RESUMO
We found that 5'-O-trityl-inosine (KIN59) inhibits recombinant bacterial (E. coli) and human thymidine phosphorylase (TPase) with an IC50 of 44 microM and 67 microM, respectively. In contrast to previously described TPase inhibitors, KIN59 does not compete with thymidine (dThd) at the pyrimidine nucleoside-binding site or with inorganic phosphate (Pi) at the phosphate-binding site of the enzyme. These findings are strongly suggestive for the presence of an allosteric binding site at the enzyme. TPase is identical to the angiogenic protein platelet-derived endothelial cell growth factor (PD-ECGF). As such, PD-ECGF stimulates angiogenesis in the chick chorioallantoic membrane (CAM) assay. This angiogenic response was completely inhibited by KIN59. Inosine did not inhibit the enzyme or the angiogenic effect of TPase, confirming that the 5'-O-trityl group in KIN59 is essential for the observed effect. Our observations indicate that allosteric sites in TPase may regulate its biological activity.
Assuntos
Inibidores Enzimáticos/farmacologia , Inosina/análogos & derivados , Timidina Fosforilase/antagonistas & inibidores , Compostos de Tritil/farmacologia , Sítio Alostérico , Animais , Galinhas , Córion/metabolismo , Escherichia coli/enzimologia , Humanos , Concentração Inibidora 50 , Inosina/farmacologia , Neoplasias/tratamento farmacológico , Neovascularização Patológica , Fosfatos , Pirimidinas/química , Timidina Fosforilase/metabolismoRESUMO
Computational studies have been conducted to built a closed form of TPase and to characterize the transition state of the phosphorylisis reaction catalyzed by TPase. The results obtained point to a crucial role of His-85 and the O2 of thymine in the catalysis. This modelled transition state forms the basis for the design of new TPase inhibitors.