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 nucleosidebinding 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.Angiogenesis is the process by which new blood vessels arise from pre-existing vessels (1). It is essential during embryogenesis for the formation of a vascular plexus, which ensures an adequate blood supply to all developing tissues. In adults, neovascularization is limited to wound healing and the female reproductive cycle. During these processes, angiogenesis is tightly regulated. Unregulated angiogenesis may lead to the progression of several inflammatory diseases and is an essential component of solid tumor growth and metastasis. The formation of new blood vessels requires several sequential steps, which are regulated by a number of angiogenic factors, including chemokines, growth factors, integrins, and enzymes, such as proteases and thymidine phosphorylase (TPase) 1 (1).TPase is an enzyme that catalyzes the reversible phosphorolysis of pyrimidine 2Ј-deoxynucleosides to 2-deoxyribose 1-phosphate and their respective pyrimidine bases. TPase also recognizes several nucleoside analogues that are being used clinically as antiviral

at. tocol YER-2002245-031416GN). The protocol was also carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011). The project design to obtain human samples was approved by the ethical committee of the Instituto de Salud Carlos III (Madrid, Spain). In addition, samples and/or data from patients included in this study were provided by the Biobanco i+12 in the Hospital 12 de Octubre integrated in the Spanish Hospital Biobanks Network (RetBioH; www. redbiobancos.es) following standard operation procedures, with appropriate approval of the Ethical and Scientific Commitees, Madrid Spain. Paraffin-embedded tissue sections and OCT-embedded fresh tissue from 11 pulmonary fibrosis patients and 3 controls without any lung pathology were obtained. All patients provided informed consent.

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