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 kinetically characterized. The P1 transporter displayed a higher affinity and capacity for adenosine (K(m) = 0.38 +/- 0.10 microM, V(max) = 2.8 +/- 0.4 pmol x 10(7) cells(-1) x s(-1)) than the P2 transporter (K(m) = 0.92 +/- 0.06 microM, V(max) = 1.12 +/- 0.08 4 pmol x 10(7) cells(-1) x s(-1)). To formulate a structure-activity relationship for the interaction of adenosine with the transporters, a series of analogs were evaluated as potential inhibitors of adenosine transport, and the K(i) values were converted to binding energy. The P1 transporter was found to be selective inhibited by purine nucleosides (K(i) approximately 1 microM for inosine and guanosine), but nucleobases and pyrimidines had little effect on P1-mediated transport. The P1 transporter appears to form hydrogen bonds with N3 and N7 of the purine ring as well as with the 3' and 5' hydroxyl groups of the ribose moiety, with apparent bond energies of 12.8 to 15.8 kJ/mol. The P2 transporter, in contrast, had high-affinity (K(i) = 0.2-4 microM) for 6-aminopurines, including adenine, 2'-deoxyadenosine, and tubercidin, but not for any oxopurines. The main interaction of adenosine with the P2 transporter is suggested to be via hydrogen bonds to N1 and the 6-amino group. Additional pi-pi interactions of the purine ring and electrostatic interactions with N9 may also be important. The predicted substrate recognition motif of P2, but not of P1, corresponds to parts of the melaminophenylarsenical and diamidine molecules, confirming the potent inhibition observed with these trypanocides for P2-mediated adenosine transport (K(i) = 0.4-2.4 microM).
de Koning
H P
HP
Research School of Biosciences, University of Kent at Canterbury, Canterbury, Kent, UK.
Jarvis
S M
SM
D000241:Adenosine / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009687:Nuclear Proteins / Q000378:metabolism*; D010419:Pentamidine / Q000494:pharmacology; D015800:Protozoan Proteins; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D014344:Trypanocidal Agents / Q000494:pharmacology*; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000378:metabolism*
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D009687:Nuclear Proteins; D015800:Protozoan Proteins; D018047:Receptors, Purinergic P1; C400023:TbNT2 protein, Trypanosoma brucei; D014344:Trypanocidal Agents; D010419:Pentamidine; D000241:Adenosine