Nucleoside transporters (NTs) are integral membrane proteins that are designed to transport nucleosides into the human body. Nucleoside transport across cell membranes is physiologically important as it provides the major source of nucleosides for DNA/RNA synthesis in cells and it is responsible for the termination of adenosine signaling. Another important fact about NTs is that many nucleoside-derived anticancer and antiviral drugs are transported into cells by way of NTs. Despite their importance in physiology and pharmacology, the molecular mechanism of NTs is largely unknown due to the lack of atomic structures of the transporters. Great advances in the field depend on the structure determination and illumination of the transport mechanism of NTs. Our long- term goal is to understand the fundamental mechanism of selective nucleoside transport by concentrative nucleoside transporters (CNTs) with structural and functional studies. CNTs belong to a family of the solute carrier transporter superfamily (SLC28) and use an ion gradient to transport nucleosides actively against their chemical gradients. We have chosen to use a CNT homolog from Vibrio cholerae (vcCNT) as a model system for our structural and functional studies. vcCNT is an excellent model system to study hCNTs that shares significant sequence homology and functional properties with hCNTs. We solved the crystal structure of vcCNT in complex with uridine. The structure reveals the overall architecture and the nucleoside-binding site of the transporter, providing mechanistic insights of selective nucleoside transport by vcCNT and CNT in general. These new results have led us to propose a combined structure-function study with the following three aims: 1) Determination of the crystal structures of vcCNT in complex with different nucleosides and nucleoside drugs, 2) Functional characterization and comparison of vcCNT with hCNTs, and 3) Structure determination of vcCNT in alternate conformations. The proposed studies will significantly advance our understanding of the mechanism of nucleoside and nucleoside drug transport by prokaryotic and eukaryotic CNTs, which eventually will facilitate the development of new therapeutic reagents.
In humans, CNTs are expressed in a wide array of organs, such as kidney, intestine, and liver. The expression of NTs is upregulated in cancer cells in a manner specific to the type of cancer. NTs are essential for the transport of many nucleoside-derived anticancer and antiviral drugs across cell membranes as cancer cell lines that do not express NTs show resistance to anticancer drug treatment while cancer cell lines expressing NTs show cytotoxic responses upon drug treatment. A wide range of nucleoside-derived drugs utilizes NTs for transport across cell membranes including anticancer drugs (e.g. Ara-C and gemcitabine) and antiviral drugs. Therefore the CNT is a potential drug target to improve bioavailability of anticancer and antiviral drugs and reduce side effects associated with these drugs.
