Uridine Recognition Motifs of Human Equilibrative Nucleoside Transporters 1 and 2 Produced in Saccharomyces cerevisiae†

Abstract

The sugar moiety of nucleosides has been shown to play a major role in permeant‐transporter interaction with human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2). To better understand the structural requirements for interactions with hENT1 and hENT2, a series of uridine analogs with sugar modifications were subjected to an assay that tested their abilities to inhibit [3H]uridine transport mediated by recombinant hENT1 and hENT2 produced in Saccharomyces cerevisiae. hENT1 displayed higher affinity for uridine than hENT2. Both transporters barely tolerated modifications or inversion of configuration at C(3′). The C(2′)‐OH at uridine was a structural determinant for uridine‐hENT1, but not for uridine‐hENT2, interactions. Both transporters were sensitive to modifications at C(5′) and hENT2 displayed more tolerance to removal of C(5′)‐OH than hENT1; addition of an O‐methyl group at C(5′) greatly reduced interaction with either hENT1 or hENT2. The changes in binding energies between transporter proteins and the different uridine analogs suggested that hENT1 formed strong interactions with C(3′)‐OH and moderate interactions with C(2′)‐OH and C(5′)‐OH of uridine, whereas hENT2 formed strong interactions with C(3′)‐OH, weak interactions with C(5′)‐OH, and no interaction with C(2′)‐OH. †In honor and celebration of the 70th birthday of Professor Leroy B. Townsend. #These authors contributed equally to this work.