Electrophysiological Characterization and Modeling of the Structure Activity Relationship of the Human Concentrative Nucleoside Transporter 3 (hCNT3)

Abstract

We characterized the electrophysiology, kinetics, and quantitative structure-activity relationship (QSAR) of the human concentrative nucleoside transporter 3 (hCNT3) expressed in Xenopus laevis oocytes by measuring substrate-induced inward currents using a two-microelectrode voltage-clamp system. At membrane potentials between Ϫ30 and Ϫ150 mV, sodium activation of gemcitabine transport was sigmoidal, with a K 0.5 of 8.5 Ϯ 0.3 mM for Na ϩ and a Hill coefficient of 2.2 Ϯ 0.25 independent of membrane potential. We measured the I max and K 0.5 for substrate at Ϫ50 mV for the nucleoside analog drugs gemcitabine (638 Ϯ 58 nA, 59.7 Ϯ 17.5 M), ribavirin (546 Ϯ 37 nA, 61.0 Ϯ 13.2 M), AZT (420 Ϯ 4 nA, 310 Ϯ 9 M), and 3-deazauridine (506 Ϯ 30 nA, 50.8 Ϯ 9.90 M). K 0.5 and I max for substrate were dependent on membrane potential (both increasing as the membrane became more hyperpolarized) for all four drugs. hCNT3 also exhibited pre-steady-state currents. The quantitative structure-activity relationship (QSAR) was examined using comparative molecular field analysis and comparative molecular similarity indices analysis of the inward currents induced by 27 nucleoside analogs with substitutions at both the ribose and the nucleobase. Two statistically significant QSAR models identified electrostatic interaction as the major force in hCNT3 transport and attributed a critical role to the 3Ј-hydroxyl position of hCNT3 substrates. Steric hindrance at the 3-position and positive charge at the 5-position of the pyrimidine ring were favorable for transport. Two hCNT3 pharmacophore models revealed the minimal features required for hCNT3 transport as two hydrogen bond acceptors at 3Ј-OH and 5Ј-O and the hydrophobic center occupied by the base ring.

Human Small Intestinal Epithelial Cells Differentiated from Adult Intestinal Stem Cells as a Novel System for Predicting Oral Drug Absorption in Humans

“…In contrast with the Caco-2 monolayer, the significantly high expression of CNT3 mRNA (16% of human small intestine) in HIECs may have conferred an absorption capability for didanosine, ribavirin, and doxifluridine to the HIEC monolayer (Table 2). Ribavirin and doxifluridine, which are good substrates for CNT3 (Hu et al, 2006), had higher P app values than didanosine, a relatively poor substrate for CNT3 (Hu et al, 2006), in the HIEC monolayer, and the rank order of their P app values was in agreement with that of Fa values.…”

The Role of Nucleoside Transporters in the Erythrocyte Disposition and Oral Absorption of Ribavirin in the Wild-Type and Equilibrative Nucleoside Transporter 1(−/−) Mice

“…These concentrations were chosen to investigate the role of both the CNTs and ENTs on the intestinal absorption or ribavirin. In humans, the K m of CNT2 for ribavirin is approximately 18 M (Yamamoto et al, 2007), whereas the K m of CNT3 is ϳ14 to 61 M (Hu et al, 2006;Yamamoto et al, 2007). We previously found that the K m of mouse Ent1 for ribavirin is 382 M (Endres et al, 2009).…”

“…There are two families of the nucleoside transporters, the equilibrative nucleoside transporters (ENTs) and the sodium-dependent concentrative nucleoside transporters (CNTs). Ribavirin is a substrate of the human nucleoside transporters ENT1 (Jarvis et al, 1998), ENT2 (Yamamoto et al, 2007), CNT2 (Yamamoto et al, 2007), and CNT3 (Hu et al, 2006). The active metabolites of ribavirin, ribavirin 5Ј-mono and 5Ј-triphosphate (RMP and RTP), are formed intracellularly (Russmann et al, 2006); thus, this metabolic activation is in part dependent on the transport of ribavirin into the cell.…”

Functional Characterization of a Nucleoside-Derived Drug Transporter Variant (hCNT3C602R) Showing Altered Sodium-Binding Capacity

“…Its role in drug transport is not well known, being involved in the uptake of cladribine and fludarabine (Mangravite et al, 2003) and of cytosine arabinoside (Sarkar et al, 2005), as well as other non-nucleosidic drugs, such as the anthracycline pirarubicin (Nagai et al, 2005). CNT3 has also been shown to transport gemcitabine and the antiviral drugs azidothymidine and ribavirin (Hu et al, 2006;Yamamoto et al, 2007). Its location at the apical membrane of a polarized cell model (Mangravite et al, 2003;Errasti-Murugarren et al, 2007) together with its presence along the rat and human renal tubule (Rodríguez-Mulero et al, 2005;Damaraju et al, 2007) and human intestine (Ritzel et al, 2001) suggest that CNT3 plays an important role in the absorption and disposition of nucleosides and synthetic nucleoside analogs.…”