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

Abstract

The equilibrative nucleoside transporters of mammalian cells play an important role in the regulation of extracellular adenosine concentrations, and inhibition of these transporters potentiates the biological effects of adenosine. 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 species differences have been noted in es/ENT1 transporter affinity for a subset of inhibitors including draflazine and dipyridamole. Draflazine and a series of 15 chemically related compounds were compared for their abilities to: (a) inhibit the binding of [3H]NBMPR to the es/ENT1 transporter in mouse Ehrlich cell and human erythrocyte membranes, and (b) inhibit the es/ENT1 and ei/ENT2 transporter-mediated uptake of [3H]uridine in Ehrlich cells. Compounds within this series represented over a 1000-fold range of affinities for the es/ENT1 and ei/ENT2 transporters with subtype selectivities (ENT1/ENT2) ranging from 370 for R70527 to 0.17 for soluflazine. Five other analogues were identified, in addition to soluflazine, that had significantly higher affinity for the ei/ENT2 transporter compared with es/ENT1. Structure activity analyses of these data identified the requirement of a hydrophobic group connected to a 2-aminocarbonyl piperazine by a 5-carbon chain for high-affinity interactions with es/ENT1. This hydrophobic moiety was not as important for ei/ENT2 affinity and, in contrast to es/ENT1, a shorter alkyl chain 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 position of the aminocarbonyl group on the piperazine ring. This information, combined with structural data derived from molecular studies with ENT1 and ENT2 recombinant proteins, should guide further development of subtype-selective inhibitors of the equilibrative nucleoside transporters.

Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain

“…This suggests that there are multiple structural determinants for dipyridamole binding to the transporter, and that the mouse transporter shares at least one additional interaction site for dipyridamole with the human transporter that is not found in rENT1. We have also screened a variety of analogues of draflazine and have identified functional groups important for ENT1 versus ENT2 selectivity in the mouse, as well as chemical features that contribute to hENT1 versus mENT1 binding affinity [20,34]. These structure-activity data, in conjunction with the amino acid sequences of mENT1 and mENT2 determined in the present study, will assist in the delineation of the protein domains involved in the binding of draflazine, and other cardioprotective agents, to nucleoside transporters [11,35,36].…”

“…Relative to hENT1, mENT1 was also missing two amino acids (Lys-Gly) from the large central intracellular loop ; however, another mENT1 isoform (mENT1b ; Figure 1 and see below) was also identified in the present study that did not have this particular two-amino-acid deletion. In general, the major differences in ENT1 sequence between species (rat, mouse and human) occurred in the C-terminal half of the large extracellular loop between TM1 and TM2 and in the fifth extracellular loop between TM9 and TM10 ; these differences may contribute to the distinctive affinities of the transporters from the three species for the cardioprotective agents dipyridamole, dilazep and draflazine [21][22][23][24][25][26]34]. In this regard, Sundaram et al [37] have used hENT1\rENT1 chimaeric constructs to identify a region bordered by residues 100 and 231 in hENT1 as being critical for the interaction of dipyridamole and dilazep with the transport protein.…”

“…It is noteworthy that the amino acid sequence between residues 99 and 231 is highly conserved between species (Figure 2), suggesting that differences in dipyridamole sensitivity may be due to discrete amino acid substitutions in this region. Structure-activity studies [19,34,38] have led to the hypothesis that interactions with discrete hydrophobic elements of the transporter are required for high-affinity binding of inhibitors to hENT1. Comparative hydropathy analysis showed that residues 50-67 of the first extracellular loop of hENT1 are significantly more hydrophobic than the corresponding residues in mENT1 and rENT1 ; indeed, this region is the only one that displays a marked difference in hydrophobicity among the species examined.…”

Synthesis, Flow Cytometric Evaluation, and Identification of Highly Potent Dipyridamole Analogues as Equilibrative Nucleoside Transporter 1 Inhibitors

“…Draflazine, a lidoflazine analog, also exhibits high ENT1 inhibitory activity (IC 50 = 0.28-10 nM). 18 However, NBMPR and the flazine compounds like draflazine are poor candidates for further exploration. NBMPR has immunosuppressive and mutagenic activities deriving from its 6-mercaptopurine metabolite.…”

Determinants of sensitivity of human T-cell leukemia CCRF-CEM cells to immucillin-H

“…We observed that the rapid uptake of ImmH was facilitated by one of the equilibrative nucleoside transporters (ENTs). ImmH uptake was significantly enhanced in PK15 cells expressing ENT1 or ENT2 compared to ENT-deficient PK15 cells and the uptake of ImmH into CCRF-CEM cells was blocked by incubation with DP, an ENT1 and ENT2 inhibitor 8,22 . Based on ENT expression analysis, our data suggests that ENT1 is predominantly responsible for ImmH uptake in CCRF-CEM cells.…”