Abstract

Pharmacologically important drugs were examined as potential inhibitors or permeants of human concentrative nucleoside transporters 1 (hCNT1)- and 2 (hCNT2)-producing stable transfectants by assessing their abilities to inhibit uridine transport. hCNT1 exhibited high affinities for uridine analogs (5-fluorouridine, 2′-deoxyuridine, 5-fluoro-2′-deoxyuridine, and 5-fluoro-5′-deoxyuridine) with K_i values of 22 to 33 μM, whereas hCNT2 exhibited moderate affinities for 5-fluoro-2′-deoxyuridine, high affinities for 2′-deoxyuridine and 5-fluorouridine, and low affinity for 5-fluoro-5′-deoxyuridine. The uridine analogs were transported at 2-fold higher rates (at 10 μM) by hCNT1 than by hCNT2. Enantiomeric configuration and the 3′-hydroxyl group of the ribose ring were important determinants for interaction with hCNTs, whereas the 2′-hydroxyl group was less important. Both transporters bound N⁶-(p-aminobenzyl)adenosine with affinities similar to those of adenosine (K_i = 28-39 μM). Other adenosine receptor ligands, including caffeine, bound better to hCNT1 than to hCNT2 (K_i = 46 versus 103 μM, respectively), whereas 2-chloroadenosine bound better to hCNT2 than to hCNT1 (K_i = 37 and 101 μM, respectively). There was a greater than 3-fold difference in binding affinities between hCNT1 and hCNT2 for nicotine (K_i = 63 versus 227 μM). However, direct measurements of nicotine and caffeine uptake rates (10 μM) failed to demonstrate mediated uptake by either transporter. Although hCNT1 bound several adenosine analogs relatively well, it did not transport 2-chloro-2′-deoxyadenosine (cladribine) or 2-fluoro-9-β-d-arabinofuranosyladenine (fludarabine), whereas hCNT2 transported both, albeit with low activities. The results indicated that although hCNT1 and hCNT2 possess some overlap in transport of several uridine and adenosine analogs, they also exhibit distinct differences in capacity to interact with some adenosine receptor ligands, adenosine-based drugs, and nicotine.