Uridine binding and transportability determinants of human concentrative nucleoside transporters.
Human concentrative nucleoside transporters 1, 2, and 3 (hCNT1, hCNT2, and hCNT3) exhibit different functional characteristics, and a better understanding of their permeant selectivities is critical for development of nucleoside analog drugs with optimal pharmacokinetic properties. In this study, the sensitivity of a high-throughput yeast expression system used previously for hCNT1 and hCNT3 was improved and used to characterize determinants for interaction of uridine (Urd) with hCNT2. The observed changes of binding energy between hCNT2 and different Urd analogs suggested that it interacts with C3'-OH, C5'-OH, and N3-H of Urd. The C2' and C5 regions of Urd played minor but significant roles for Urd-hCNT2 binding, possibly through Van der Waals interactions. Because the yeast assay only provided information about potential transportability, the permeant selectivities of recombinant hCNT1, hCNT2, and hCNT3 produced in Xenopus laevis oocytes were investigated using a two-electrode voltage clamp assay. hCNT1-mediated transport was sensitive to modifications of the N3, C3', and C5' positions of Urd. hCNT2 showed some tolerance for transporting Urd analogs with C2' or C5 modifications, little tolerance for N3 modifications, and no tolerance for any modifications at C3' or C5' of Urd. Although hCNT3 was sensitive to C3' modifications, it transported a broad range of variously substituted Urd analogs. The transportability profiles identified in this study, which reflected the binding profiles well, should prove useful in the development of anticancer and antiviral therapies with nucleoside drugs that are permeants of members of the hCNT protein family.
Zhang
Jing
J
Department of Oncology, University of Alberta, Cross Cancer Institute, 11540 University Ave., Edmonton, Alberta, Canada T6H 1Z2.
Smith
Kyla M
KM
Tackaberry
Tracey
T
Visser
Frank
F
Robins
Morris J
MJ
Nielsen
Lars P C
LP
Nowak
Ireneusz
I
Karpinski
Edward
E
Baldwin
Stephen A
SA
Young
James D
JD
Cass
Carol E
CE
D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D014529:Uridine / Q000378:metabolism*
D017931:DNA Primers; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C065171:cif nucleoside transporter; D014529:Uridine