Received for publication October 10, 2006.
Revised December 5, 2006.
Accepted for publication December 18, 2006.

Molecular interactions underlying the unusually high adenosine affinity of a novel Trypanosoma brucei nucleoside transporter

Abstract

Trypanosoma brucei encode a relatively high number of genes of the Equilibrative Nucleoside Transporter (ENT) family. We report here the cloning and in-depth characterization of one T. b. brucei ENT member, TbNT9/AT-D. This transporter was expressed in Saccharomyces cerevisiae and displayed a uniquely high affinity for adenosine (K_m = 0.068 ± 0.013 µM), as well as broader selectivity for other purine nucleosides in the low micromolar range, but was not inhibited by nucleobases or pyrimidines. This selectivity profile is consistent with the P1 transport activity observed previously in procyclic and long-slender bloodstream T. brucei, apart from the 40-fold higher affinity for adenosine than for inosine. We found that, like the previously investigated P1 activity of long/slender bloodstream trypanosomes, the 3'-hydroxy, 5'-hydroxy, N3 and N7 functional groups contribute to transporter binding. In addition, we show that the 6-position amine group of adenosine, but not the inosine 6-keto group, makes a major contribution to binding (G⁰ = 12 kJ/mol), explaining the different K_m values of the purine nucleosides. We further found that P1-activity in procyclic and long-slender trypanosomes are pharmacologically distinct and identified the main gene encoding this activity in procyclics as NT10/AT-B. The presence of multiple P1-type nucleoside transport activities in T. b. brucei facilitates the development of nucleoside-based treatments for African trypanosomiasis and would delay the onset of uptake-related drug resistance to such therapy. We show that both TbNT9/AT-D and NT10/AT-B transport a range of potentially therapeutic nucleoside analogues.

Key words: Adenosine, Nucleoside/Nucleotide, Structure-activity relationships and modeling, Antiprotozoal drugs

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