridine hepatotoxicity.
1997,
Pubmed
Koczor,
The role of transporters in the toxicity of nucleoside and nucleotide analogs.
2012,
Pubmed
Kwong,
Enzymic cleavage as a probe of the molecular structures of mammalian equilibrative nucleoside transporters.
1993,
Pubmed
Lang,
Interactions of nucleoside analogs, caffeine, and nicotine with human concentrative nucleoside transporters 1 and 2 stably produced in a transport-defective human cell line.
2004,
Pubmed
Lee,
Identification of the mitochondrial targeting signal of the human equilibrative nucleoside transporter 1 (hENT1): implications for interspecies differences in mitochondrial toxicity of fialuridine.
2006,
Pubmed
Lum,
Human intestinal es nucleoside transporter: molecular characterization and nucleoside inhibitory profiles.
2000,
Pubmed
,
Xenbase
Maenza,
Combination antiretroviral therapy for HIV infection.
1998,
Pubmed
Mangravite,
Nucleoside transporters in the disposition and targeting of nucleoside analogs in the kidney.
2003,
Pubmed
Molho-Pessach,
The H syndrome is caused by mutations in the nucleoside transporter hENT3.
2008,
Pubmed
Morgan,
Mutations in SLC29A3, encoding an equilibrative nucleoside transporter ENT3, cause a familial histiocytosis syndrome (Faisalabad histiocytosis) and familial Rosai-Dorfman disease.
2010,
Pubmed
Moss,
Role of the equilibrative and concentrative nucleoside transporters in the intestinal absorption of the nucleoside drug, ribavirin, in wild-type and Ent1(-/-) mice.
2012,
Pubmed
Pastor-Anglada,
SLC28 genes and concentrative nucleoside transporter (CNT) proteins.
2008,
Pubmed
Quan,
Antiviral therapy: nucleotide and nucleoside analogs.
2004,
Pubmed
Rahman,
Molecular determinants of acidic pH-dependent transport of human equilibrative nucleoside transporter 3.
2017,
Pubmed
,
Xenbase
Sanders,
Immune-associated toxicities induced by in vivo and in vitro exposure to interferon-alpha alone or in combination with nucleoside analogs.
1991,
Pubmed
Singh,
ENT3 utilizes a pH Sensing Mechanism for Transport.
2018,
Pubmed
Sun,
Down-regulation of mitochondrial thymidine kinase 2 and deoxyguanosine kinase by didanosine: implication for mitochondrial toxicities of anti-HIV nucleoside analogs.
2014,
Pubmed
Sundaram,
Chimeric constructs between human and rat equilibrative nucleoside transporters (hENT1 and rENT1) reveal hENT1 structural domains interacting with coronary vasoactive drugs.
1998,
Pubmed
,
Xenbase
Yao,
Functional and molecular characterization of nucleobase transport by recombinant human and rat equilibrative nucleoside transporters 1 and 2. Chimeric constructs reveal a role for the ENT2 helix 5-6 region in nucleobase translocation.
2002,
Pubmed
,
Xenbase
Yao,
Transport of the antiviral nucleoside analogs 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine by a recombinant nucleoside transporter (rCNT) expressed in Xenopus laevis oocytes.
1996,
Pubmed
,
Xenbase
Yao,
Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative, nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes.
2001,
Pubmed
,
Xenbase
Young,
Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins.
2008,
Pubmed
Zhou,
Molecular determinants of substrate selectivity of a novel organic cation transporter (PMAT) in the SLC29 family.
2007,
Pubmed
Zimmerman,
3'-azido-3'-deoxythymidine. An unusual nucleoside analogue that permeates the membrane of human erythrocytes and lymphocytes by nonfacilitated diffusion.
1987,
Pubmed