Adenosine transporters in bloodstream forms of Trypanosoma brucei brucei: Substrate recognition motifs and affinity for trypanocidal drugs

Adenosine influx by Trypanosoma brucei brucei P1 and P2 transporters was ki netically characterized. The P1 transporter displayed a higher affinity and capacity for adenosine (K-m = 0.38 +/- 0.10 mM, V-max = 2.8 +/- 0.4 pmol . 10(7) cells(-1) . s(-1)) than the P2 transporter (K-m = 0.92 +/- 0.06 mu M , V-max = 1.12 +/- 0.08 4 pmol . 10(7) cells(-1) . s(-1)). To formulate a s tructure-activity relationship for the interaction of adenosine with the tr ansporters, a series of analogs were evaluated as potential inhibitors of a denosine transport, and the K-i values were converted to binding energy. Th e P1 transporter was found to be selective inhibited by purine nucleosides (K-i similar to 1 mu M for inosine and guanosine), but nucleobases and pyri midines had little effect on P1-mediated transport. The P1 transporter appe ars to form hydrogen bonds with N3 and N7 of the purine ring as well as wit h the 3' and 5' hydroxyl groups of the ribose moiety, with apparent bond en ergies of 12.8 to 15.8 kJ/mol. The P2 transporter, in contrast, had high-af finity (K-i = 0.2-4 mu M) for 6-aminopurines, including adenine, 2'-deoxyad enosine, and tubercidin, but not for any oxopurines. The main interaction o f adenosine with the P2 transporter is suggested to be via hydrogen bonds t o N1 and the 6-amino group. Additional pi-pi interactions of the purine rin g and electrostatic interactions with N9 may also be important. The predict ed substrate recognition motif of P2, but not of P1, corresponds to parts o f the melaminophenylarsenical and diamidine molecules, confirming the poten t inhibition observed with these trypanocides for P2-mediated adenosine tra nsport (K-i = 0.4-2.4 mu M).