Nitrobenzylthioinosine (
NBTI) was systematically modified by attachment of substituents at the 2-, 5'-, 3'- and 2'-positions in order to assess the importance of these positions in the binding of
NBTI to high-affinity membrane binding sites (Kd < or = 1 nM) and the inhibition of
NBTI-sensitive, equilibrative
nucleoside transport by mammalian cells. We determined the effect of the derivatives on the equilibrium binding of 1 nM [3H]
NBTI to human erythrocytes and mouse
P388 leukemia cells and on the inhibition of zero-trans influx of
formycin B in P388 cells and equilibrium exchange of
uridine in human erythrocytes. Placement of substituent groups at the 5'-position of
NBTI had relatively little effect on its binding to high-affinity binding sites or its inhibition of
nucleoside transport, regardless of the size of the substituent group (up to about 1000 kDa). All substituents at the 2-position considerably reduced the affinity of
NBTI to membrane binding sites and its potency as an inhibitor of
nucleoside transport, but some substituent groups reduced the affinity of binding more than the inhibition of
nucleoside transport. The effect of the 2-substituents was not directly related to their size. Attachment of a
succinate at the 3'- or 5'-position also reduced to a greater extent the binding of
NBTI than its inhibition of
nucleoside transport, which was relatively little affected. Attachment of
succinates at both the 3' and 5'-positions almost completely abolished both binding to high-affinity sites and inhibition of
nucleoside transport. Both functions of
NBTI were abolished completely by the simultaneous blockage of the 2'- and 3'-positions. None of the
NBTI derivatives significantly inhibited
NBTI-resistant equilibrative
formycin B transport in P388 and Novikoff rat
hepatoma cells at concentrations of < or = 1 microM.