Interaction of a series of draflazine analogues with equilibrative nucleoside transporters: species differences and transporter subtype selectivity

The equilibrative nucleoside transporters of mammalian cells play an import ant role in the regulation of extracellular adenosine concentrations, and i nhibition of these transporters potentiates the biological effects of adeno sine. Two subtypes of equilibrative transporters have been defined by their differential sensitivities to inhibition by nitrobenzylthioinosine (NBMPR; es/ENT1, sensitive; ei/ENT2, insensitive). In addition, significant specie s differences have been noted in es/ENT1 transporter affinity for a subset of inhibitors including draflazine and dipyridamole. Draflazine and a serie s of 15 chemically related compounds were compared for their abilities to: (a) inhibit the binding of [H-3]NBMPR to the es/ENT1 transporter in mouse E hrlich cell and human erythrocyte membranes, and (b) inhibit the es/ENT1 an d ei/ENT2 transporter-mediated uptake of [3H]uridine in Ehrlich cells. Comp ounds within this series represented over a 1000-fold range of affinities f or the es/ENT1 and ei/ENT2 transporters with subtype selectivities (ENT1/EN T2) ranging from 370 for R70527 to 0.17 for soluflazine. Five other analogu es were identified, in addition to soluflazine, that had significantly high er affinity for the ei/ENT2 transporter compared with es/ENT1. Structure ac tivity analyses of these data identified the requirement of a hydrophobic g roup connected to a 2-aminocarbonyl piperazine by a 5-carbon chain for high -affinity interactions with es/ENT1. This hydrophobic moiety was not as imp ortant for ei/ENT2 affinity and, in contrast to es/ENT1, a shorter alkyl ch ain enhanced binding to ei/ENT2. These draflazine analogues also varied in their differential affinities for mouse vs. human es/ENT1 transporters, and the degree of species discrimination was strongly dependent on the positio n of the aminocarbonyl group on the piperazine ring. This information, comb ined with structural data derived from molecular studies with ENT1 and ENT2 recombinant proteins, should guide further development of subtype-selectiv e inhibitors of the equilibrative nucleoside transporters.