Study of FPMINT derivative acting on human equilibrative nucleoside transporters

Abstract

Equilibrative nucleoside transporters (ENTs) are the membranous proteins responsible for transporting nucleosides. The role of ENT1 in cardioprotection has been reported but the biological functions of ENT2 remains unclear. Pharmacological approach such as using ENT2-selective inhibitor is useful for studying the biological role of ENT2 in human, but such inhibitor is currently not available. My laboratory has recently developed a compound known as FPMINT, which has a slightly higher affinity to ENT2 than ENT1 but the difference between its IC50 on ENT1 and ENT2 is not very distinctive. Therefore, structural optimization of FPMINT to enhance its selectivity to ENT2 is required. In this study, the effects of a FPMINT derivative was examined. PK15 nucleoside transporter-deficient cells transfected with ENT1 or ENT2 were adopted as the cell models. Cell viability and levels of ENT1 and ENT2 protein expression were testified by MTT assay and Western blot, respectively. The nucleoside transport was studied by measuring the intracellular [3H]uridine and [3H]adenosine uptake. The results showed that the viabilities of PK15NTD/ENT1 and PK15NTD/ENT2 cells were not affected after incubation with FPMINT derivative (0.5 μM to 50 μM) for 24 to 48 hr. The levels of both ENT1 and ENT2 protein expression were also unchanged. The ENT1-mediated [3H]uridine and [3H]adenosine transport were inhibited by FPMINT derivative in a dosage-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 in the case of ENT2, respectively. Kinetic study showed that FPMINT derivative reduced the Vmax of ENT1 and ENT2-mediated [3H]uridine transport without significant change on the apparent Km. Furthermore, the effects of FPMINT derivative inhibition on both ENT1 and ENT2 could only be partially rinsed out. In conclusion, our study has provided evidence that FPMINT derivative is an irreversible and non-competitive inhibitor of ENT1 and ENT2. It is more effective than its parent compound FPMINT, but its selectivity is not superior than FPMINT.