Molecular Interactions Underlying the Unusually High Adenosine Affinity of a NovelTrypanosoma bruceiNucleoside Transporter

Abstract

Trypanosoma brucei encodes 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. brucei brucei ENT member, TbNT9/AT-D. This transporter was expressed in Saccharomyces cerevisiae and displayed a uniquely high affinity for adenosine (K m ϭ 0.068 Ϯ 0.013 M), 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 longslender 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 3Ј-hydroxy, 5Ј-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 (⌬G 0 ϭ 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 is pharmacologically distinct, and we identified the main gene encoding this activity in procyclic cells as NT10/AT-B. The presence of multiple P1-type nucleoside transport activities in T. brucei 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 analogs.

2,N 6 -Disubstituted Adenosine Analogs with Antitrypanosomal and Antimalarial Activities

“…The relatively low affinity for the T. b. brucei nucleoside transporters is clearly the result of steric hindrance, particularly at position 2. It should be noted that similar K i values for inhibition of the P2 transporter by 2-substituted aminopurines have been reported, whereas for P1 the substitutions on positions 2 and N 6 are both likely to contribute to steric hindrance and hence the lack of binding (2,11).…”

Trypanocidal Furamidine Analogues: Influence of Pyridine Nitrogens on Trypanocidal Activity, Transport Kinetics, and Resistance Patterns

“…Using the requirement for reciprocal inhibition, we investigated whether P2 actually mediates the uptake of these compounds and what percentage of their uptake might be mediated by this adenosine-adenine transporter. Ex vivo trypanosomes (strain 427) were incubated with 10 M each furamidine analog in the presence of 100 M adenine, a highaffinity P2 substrate that does not interact with the P1 class of adenosine transporters at that concentration (1,10,21). Over a period of 60 min, uptake of all three diamidines was reduced by ϳ80% in the presence of 100 M adenine throughout the observation period (Fig.…”

The Diamidine Diminazene Aceturate Is a Substrate for the High-Affinity Pentamidine Transporter: Implications for the Development of High Resistance Levels in Trypanosomes

“…Transport assays with procyclic and bloodstream form trypanosomes were performed as described previously (Wallace et al, 2002;Natto et al, 2005;Al-Salabi et al, 2007). In brief, cell cultures were harvested at late-log stage of growth, washed in assay buffer (AB; 33 mM HEPES, 98 mM NaCl, 4.6 mM KCl, 0.55 mM CaCl 2 , 0.07 mM MgSO 4 , 5.8 mM NaH 2 PO 4 , 0.3 mM MgCl 2 , 23 mM NaHCO 3 , and 14 mM glucose, pH 7.3), and resuspended at a concentration of ϳ10 8 cells/ml.…”