formycin-b and draflazine

We studied the binding of [3H]nitrobenzylthioinosine (NBMPR) and the uptake of [3H]formycin B by the es (equilibrative inhibitor-sensitive) nucleoside transporter of Madin Darby Canine Kidney (MDCK) cells. NBMPR inhibited [3H]formycin B uptake with a Ki of 2.7+/-0.6 nM, and [3H]NBMPR had a KD of 1.3+/-0.3 nM for binding to these cells; these values are significantly higher than those obtained in human and mouse cell models. In contrast, other recognized es inhibitors, such as dipyridamole, were significantly more effective as inhibitors of [3H]NBMPR binding and [3H]formycin B uptake by MDCK cells relative to that seen for human cells. We isolated a cDNA encoding the canine es nucleoside transporter (designated cENT1), and assessed its function by stable expression in nucleoside transport deficient PK15NTD cells. The PK15-cENT1 cells displayed inhibitor sensitivities that were comparable to those obtained for the endogenous es nucleoside transporter in MDCK cells. These data indicate that the dog es/ENT1 transporter has distinctive inhibitor binding characteristics, and that these characteristics are a function of the protein structure as opposed to the environment in which it is expressed.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Carrier Proteins; Cell Line; Cloning, Molecular; Dilazep; Dipyridamole; DNA, Complementary; Dogs; Dose-Response Relationship, Drug; Equilibrative Nucleoside Transporter 1; Formycins; Kinetics; Molecular Sequence Data; Piperazines; Protein Binding; Protein Conformation; Radioligand Assay; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Structure-Activity Relationship; Thioinosine; Tritium