Abstract
Nucleoside transporters that mediate cellular uptake of therapeutic nucleoside analogs are major determinants of the pharmacokinetic properties of these compounds. Understanding the substrate selectivity of these transporters is critical in the development of therapeutic nucleoside analogs with optimal pharmacokinetic properties, including high oral bioavailability and tissue-specific distribution. In general, substrate selectivity of nucleoside transporters has been evaluated indirectly by inhibition studies. The purpose of this study was to directly measure the transport of nucleoside analogs by the sodium-coupled pyrimidine-selective transporter rCNT1 using electrophysiology methods. We used a two-electrode voltage clamp assay to investigate the substrate selectivity of rCNT1; 19 structurally diverse nucleosides and nucleoside analogs were studied. Uridine-induced currents in voltage-clamped oocytes expressing rCNT1 were sodium-, voltage-, and concentration-dependent (K0.5 = 21 μM), and were blocked by adenosine. Uridine-induced currents increased ∼5-fold upon hyperpolarization of membrane potential from −10 to −150 mV. Uridine, thymidine, and cytidine induced currents in rCNT1-expressing oocytes, whereas guanosine, inosine, and adenosine did not. Uridine, deoxyuridine, and cytidine analogs with modifications at the 3-, 4-, or 5-position were found to be substrates of rCNT1, whereas uridine and cytidine analogs modified at the 6-position were not. In addition, it was found that the 5′-hydroxyl group of the sugar is not required for transport by rCNT1. These results enhance our understanding of the structural basis for substrate selectivity of nucleoside transporters and should prove useful in the development of therapeutic nucleoside analogs.
Footnotes
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Send reprint requests to: Kathleen M. Giacomini, Ph.D., Department of Biopharmaceutical Sciences, University of California-San Francisco, 513 Parnassus Ave., S-926, San Francisco, CA 94143-0446. E-mail: kmg{at}itsa.ucsf.edu
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This work was supported by grants from the National Institutes of Health (GM42230, DK19567), the U.S. Department of Agriculture (99-35304-7975), and the Committee on Research of the Academic Senate at the University of California-San Francisco. Part of this work was presented at the Millennial World Congress of Pharmaceutical Sciences in San Francisco, CA (April 16–20, 2000).
- Abbreviations used are::
- U
- uridine
- 4TU
- 4-thiouridine
- A
- adenosine
- 5AC
- 5-azacytidine
- 6AC
- 6-azacytidine
- 6AU
- 6-azauridine
- BrUdR
- 5-bromo-2′-deoxyuridine
- C
- cytidine
- ClUdR
- 5-chloro-2′-deoxyuridine
- 3DAU
- 3-deazauridine
- 5′dFU
- 5-fluoro-5′-deoxyuridine
- FUdR
- 5-fluoro-2′-dideoxyuridine (floxuridine)
- G
- guanosine
- HMUdR
- 5-hydroxymethyl-2′-deoxyuridine
- I
- inosine
- IUdR
- 5-iodo-2′-deoxyuridine (idoxuridine)
- R
- ribavirin
- T
- thymidine
- UdR
- 2′-deoxyuridine
- Vm
- membrane potential
- Received April 11, 2000.
- Accepted June 9, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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