Membrane permeation characteristics of 5'-modified thymidine analogs

The membrane permeation characteristics of 5'-deoxythymidine (5'-ddThd) and 5'-azido-5'-deoxythymidine (5'-N3-5'-ddThd) were investigated in human erythrocytes, with an inhibitor-stop assay, at 20 degrees. Uptake of both nucleoside analogs occurred without metabolism, was nonconcentrative, and was partially inhibited by nucleosides or inhibitors of nucleoside transport at micromolar permeant concentrations. At higher permeant concentrations (greater than 1.0 mM), the influx rate of each analog was linearly dependent on concentration and insensitive to inhibition by nucleosides, inhibitors of nucleoside transport, and nucleobases. Kinetic analyses using nonlinear regression revealed that a saturable component of 5'-ddThd influx (Km = 200 microM) was competitively inhibited by thymidine (dThd) (Ki = 86 microM) or 5-iodo-2'-deoxyuridine (Ki = 84 microM). Similarly, a saturable component of 5'-N3-5'-ddThd influx (Km = 220 microM) was competitively inhibited by 2-chloroadenosine (Ki = 18 microM). The Ki values for these nucleoside inhibitors were similar to their reported Km values as permeants of the nucleoside transporter. Both 5'-ddThd and 5'-N3-5'-ddThd competitively inhibited the influx of dThd (Km = 60 microM), with similar Ki values (150 and 200 microM, respectively). We conclude that these two 5'-modified dThd analogs enter human erythrocytes both by nonfacilitated diffusion and by the nucleoside transporter. The absence of the 5'-hydroxyl group of dThd (5'-ddThd) resulted in a large increase in the octanol/buffer partition coefficient, in an ability to permeate human erythrocytes by nonfacilitated diffusion, and in a 3-fold diminished binding to the nucleoside transporter. The 5'-azido group (5'-N3-5'-ddThd) resulted in an additional 1.4-fold increase in the octanol/buffer partition coefficient and in a 2-fold increase in the rate of nonfacilitated diffusion.