The Nucleoside Derivative 5′-O-Trityl-inosine (KIN59) Suppresses Thymidine Phosphorylase-triggered Angiogenesis via a Noncompetitive Mechanism of Action

Abstract

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

Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule “crystallization chaperone” 5′-O-tritylinosine (KIN59)

“…and (:) conserved and semi-conserved substitutions, respectively. Asp-203 is shown in red, amino acids participating in substrate binding are shown in blue, and residues proposed to be involved in 5 0 -Otritylinosine (previously designated KIN59 [20]) are shown in green.…”

“…7, panel A). In sharp contrast, the competitive inhibitors 6AT and 6A5BU, which have been shown to interact with the dThdbinding site of TP [20], were able to dose-dependently decrease the enzymatic activity of wild-type and mutant D203A TP to a similar extent (Fig. 7, panels B and C) (6AT and 6A5BU inhibitor concentrations used: 50, 25 and 5 mM for wild-type TP and 1250, 625 and 125 mM for mutant D203A TP in the presence of 100 mM dThd for wild-type TP and 2500 mM dThd for mutant D203A TP).…”

“…However, we previously showed that 5 0 -O-tritylinosine inhibits TP in a non-competitive fashion [20]. If Asp-203 is part of a putative allosteric site to which this inhibitor binds one would expect that mutation of this amino acid residue to alanine would affect the inhibitory activity of this compound against TP.…”

“…5 0 -O-Tritylinosine (earlier designated KIN59) was synthesized as described previously [20,27]. The synthesis of 6AT and 6A5BU was performed as described [13,28].…”

“…Our research groups have described 7-deazaxanthine (7-DX), the first purine derivative with inhibitory activity against TP and angiogenesis in the 'chicken chorioallantoic membrane' (CAM) assay [17], and also TP65 (9-[8-phosphonooctyl]-7-deazaxanthine), the first multisubstrate inhibitor of TP that was shown to interact with both the thymidine and the phosphate binding site of TP [18,19]. More recently, we identified the purine nucleoside 5 0 -O-tritylinosine as a novel TP inhibitor [20,21], in which the presence of the trityl moiety was proven to be crucial for both its inhibitory activity against TP and its anti-angiogenic effect in the CAM assay. 5 0 -OTritylinosine is also a rather unique TP inhibitor because (i) it promotes the degradation of pre-existing immature vessels in the CAM assay, and (ii) in contrast to all previously described TP inhibitors, it inhibits TP in a non-competitive fashion with respect to both thymidine and phosphate.…”