Journal of Biological Chemistry vol:279 issue:28 pages:29598-605
Thymidine phosphorylase (TPase) catalyzes the reversible phosphorolysis of pyrimidine deoxynucleosides to 2-deoxy-d-ribose-1-phosphate and their respective pyrimidine bases. The enzymatic activity of TPase was found to be essential for its angiogenesis-stimulating properties. All of the previously described TPase inhibitors are either pyrimidine analogues that interact with the nucleoside-binding site of the enzyme or modified purine derivatives that mimic the pyrimidine structure and either compete with thymidine or act as a multisubstrate (competitive) inhibitor. We now describe the inhibitory activity of the purine riboside derivative KIN59 (5'-O-tritylinosine) against human and bacterial recombinant TPase and TPase-induced angiogenesis. In contrast to previously described TPase inhibitors, KIN59 does not compete with the pyrimidine nucleoside or the phosphate-binding site of the enzyme but noncompetitively inhibits TPase when thymidine or phosphate is used as the variable substrate. In addition, KIN59 was far more active than other TPase inhibitors, previously tested by us, against TPase-induced angiogenesis in the chorioallantoic membrane assay. The observed anti-angiogenic effect of KIN59 was not accompanied by inflammation or any visible toxicity. Inosine did not inhibit the enzymatic or angiogenic activity of the enzyme, indicating that the 5'-O-trityl group in KIN59 is essential for the observed effects. In contrast with current concepts, our data indicate that the angiogenic activity of TPase is not solely directed through its functional nucleoside and phosphate-binding sites. Other regulatory (allosteric) site(s) in TPase may play an important role in the mechanism of TPase-triggered angiogenesis stimulation and apoptosis inhibition. Identification of these site(s) is important to obtain a better insight into the molecular role of TPase in the progression of cancer and angiogenic diseases.