Effects of FPMINT derivative on Human Equilibrative Nucleoside Transporters

Abstract

Equilibrative nucleoside transporters (ENTs) are the proteins responsible for transporting nucleosides across cell membrane. The role of ENT 1 in cardioprotection has been reported but the biological functions of ENT 2 remains unclear. To study the functions of ENT 2, pharmacological approach using selective ENT 2 inhibitors is an option but such inhibitors are not available. Our laboratory has recently developed a compound called FPMINT, which is slightly more selective to ENT2 than ENT1 but the difference between its IC50 on ENT1 and ENT2 is not very distinctive. Therefore, modification of the structure of FPMINT to increase its selectivity to ENT 2 is required. In this study, the effects of a FPMINT derivative was examined. PK15 nucleoside transporter‐deficient cells transfected with ENT1 or ENT2 were used as the models. Cell viability and protein expression level were studied by MTT assay and Western blotting, respectively. The nucleoside transport was studied by measuring the [3H]uridine and [3H]adenosine uptake in the cells. The results showed that the viability of PK15NTD/ENT1 and PK15/NTD/ENT 2 cells were not affected after incubation with FPMINT derivative (0.5 μM to 50 μM) for 24 to 48 hr. The protein expression level of both ENT 1 and ENT 2 was also unchanged. The ENT1‐mediated [3H]uridine and [3H]adenosine transport were inhibited by FPMINT derivative in a concentration‐dependent manner with IC50 values of 2.458 μM and 7.113 μM, respectively, compared to the IC50 values of 0.5697 μM and 2.571 μM respectively in the case of ENT 2. In addition, FPMINT derivative reduced the Vmax of ENT1 and ENT2‐mediated [3H]uridine transport without significant change on the apparent Km. Furthermore, the inhibitory effects of FPMINT derivative on both ENT 1 and ENT 2 could not be washed away. In conclusion, our study has provided evidence that FPMINT derivative is an irreversible and non‐competitive inhibitor which is more selective to ENT2 than ENT1, when compared to its parent compound FPMINT.