Abstract
Nucleoside reverse transcriptase inhibitors (NRTIs) need to enter cells to act against the HIV-1. Human organic cation transporters (hOCT1–3) are expressed and active in CD4+ T cells, the main target of HIV-1, and have been associated with antiviral uptake in different tissues. In this study, we examined whether NRTIs interact and are substrates of hOCT in cells stably expressing these transporters. Using [3H]N-methyl-4-phenylpyridinium, we found a high-affinity interaction among abacavir [[(1S,4R)-4-[2-amino-6-(cyclopropylamino)purin-9-yl]-cyclopent-2-enyl]methanol sulfate] (ABC); <0.08 nM], azidothymidine [3′-azido-3′-deoxythymidine (AZT); <0.4 nM], tenofovir disoproxil fumarate (<1.0 nM), and emtricitabine (<2.5 nM) and hOCTs. Using a wide range of concentrations of lamivudine [(-)-β-l-2′,3′-dideoxy-3′-thiacyitidine (3TC)], we determined two different binding sites for hOCTs: a high-affinity site (Kd1 = 12.3–15.4 pM) and a low-affinity site (Kd2 = 1.9–3.4 mM). Measuring direct uptake of [3H]3TC and inhibition with hOCT substrates, we identified 3TC as a novel substrate for hOCT1, 2, and 3, with hOCT1 as the most efficient transporter (Km = 1.25 ± 0.1 mM; Vmax = 10.40 ± 0.32 nmol/mg protein/min; Vmax/Km = 8.32 ± 0.40 μl/mg protein/min). In drug-drug interaction experiments, we analyzed cis-inhibition of [3H]3TC uptake by ABC and AZT and found that 40 to 50% was inhibited at low concentrations of the drugs (Ki = 22–500 pM). These data reveal that NRTIs experience a high-affinity interaction with hOCTs, suggesting a putative role for these drugs as modulators of hOCT activity. Finally, 3TC is a novel substrate for hOCTs and the inhibition of its uptake at low concentrations of ABC and AZT could have implications for the pharmacokinetics of 3TC.
Footnotes
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This work was supported by Fundación para la Investigación y la Prevención del SIDA en España (FIPSE) [Grants FIPSE36372/03 and FIPSE36621/06] (to J.M.-P.). Additional support was provided by Plan Nacional de Salud from Ministerio de Ciencia e Innovación (MICINN) of Gobierno de España [Research Grants SAF2004-01259 and SAF2008-00577] (to M.P.-A.) and the Spanish AIDS network “Red Temática Cooperativa de Investigación en SIDA” (RIS) [Grants G03/173 and RD06/006]. G.M. was supported by Agència de Gestió d'Ajuts Universitaris i de Recerca from Generalitat de Catalunya [Grant 2005FI-00314].
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The measurements were performed by G.M. during a visit to the laboratory of H.K. and were supported by the Agència de Gestió d'Ajuts Universitaris i de Recerca from Generalitat de Catalunya [Grant 2006BE-2 00228], the Spanish AIDS network “Red Temática Cooperativa de Investigación en SIDA” (RIS) and Deutsche Forschungsgesellschaft, Sonderforschungsbereich 487/A4.
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H.K. and J.M.-P. contributed equally to this work.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.146225.
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ABBREVIATIONS: NRTI, nucleoside reverse transcriptase inhibitor; 3TC, lamivudine, (-)-β-l-2′,3′-dideoxy-3′-thiacyitidine; ABC, abacavir, [[(1S,4R)-4-[2-amino-6-(cyclopropylamino)purin-9-yl]cyclopent-2-enyl]methanol sulfate]; AZT, azidothymidine, 3′-azido-3′-deoxythymidine; FTC, emtricitabine; TDF, tenofovir disoproxil fumarate; SLC, solute carrier; h, human; OAT, organic anion transporter; OCT, organic cation transporter; CHO, Chinese hamster ovary; MPP+, N-methyl-4-phenylpyridinium; metformin, 1,1-[dimethyl]-biguanide hydrochloride; TEA, tetraethylammonium; TBuA, tetrabutylammonium; Rani, ranitidine; Atrop, atropine; D-22, 1,1′-diethyl-2,2′-cyanine iodide; HAART, highly active antiretroviral therapy; PI, protease inhibitor; r, rat.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received September 16, 2008.
- Accepted January 12, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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