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
-
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].
-
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.
-
H.K. and J.M.-P. contributed equally to this work.
-
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
-
doi:10.1124/jpet.108.146225.
-
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.
-
↵
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
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|
Log in using your username and password
Purchase access
In this issue
Transport of Lamivudine [(-)-β-l-2′,3′-Dideoxy-3′-thiacytidine] and High-Affinity Interaction of Nucleoside Reverse Transcriptase Inhibitors with Human Organic Cation Transporters 1, 2, and 3
