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Uridine Binding Motifs of Human Concentrative Nucleoside Transporters 1 and 3 Produced in Saccharomyces cerevisiae

Membrane Protein Research Group (J.Z., F.V., M.F.V., T.L., J.D.Y., C.E.C.), Departments of Oncology (J.Z., F.V., M.F.V., T.L., C.E.C.) and Physiology (J.D.Y.), University of Alberta, and the Cross Cancer Institute (J.Z., F.V., M.F.V., T.L., C.E.C.), Edmonton, Alberta, Canada; Department of Chemistry and Biochemistry (M.J.R., L.P.C.N., I.N.), Brigham Young University, Provo, Utah; and the School of Biochemistry and Molecular Biology (S.A.B.), University of Leeds, Leeds, United Kingdom

An extensive series of structural analogs of uridine that differed in substituents in the sugar and/or base moieties were subjected to inhibitor-sensitivity assays in a yeast expression system to define uridine structural determinants for inhibitors of human concentrative nucleoside transporters 1 and 3 (hCNT1 and hCNT3). The production of recombinant hCNT1 and hCNT3 in a nucleoside-transporter deficient strain of yeast was confirmed by immunoblotting, and uridine transport parameters (K_m, V_max) were determined by defining the concentration dependence of initial rates of uptake of [³H]uridine by intact yeast. The K_i values of uridine analogs were obtained from inhibitory-effect curves and converted to binding energies. hCNT1 and hCNT3 recognized uridine through distinguishable binding motifs. hCNT1 was sensitive to modifications at C(3), less sensitive at C(5') or N(3), and much less sensitive at C(2'). hCNT3 was sensitive to modifications at C(3'), but much less sensitive at N(3), C(5') or C(2'). The changes of binding energy between transporter proteins and different uridine analogs suggested that hCNT1 formed hydrogen bonds (H-bonds) with C(3')-OH, C(5')-OH, or N(3)-H of uridine, but not with C(2')-OH, whereas hCNT3 formed H-bonds to C(3')-OH, but not to N(3)-H, C(5')-OH, and C(2')-OH. Both transporters barely tolerated modifications at C(3') or inversion of configurations at C(2')orC(3'). The binding profiles identified in this study can be used to predict the potential transportability of nucleoside analogs, including anticancer or antiviral nucleoside drugs, by hCNT1 and hCNT3.

Address correspondence to: Dr. Carol E. Cass, Department of Oncology, University of Alberta, Cross Cancer Institute, 11540 University Ave., Edmonton, AB, Canada T6H 1Z2. E-mail: carol.cass{at}cancerboard.ab.ca

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