Abstract
Human nucleoside transporters (hNTs) mediate cellular influx of anticancer nucleoside drugs, including cytarabine, cladribine, and fludarabine. BCR-ABL tyrosine kinase inhibitors (TKIs) imatinib and dasatinib inhibit fludarabine and cytarabine uptake. We assessed interactions of bosutinib, dasatinib, imatinib, nilotinib, and ponatinib with recombinant hNTs (hENT1, 2; hCNT1, -2, and -3) produced individually in yeast Saccharomyces cerevisiae Nilotinib inhibited hENT1-mediated uridine transport most potently (IC50 value, 0.7 μm) followed by ponatinib > bosutinib > dasatinib > imatinib. Imatinib inhibited hCNT2 with an IC50 value of 2.3 μm Ponatinib inhibited all five hNTs with the greatest effect seen for hENT1 (IC50 value, 9 μm). TKIs inhibited [(3)H]uridine uptake in a competitive manner. Studies in yeast with mutants at two amino acid residues of hENT1 (L442I, L442T, M33A, M33A/L442I) previously shown to be involved in uridine and dipyridamole binding, suggested that BCR-ABL TKIs interacted with Met(33) (TM1) and Leu(442) (TM11) residues of hENT1. In cultured human CEM lymphoblastoid cells, which possess a single hNT type (hENT1), accumulation of [(3)H]cytarabine, [(3)H]cladribine, or [(3)H]fludarabine was reduced by each of the five TKIs, and also caused a reduction in cell surface expression of hENT1 protein. In conclusion, BCR-ABL TKIs variously inhibit five different hNTs, cause a decrease in cell surface hENT1 expression, and decrease uridine accumulation when presented together with uridine or when given before uridine. In experiments with mutant hENT1, we showed for the first time interaction of Met(33) (involved in dipyridamole binding) with BCR-ABL inhibitors and reduced interaction with M33A mutant hENT1.
Highlights
Summary of IC50 values for inhibition of uridine transport in yeast and CEM cells Inhibition of [3H]uridine uptake by BCR-ABL TKIs was assessed in yeast producing each of the five recombinant hNTs and in CEM cells, which have only hENT1 activity, in concentration-effect experiments as described under “Experimental Procedures.”
Summary of IC50 values for inhibition of uridine transport in yeast containing recombinant hENT1 or one of four hENT1 mutants Inhibition of [3H]uridine uptake by BCR-ABL TKIs was assessed in concentration-effect experiments as described under ”Experimental Procedures.“ IC50 values are listed below
We have shown that all five BCR-ABL inhibitors inhibit hENT1, a ubiquitous hNT that is necessary for activity of many nucleoside chemotherapy drugs, and other classes of nucleoside transporters to varying extents
Summary
Effects of BCR-ABL TKIs on Uridine Uptake Mediated by Recombinant hNTs Produced in Saccharomyces cerevisiae— Bosutinib, dasatinib, imatinib, nilotinib, and ponatinib (chemical structures shown in Fig. 1) were assessed for their relative abilities to inhibit [3H]uridine uptake by each of five hNTs in concentration-dependent inhibition experiments that yielded IC50 values (inhibitor concentration that produced 50% inhibition of transport). Summary of IC50 values for inhibition of uridine transport in yeast and CEM cells Inhibition of [3H]uridine uptake by BCR-ABL TKIs was assessed in yeast producing each of the five recombinant hNTs and in CEM cells, which have only hENT1 activity, in concentration-effect experiments as described under “Experimental Procedures.”. Summary of IC50 values for inhibition of uridine transport in yeast and CEM cells Inhibition of [3H]uridine uptake by BCR-ABL TKIs was assessed in yeast producing each of the five recombinant hNTs and in CEM cells, which have only hENT1 activity, in concentration-effect experiments as described under “Experimental Procedures.” IC50 values (mean Ϯ S.E.) are listed below
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