The relationship between ionization and affinity of nucleoside transport inhibitors.

The displacement of [3H]nitrobenzylthioinosine from nucleoside transporter-associated binding sites on calf lung tissue membranes by several transport inhibitors appeared to be pH dependent. These blockers, viz. dilazep, hexobendine, soluflazine and ST7092, provided to be more potent in this respect at pH 9.5 than at pH 6.6, although to differing extents. A further analysis showed that this behaviour could be related to the ionization characteristics of the compounds. For a quantitative evaluation of the findings presented in this study, we paid attention to the degradation of these labile compounds by esterases present in the membrane preparation. Degradation was almost fully prevented by the use of 10 mumol/l physostigmine, a potent ester hydrolase inhibitor, in the binding experiments. This concentration of physostigmine did not displace any [3H]NBI binding. From the aqueous ionization constants of the compounds the relative distribution of the diprotonated, monoprotonated and uncharged molecules at pH 6.6 and pH 9.5 was calculated. Combination of these data with the Ki values of the compounds, obtained from [3H]NBI displacement studies at both pH values and corrected for degradation, yielded the 'true' affinities of the monoprotonated and uncharged species. The relative paucity of the diprotonated species at pH 6.6 and its virtual absence at pH 9.5 made us assume that this species has only negligible affinity for the nucleoside transport protein. For all compounds it was found that the uncharged species has higher affinity than the monoprotonated molecule. This phenomenon was most pronounced for hexobendine, the difference in 'true' Ki values being more than 80-fold.