TABLE 1

Affinity of natural nucleosides (purine and pyrimidine) and nucleoside-derived drugs for hCNT3, hCNT3C602R, hCNT3C602S, and hCNT3C602K measured in transiently transfected HeLa cells

Transporter-mediated uptake of [3H]uridine (1 μM, 1 min) by hCNT3, hCNT3C602R, hCNT3C602S, and hCNT3C602K was inhibited with increasing concentrations of natural nucleosides or nucleoside-derived drugs. Ki values were determined by fitting data by nonlinear regression using GraphPad Prism 4.0 software. For hCNT3C602S and hCNT3C602K mutants, Ki values were only obtained for natural nucleosides and the most commonly used nucleoside analogs. Data are expressed as the mean ± S.E.M. of three experiments carried out on different days using different batches of cells. Statistical analysis was carried out comparing Ki values for hCNT3 versus those obtained for mutated transporters.

SubstrateKi
hCNT3hCNT3C602RhCNT3C602ShCNT3C602K
μM
Adenosine2.4 ± 0.22.2 ± 0.32.3 ± 0.84.3 ± 0.2**
2-Fluoroadenosine12.4 ± 0.916.6 ± 3.4
5′-Deoxyadenosine12.4 ± 1.149.3 ± 6.6**
Fludarabine390 ± 9.2405 ± 25.4333.7 ± 43.21200 ± 210**
Cytidine3.5 ± 0.93.9 ± 0.83.7 ± 0.87.7 ± 0.4*
2′-Deoxycytidine6.2 ± 1.15.8 ± 0.7
2′,3′-Dideoxycytidine3400 ± 8003700 ± 11003500 ± 500N.D.
Cytarabine16,200 ± 230013,400 ± 280015,600 ± 2100N.D.
Gemcitabine14.7 ± 1.1119 ± 13.2**12.6 ± 1.3410 ± 20.2***
Thymidine10.6 ± 1.29.5 ± 1.211.6 ± 1458.8 ± 6.6**
AZT414 ± 205340 ± 965**435 ± 40.2N.D.
Uridine3.5 ± 0.53.3 ± 0.43.9 ± 0.86.2 ± 0.9
5′-DFUR22.6 ± 3.4114 ± 13.2**23.0 ± 2.2422 ± 32.2***
5-Fluorouridine5.7 ± 0.86.5 ± 0.9
2′-Deoxyuridine2.9 ± 0.721.0 ± 3.3**
Guanosine11.3 ± 2.19.3 ± 1.110.5 ± 1.522.4 ± 2.2*
Ribavirin21.3 ± 3.455.1 ± 6.4**21.8 ± 2.2102.9 ± 11.2**

  • N.D., not determined because of insufficient interaction of analog with transporter protein.

  • ↵* p ≤ 0.05.

  • ↵** p ≤ 0.01.

  • ↵*** p ≤ 0.001.