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Originally published In Press as doi:10.1074/jbc.M402602200 on May 3, 2004

J. Biol. Chem., Vol. 279, Issue 28, 29598-29605, July 9, 2004
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The Nucleoside Derivative 5'-O-Trityl-inosine (KIN59) Suppresses Thymidine Phosphorylase-triggered Angiogenesis via a Noncompetitive Mechanism of Action*

Sandra Liekens{ddagger}§, Ana-Isabel Hernández¶||, Domenico Ribatti**, Erik De Clercq{ddagger}, María-José Camarasa¶, María-Jesús Pérez-Pérez¶, and Jan Balzarini{ddagger}

From the {ddagger}Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium, the **Department of Human Anatomy and Histology, Piazza Giulio Cesare, 11 Policlinico, I-70124 Bari, Italy, and the Instituto de Química Médica, Consejo Superior de Investigaciones Científicas, Juan de la Cierva 3, 28006 Madrid, Spain

Received for publication, March 8, 2004 , and in revised form, April 28, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 2'-deoxynucleosides to 2-deoxyribose 1-phosphate and their respective pyrimidine bases. TPase also recognizes several nucleoside analogues that are being used clinically as antiviral (i.e. 5-(E)-(2-bromovinyl)-2'-deoxyuridine, 5-trifluoromethyl-2'-deoxyuridine, 5-iodo-2'-deoxyuridine) or anti-tumor (i.e. 5-fluoro-2'-deoxyuridine) agents (2). As such, TPase plays a key role in the pyrimidine nucleoside salvage pathway, as well as in the inactivation of cytotoxic pyrimidine nucleoside analogues.

The angiogenic protein, platelet-derived endothelial cell growth factor, isolated from platelets in 1987, was shown to be identical to TPase (3). Mutational analysis revealed that the enzymatic activity of TPase is essential for its angiogenic effect (4). In vitro, TPase/platelet-derived endothelial cell growth factor stimulates endothelial cell migration and is therefore not an endothelial cell growth factor (5). However, endothelial cell migration, in the absence of proliferation, is sufficient to induce an angiogenic response. The mechanism by which TPase induces angiogenesis is still unclear, although significant progress has been made recently. In contrast to other angiogenesis stimulators, TPase does not contain a signal sequence required for cell secretion. Also, an endothelial cell receptor for TPase has never been identified. Therefore, it is most likely that the products of its enzymatic activity, rather than TPase itself, possess the angiogenic properties.

Recent observations suggest that 2-deoxy-D-ribose induces angiogenesis by generating oxygen radical species, which induce the secretion of oxidative stress-responsive angiogenic factors, like vascular endothelial cell growth factor, interleukin-8, and matrix metalloproteinase-1 (6). Moreover, Hotchkiss et al. (7) have shown that TPase and 2-deoxy-D-ribose activate specific integrins, which directly links TPase-induced endothelial cell migration to intracellular signal transduction pathways.

TPase is overexpressed in many solid tumors. Moreover, TPase levels correlate well with microvessel density in breast, ovarian, colorectal, endometrial, and esophageal cancers (8-12), pointing to an important role for this enzyme in tumor vascularization. In addition, TPase has been shown to inhibit tumor cell apoptosis (12). Therefore, there is an anti-cancer potential for potent and specific TPase inhibitors.

So far, few potent TPase inhibitors have been described, most of which are pyrimidine analogues, including 6-aminothymine, 6-amino-5-bromouracil, and 5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]uracil hydrochloride (13). Based on the structure of Escherichia coli TPase (14), which shows 40% sequence identity with human TPase (15), we recently designed and synthesized the first purine derivative (7-deazaxanthine (7DX)) with inhibitory activity against E. coli TPase (16). 7DX was then used as a lead compound to develop more potent purine-based multisubstrate inhibitors of TPase (17). The prototype compound (TP65) contains an alkyl phosphonate moiety, covalently linked to 7DX (Fig. 1). We proved that TP65 is able to concomitantly interact at the nucleoside and phosphate-binding sites (in a competitive manner with regard to the natural substrates dThd and phosphate), thus immobilizing the enzyme in an open, inactive conformation (18). In addition, TP65 inhibited TPase-induced angiogenesis in vitro and in the chick chorioallantoic membrane (CAM) assay (19).



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  		FIG. 1.
Structural formulae of the TPase inhibitors TP65, KIN59, and KIN56.

 
In the present study, we describe the inhibitory activity of the inosine analogue KIN59 (Fig. 1) against human and bacterial recombinant TPase. KIN59 is an unusual TPase inhibitor; it is a purine nucleoside with an intact ribose moiety. Extensive kinetic studies show that, in contrast to previously described TPase inhibitors, KIN59 does not compete with the phosphate or the nucleoside-binding site of the enzyme. Moreover, KIN59 was far more active than other TPase inhibitors (tested in our laboratory) in the CAM assay, indicating that other, yet unrevealed sites on the TPase enzyme may be essential to confer certain biological properties to TPase, such as its angiogenic potential. Identification of these sites might help to clarify the mechanism of angiogenesis stimulation by TPase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—KIN56 (5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine; Fig. 1), 2'-deoxyadenosine, hypoxanthine, 5'-deoxy-5'-(methylthio)adenosine, and 5'-deoxyadenosine were purchased from Sigma. Inosine was from Fluka (Switzerland), and 5'-chloro-5'-deoxyadenosine was from Berry and Associates (Dexter, MI).

Compound Synthesis—We have employed an improved synthetic procedure compared with the reported method (20) for the synthesis of 5'-O-tritylinosine (KIN59; Fig. 1), as follows: To a solution of inosine (1.0 g, 3.73 mmol) in dry pyridine (19 ml), trityl chloride (1.8 g, 6.34 mmol), and 4-N,N'-dimethylaminopyridine (18.2 mg, 0.15 mmol) were added. The mixture was stirred at 80 °C for 15 h. The reaction mixture was diluted with ethyl acetate (20 ml) and a 0.1 N HCl solution (20 ml). The organic phase was washed with water (20 ml) and brine (20 ml). The organic layer was dried on anhydrous Na2SO4, filtered, and evaporated. The residue was purified by flash column chromatography (CH2Cl2:MeOH, 10:1) to yield 780 mg (41%) of 5'-O-trityl-inosine as a white solid: melting point, 230-232 °C; melting point described in the literature, (20) 207-210 °C; mass spectrometry (electrospray, positive mode), m/z 511 (M +1)+; 1H NMR (Me2SO-d6) {delta} 3.43 (dd, J = 6.2, 4.2 Hz, 2H, H-5'), 4.06 (q, J = 5.4 Hz, 1H, H-4'), 4.26 (m, 1H, H-3'), 4.62 (m, 1H, H-2'), 5.23 (d, J = 5.9 Hz, 1 H, OH), 5.60 (d, J = 5.7 Hz, 1 H, OH), 5.90 (d, J = 4.6 Hz, 1H, H-1'), 7.29 (m, 14H, Ph), 7.99 (s, 1H, H-8), 8.21 (s, 1H, H-2), 12.20 (br s, 1H, NH-1); 13C NMR (Me2SO-d6) {delta} 63.94 (C-5'), 70.20 (C-3'), 73.34 (C-2'), 83.42 (C-4'), 86.07 (CPh3), 88.00 (C-1'), 124.58 (C-5), 127.06, 127.89, 128.26, 143.60 (Ph), 138.85 (C-8), 145.77 (C-2), 148.15 (C-4), 156.58 (C-6. Analysis for C29H26N4O5: C, 68.22; H, 5.13; N, 10.97. Found: C, 68.05; H, 5.29; N, 10.76. The synthesis of TP65 has been described by Esteban-Gamboa et al. (17). 5'-O-(t-Butyldimethylsilyl)inosine was synthesized according to the published procedure (21).

Cell Cultures—BALB/c mouse brain microvascular endothelial 10027 cells (MBECs) were kindly provided by Prof. M. Presta (Brescia, Italy). MBECs were grown in Dulbecco's modified minimum essential medium supplemented with 10% fetal calf serum. This spontaneously immortalized cell line was identified as endothelial on the basis of different phenotypic markers (22).

Purification of Recombinant TPase—The E. coli TPase gene was expressed in E. coli as a glutathione S-transferase fusion protein, as described previously (19). The pMOAL-10T vector containing the human TPase gene (fused to glutathione S-transferase) was kindly provided by Prof. R. Bicknell (Oxford, UK). Protein purification was performed as described (19).

TPase Enzyme Assays—The phosphorolysis of thymidine (dThd) by human or E. coli glutathione S-transferase-TPase was measured by HPLC analysis. The incubation mixture (500 µl) contained 10 mM Tris-HCl (pH 7.6), 1 mM EDTA, 2 mM potassium phosphate (unless otherwise stated in the kinetic experiments), 150 mM NaCl, and 100 µM of dThd in the presence of 0.025 U TPase. Incubations were performed at room temperature. At different time points (i.e. 0, 20, 40, and 60 min), 100-µl fractions were withdrawn, transferred to an Eppendorf tube thermo block, and boiled at 95 °C for 5 min. Next, the samples were rapidly cooled on ice, and dThd was separated from thymine (Thy) and quantified in the samples on a reverse phase RP-8 column (Merck) by HPLC analysis. The separation of Thy and dThd was performed by a linear gradient from 98% buffer B (50 mM NaH2PO4 with 5 mM heptane sulfonic acid, pH 3.2) and 2% acetonitrile to 50% buffer B and 50% acetonitrile. The retention times of Thy and dThd were 4.2 and 8.5 min, respectively. UV-based detection of Thy and dThd was performed at 267 nm.

To evaluate the inhibitory effect of the compounds, a variety of inhibitor concentrations, including 1 mM, 100 µM, 10 µM, and 0 µM (control) were added to the reaction mixture (500 µl) containing 100 µM of dThd. Aliquots of 100 µl were withdrawn from the reaction mixture at several time points, as described above, heated at 95 °C to inactivate the enzyme, and analyzed by HPLC.

To discriminate between reversible and irreversible inhibition of TPase, 150, 100, and 50 µM KIN59 were exposed to different TPase concentrations (0.005, 0.004, 0.003, 0.0025, 0.002, 0.001, and 0.0005 unit) in a 100-µl reaction mixture and incubated for 30 min at room temperature. Conversion of dThd to Thy was evaluated as described above.

In the kinetic assays, where the inhibitory effect of the test compounds was evaluated at varying concentrations of inorganic phosphate (Pi), the compounds were tested at concentrations ranging from 75 µM to 400 µM, in the presence of 2, 3, 5, 10, and 20 mM Pi. The (saturating) dThd concentration was kept fixed at 1000 µM. The reaction mixture (100 µl) was then incubated for 20 min with 0.005 unit of TPase, after which the tubes were heated to 95 °C before cooling and HPLC analysis.

In the kinetic assays, where the inhibition of TPase enzymatic activity was evaluated at varying concentrations of dThd, the compounds were tested at concentrations ranging from 100 to 200 µM, in the presence of 125, 250, 500, 750, and 1000 µM dThd. The concentration of inorganic phosphate was kept constant at 25 mM. The analysis of the dThd-to-Thy conversion was performed as described above.

Cell Proliferation Assays—MBEC were seeded in 24-well plates at 10,000 cells/cm2. After 16 h, the cells were incubated in fresh medium in the presence of the test compounds, as indicated under "Results" (see Fig. 7). On day 5, the cells were trypsinized and counted by a Coulter counter (Harpenden Hertz, UK).



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  		FIG. 7.
Effect of the different nucleoside analogues on the proliferation of MBE cells. MBE cells were seeded in 24-well plates at 10,000 cells/cm2. After 16 h, the cells were incubated in fresh medium in the presence of various concentrations of the test compounds. On day 5, the cells were trypsinized and counted in a Coulter counter.

 
CAM Assay in Fertilized Chicken Eggs—The in vivo CAM angiogenesis model was performed as described (23). Fertilized eggs were incubated for 3 days at 37 °C when 3 ml of albumen was removed (to detach the shell from the developing CAM), and a window was opened on the eggshell exposing the CAM. The window was covered with cellophane tape, and the eggs were returned to the incubator until day 9 when the compounds were applied. The compounds were placed on sterile plastic discs (Ø, 8 mm), which were allowed to dry under sterile conditions. A solution of cortisone acetate (100 µg/disc; Sigma) was added to all discs to prevent an inflammatory response. A loaded and dried control disc was placed on the CAM ~1 cm away from the disc containing the test compound(s). Next, the windows were covered, and the eggs were further incubated until day 11 when angiogenesis was assessed. At day 11, the membranes were fixed with 10% buffered formalin (Janssen Chimica, Geel, Belgium), the plastic discs were removed, and the eggs were kept at room temperature for 2 h. A large area around the discs was cut off and placed on a glass slide. To determine the number of blood vessels, a grid containing three concentric circles with diameters of 4, 5, and 6 mm was positioned on the surface of the CAM that was previously covered by the disc. Next all of the vessels intersecting the circles were counted. A two-tailed paired Student's t test was used to assess the significance of the obtained results.

Histological Examination of Angiogenesis in the CAM Assay—For histological examination of the TPase-induced angiogenic response, a modified CAM assay was used. Fertilized White Leghorn chicken eggs (30 for each experimental series) were incubated at 37 °C at constant humidity. On day 3 of incubation a square window was opened in the eggshell after removal of 2-3 ml of albumen. The window was sealed with a glass, and the eggs were returned to the incubator. On day 8, 1-mm3 sterilized gelatin sponges (Gelfoam Upjohn, Kalamazoo, MI) were placed on top of the growing CAM, according to the method of Ribatti et al. (24). The sponges were loaded with either 10 µl of TPase (Sigma), 250 nmol of KIN59, or 10 µl of TPase with 250 nmol of KIN59. CAMs were examined daily until day 12 and photographed in ovo with a stereomicroscope equipped with a camera system (Olympus Italia, Milan, Italy). On day 12, blood vessels, entering the sponges within the focal plane of the CAM, were counted by two observers in a double-blind fashion at 50x magnification. The mean values ± standard deviation (S.D.) for vessel counts were determined for each analysis. CAMs were then fixed in ovo in Bouin's fluid, dehydrated in graded ethanol, embedded in paraffin, serially sectioned at 7 µm according to a plane parallel to their free surface, and stained with a 0.5% aqueous solution of toluidine blue (Merck). For microscopic quantification of the angiogenic response, a planimetric method of point counting was used, as previously described (24).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibitory Effect of KIN59 and Related Compounds against Purified E. coli and Human Recombinant TPase—The inhibitory activity of KIN59 and KIN56 against TPase was evaluated in the presence of 100 µM dThd as the natural substrate. The TPase-catalyzed conversion of dThd to Thy was inhibited by KIN59 at a 50% inhibitory concentration (IC50) of 44 ± 3 and 67 ± 20 µM, respectively, when purified E. coli and human TPase were used as the enzyme source. The IC50 values for the closely related compound KIN56 were 232 ± 28 and 351 ± 27 µM, respectively, that is at 5-fold higher concentrations than required for KIN59. To reveal whether KIN59 inhibits the E. coli and human TPases in a reversible or irreversible manner, KIN59 was added at fixed concentrations of 150, 100, and 50 µM to duplo-dilutions of TPase. Should the compound act as an irreversible inhibitor of TPase, equimolar drug concentrations would completely annihilate the enzymatic activity, whereas lower enzyme concentrations would be fully inactivated by KIN59, resulting in an inhibition line parallel with the control enzyme activity curve in the absence of inhibitor. If the inhibition of TPase by KIN59 is reversible, both the inhibition line and the control enzyme activity curve should converge on the intersection (zero) point of the abscissa/ordinate graph. As evident from Fig. 2, the latter kinetics was obtained. Thus, KIN59 behaved as a reversible inhibitor against both E. coli and human TPases. The kinetic parameters, including the Ki values and the nature of TPase inhibition by KIN59, were also determined in the presence of varying concentrations of dThd (at saturating concentrations of potassium phosphate) and in the presence of varying concentrations of potassium phosphate (at saturating concentrations of dThd) (Fig. 3). KIN59 emerged as a potent inhibitor of E. coli TPase with Ki values of 39 µM against dThd and 146 µM against phosphate, resulting in Ki/Km values of 0.039 and 0.10, respectively. Surprisingly, the compound inhibited the enzyme reaction in a noncompetitive fashion both with respect to dThd and to phosphate (Fig. 3). This means that KIN59 does not bind to either the dThd or phosphate-binding sites of the enzyme. A similar noncompetitive interaction was observed between human TPase and KIN59 (data not shown).



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  		FIG. 2.
Reversible inhibition of E. coli TPase (upper panel) and human TPase (lower panel) by KIN59. dThd was used as the natural substrate.

 



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  		FIG. 3.
Lineweaver-Burk plots of E. coli TPase inhibition by KIN59, in the presence of variable concentrations of dThd (upper panel) and phosphate (lower panel).

 
Inhibition of Angiogenesis in the CAM Assay—The kinetic results obtained for KIN59 against TPase indicate that this compound might interact with a yet unidentified site in the enzyme, different from the dThd- and Pi-binding sites. To evaluate whether the interaction of KIN59 with TPase is sufficient to abrogate the angiogenic activity of TPase, we studied the effect of KIN59 (and its structurally related adenosine derivative KIN56) on TPase-induced neovascularization in the CAM assay (Figs. 4 and 5). We previously showed that 10 units of a commercial batch of pure E. coli TPase significantly stimulates the formation of new blood vessels on the CAM of fertilized chicken eggs (19). After 2 days of exposure to TPase, allantoic vessels developed radially toward the stimulus in a "spoked wheel" pattern (Fig. 4, compare A with B). The effect of TPase on the CAM has also been quantified (Fig. 5A) with an average stimulation of 44 ± 10% as compared with control CAMs (p < 0.001). The addition of TPase + 250 nmol of KIN59 resulted in a complete inhibition of TPase-induced angiogenesis (Fig. 4, C and D). Indeed, KIN59 not only annihilated the stimulatory effect of TPase, it also efficiently inhibited the formation of normal CAM vessels in the absence of exogenously added TPase (i.e. normal blood vessel development that occurs between day 9 and 11) (Fig. 5A). Thus, only big veins (that were already present at day 9 when the compounds were added) were visible after microscopic evaluation of the CAM at day 11 (i.e. 44% stimulation by TPase versus 89% inhibition by TPase in the presence of KIN59) (Fig. 5A). No signs of toxicity or inflammation could be observed at and immediately around the site of drug exposure. At 100 nmol, KIN59 inhibited angiogenesis in the CAM by 32 ± 13% (Fig. 5A). A significant inhibitory effect of TPase-induced angiogenesis was still noted at 50 nmol/disc of KIN59, i.e. 24% inhibition versus 44% stimulation for TPase alone (p < 0.001). Only at 10 nmol/disc, there was no significant inhibition of TPase-induced neovascularization by KIN59 (+26% ± 9; not significant) (Fig. 5A). It should be noted that inosine did not inhibit the enzymatic activity of TPase nor TPase-induced neovascularization in the CAM assay. These results indicate that the presence of the trityl group at the 5'-position of inosine is required for the anti-TPase and anti-angiogenic activity of KIN59 (Fig. 5B).



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  		FIG. 4.
Effect of KIN59 on TPase-induced angiogenesis in the CAM assay. At day 9 of incubation, discs containing either phosphate-buffered saline (A), TPase (B), or TPase plus 250 nmol of KIN59 (C and D) were applied onto the CAM. Two days later, new blood vessels had developed toward the disc containing TPase (B), whereas a complete absence of capillaries was noted when the disc contained TPase plus KIN59 (C and D). The original magnifications were x50 for A-C and x1 for D.

 



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  		FIG. 5.
Inhibition of TPase-induced angiogenesis in the CAM assay by KIN59. At day 9 of incubation, discs containing either TPase or TPase plus test compound were applied onto the CAM. At day 11, the percentage of stimulation (positive values) or inhibition (negative values) of blood vessel formation was determined (see "Experimental Procedures"). The effect of different amounts of KIN59 on TPase-induced angiogenesis is shown in A. B, change in vascular density of CAM in the presence of 250 nmol of various test compounds.

 
Therefore, we evaluated the activity of another 5'-O-tritylated purine nucleoside, KIN56 (5'-O-4,4'-dimethoxytrityl-2'-deoxyadenosine), in the CAM assay (Fig. 5B). KIN56, at 250 nmol, was able to cause a complete inhibition of capillary formation, i.e. 53% of the treated CAMs (TPase + KIN56) were avascular. However, the remaining 47% of the CAMs showed inflammation and were characterized by many disorganized vessels. In these cases, blood vessel formation could not be reliably scored, because of the inflammation. Also here, the free nucleoside, 2'-deoxyadenosine, at a concentration of 250 nmol/disc, did not inhibit TPase-induced angiogenesis, confirming the importance of the 5'-O-trityl group for the anti-angiogenic activity of these nucleoside analogues (data not shown).

The multisubstrate analogue inhibitor TP65, which is equally as potent as KIN59 in inhibiting the enzymatic activity of TPase, was included as a reference compound (19). TP65 afforded, at 250 nmol/disc, a complete inhibition of the typical radial pattern of blood vessels, induced by exogenously added TPase, without affecting normal angiogenesis (Fig. 5B).

Histological Analysis of TPase-induced Neovascularization in the CAM Assay—Several variants of the CAM assay have been described. The method used above is reliable and quantitative but does not distinguish at day 11 between newly formed vessels and those already present at day 9, when the discs are applied. Therefore, we employed a different approach, which has the additional advantage of allowing histological examination of the newly formed microvessels. Macroscopic observation of the CAM at day 12 showed that gelatin sponges immerged with the TPase enzyme induce an angiogenic response and allantoic vessels developed radially toward the implant (Fig. 6A). Microscopically, a highly vascularized tissue was observed among the trabeculae of these sponges that consisted of newly formed blood vessels, mainly 3-10-µm-diameter capillaries growing perpendicularly to the plane of the CAM (Fig. 6B). The angiogenic response to TPase was at least reduced by 70% when TPase was administered together with 250 nmol of KIN59 (Fig. 6, C and D), whereas KIN59 alone induced the formation of an avascular area around and inside the sponge, as demonstrated at the macroscopic and microscopic level (Fig. 6, E and F). Quantification of the macroscopic and microscopic angiogenic response at day 12 is shown in Table I.



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  		FIG. 6.
Histological examination of CAMs of 12-day-old chick embryos incubated for 4 days with gelatin sponges loaded with TPase (A and B), TPase plus KIN59 (C and D), or KIN59 alone (E and F). Note that at the macroscopic level in A there are numerous blood vessels converging like spokes toward the sponge, in C there are few blood vessels, and in E an avascular area is recognizable around the sponge. These data are confirmed at the microscopic level (B, D, and F), where inside the sponge treated with KIN59 (F) no blood vessels are recognizable. The original magnifications were x 50 for A, C, and E and x400 for B, D, and F.

 


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  		TABLE I
Chick embryo CAM-sponge assay: macroscopic and microscopic assessment of vascular density on day 12 of incubation

 
Cytostatic Effect on Microvascular and Macrovascular Endothelial Cells—Because the potent anti-angiogenic activity of KIN59 could not be explained solely by its effect on the TPase enzymatic (dThd -> Thy) activity, we evaluated KIN59 and KIN56 (as well as inosine and hypoxanthine) for their anti-proliferative activity against MBECs (Fig. 7). Both inosine (the free unsubstituted nucleoside) and hypoxanthine (the base) had no effect on endothelial cell proliferation (at 300 µM). KIN59 slightly inhibited the proliferation of MBECs with an IC50 of 59.6 µM. In contrast, KIN56, which is five times less potent than KIN59 in inhibiting TPase enzymatic activity, showed a significant inhibition of endothelial cell proliferation, with an IC50 value of 1.5 µM, that is at a 40-fold lower concentration than KIN59.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
TPase is one of the key enzymes involved in the salvage biosynthesis and catabolism of pyrimidine 2'-deoxynucleosides. It catalyzes the reversible phosphorolysis of dThd and related analogues, releasing the free (thymine) base derivative and 2-deoxy-{alpha}-D-ribose-1-phosphate (25). TPase stimulates endothelial cell migration in vitro and angiogenesis in vivo and plays an important role in tumor growth and metastasis (5, 26, 27). The enzymatic activity of TPase was found to be crucial for its angiogenic effect (4). However, the mechanism by which TPase induces angiogenesis is under debate. It is assumed that the products of the enzyme reaction are angiogenic, in particular 2-deoxy-D-ribose that is obtained by dephosphorylation of 2-deoxy-{alpha}-D-ribose-1-phosphate. It has been proposed that the extracellular release of 2-deoxy-D-ribose forms a chemotactic gradient that mediates endothelial cell migration (25, 28, 29). Recent observations also suggest that 2-deoxy-D-ribose induces angiogenesis by generating oxygen radical species, which induce the secretion of oxidative stress-responsive angiogenic factors, like vascular endothelial cell growth factor, interleukin-8, and matrix metalloproteinase-1 (6, 28). Moreover, Hotchkiss et al. (7) have shown that TPase and 2-deoxy-D-ribose activate specific integrins, which directly links TPase-induced endothelial cell migration to intracellular signal transduction pathways. However, there are also recent observations where the biological actions of TPase have not been explained by the enzymatic activity of TPase and/or the release of 2-deoxy-D-ribose. For example, a metabolite of thymine ({beta}-amino-isobutyric acid) was found to stimulate microvessel growth from cultured segments of rat aorta in vitro (30). In addition, it is not clear how TPase inhibits apoptosis of tumor cells. Fas-induced apoptosis in KB cells, transfected with TPase (KB/TPase) (31), and cisplatin-induced apoptosis in Jurkat/TPase cells (32) does not seem to require TPase enzymatic activity. In contrast, the enzymatic activity of TPase is required for apoptosis induced by hypoxia in KB/TPase (33) and HL60 cells (34).

Structurally, TPase is a dimer of two identical subunits, each with a molecular mass ranging from 45 (E. coli) to 55 kDa (mammals). Human TPase shares 39% sequence identity with E. coli TPase (14) and 40% with pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus (35). Till very recently, only the structure of the bacterial enzymes had been solved and shown that each subunit of the homodimer appears as a large mixed {alpha}/{beta} domain and a smaller {alpha}-helical domain separated by a cleft. The phosphate-binding site is located in the {alpha}/{beta} domain, whereas the thymidine-binding site is located in the {alpha}-helical domain (14, 35). It is assumed that a domain closure of the cleft is necessary to generate the active site of the enzyme. This domain closure has been simulated by molecular modeling studies and the catalytic mechanism of the enzyme has been proposed (36, 37). Very recently, the structure of the human TPase has been revealed in complex with a pyrimidine derivative, TPI that appears to mimic the substrate transition state (15). The human TPase structure is proposed to be in the closed conformation.

Most TPase inhibitors identified so far are substituted uracil analogues that compete with the pyrimidine-binding site of TPase (13, 38-40). Based on the structure of E. coli TPase, we designed and synthesized the first purine derivative (7DX) with inhibitory activity against E. coli TPase (16). 7DX was used as a lead compound to develop a novel type of multisubstrate analogue inhibitors of TPase. The prototype compound (1-(8-phosphonooctyl)-7-deazaxanthine, TP65) contains a phosphonate group, covalently linked to 7DX (Fig. 1), and is able to concomitantly interact at the phosphate and pyrimidine-binding sites of TPase, thus immobilizing the enzyme in an open, inactive conformation (17, 18). We have demonstrated the inhibitory activity of TP65 against angiogenesis, induced by TPase in vitro and in the CAM assay (19). Here, we describe the potent inhibitory activity of a new purine nucleoside-based drug (KIN59) against recombinantly expressed human and bacterial TPases. TPase is known to be highly specific for 2'-deoxyribosyl-derivatives of pyrimidines, and this specificity has been explained based on structural features (14). KIN59 represents a very unusual TPase ligand, being a 5'-O-substituted purine riboside. Detailed kinetic studies showed that, in contrast to previously described TPase inhibitors, KIN59 does not compete with the nucleoside or phosphate-binding site of the enzyme. In the CAM assay, KIN59 completely prevented TPase-stimulated blood vessel formation, without any visible side effects. Although KIN59 proved as effective as TP65 in inhibiting the enzymatic activity of TPase, KIN59 was far more active than TP65 in the CAM assay. These findings point to the existence of a novel, till now unknown, allosteric binding site (different from the thymidine and phosphate-binding sites in TPase) that may also play a role in the angiogenic properties of TPase. Because the mechanism (or mechanisms) by which TPase exerts its biological actions is much under debate, the search for additional interaction sites in TPase, with substances like KIN59, might help to clarify the molecular role of TPase in various biological processes.

In contrast to KIN59, the free nucleoside inosine did not abrogate the activity of TPase, indicating that the presence of the trityl group is important to afford anti-TPase and anti-angiogenic activity to KIN59. To confirm this hypothesis, we included another 5'-O-trityl nucleoside derivative, KIN56 (5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine), in our study. Also KIN56 inhibited TPase-induced angiogenesis, whereas 2'-deoxyadenosine was inactive (data not shown), pointing again to an important role for the trityl moiety in the observed activity of these nucleoside analogue inhibitors. To assess whether the inhibition of TPase activity by KIN59 and KIN56 can be ascribed to the absence of the free 5'-OH, several commercially available purine nucleosides that lack a free 5'-OH (i.e. 5'-deoxyadenosine, 5'-deoxy-5'-methyl-thioadenosine, and 5'-chloro-5'-deoxyadenosine) were evaluated. None of these compounds showed inhibitory activity against TPase (IC50 values > 1000 µM) (data not shown). Moreover, an inosine derivative substituted at the 5'-position with a bulky lipophilic group different from trityl (i.e. 5'-O-(tert-butyldimethylsilyl)inosine) was synthesized and tested. Also this compound did not inhibit TPase enzymatic activity at 1000 µM (data not shown). Taken together, we have shown that the absence of the 5'-OH group in the purine ribosides is not sufficient to confer anti-TPase activity, confirming the requirement for the trityl group, as present in KIN59 and KIN56.

Surprisingly, KIN59 not only inhibited neovascularization, induced by exogenously added TPase, it also completely prevented the development of those blood vessels that emerged in the absence of any exogenously added TPase (i.e. basal angiogenesis in the CAM). Several hypotheses can be put forward to explain the remarkably potent anti-angiogenic activity of KIN59. First, KIN59 may inhibit the activity of endogenous TPase. Although the expression of TPase during normal CAM development has never been reported, preliminary data (obtained by Western blot analysis) indicate that TPase is present in CAM extracts at different times of development, although we have not been able to confirm the enzymatic activity in isolated CAMs yet.2 Therefore, inhibition of endogenously produced TPase by KIN59 may result in the abrogation of normal CAM angiogenesis. Second, TPase has been shown to induce the secretion of other angiogenic factors, including chemokines (i.e. interleukin-8), proteolytic enzymes (i.e. matrix metalloproteinase-1), and growth factors (i.e. vascular endothelial growth factor) (6). Therefore, although angiogenesis is a complex process, induced by a variety of molecules, inhibition of one factor (TPase) in the angiogenic cascade may also affect this process indirectly by abrogating the activity of several downstream angiogenesis stimulators. Third, several angiogenic factors may be present in the CAM at suboptimal concentrations, acting in a synergistic manner so that inhibition of one factor would result in a significant effect even in the presence of the remaining angiogenic factors. Indeed, a monoclonal antibody to FGF2 has also been shown to block basal angiogenesis in the CAM, although it only affects the activity of a single growth factor (41). Fourth, potential toxicity of KIN59 may contribute to the inhibition of normal blood vessel formation. To test this possibility, we evaluated the anti-proliferative effect of KIN59 and KIN56 against microvascular endothelial cells. KIN59 inhibited the proliferation of endothelial cells at relatively high concentrations (59.7 µM), whereas KIN56 showed pronounced toxicity (50% inhibition of cell proliferation at 1.5 µM). These findings indicate that the anti-angiogenic effect of KIN59 is unlikely to be related to endothelial cell toxicity.

Our findings do not explain why TP65 and KIN59 possess similar inhibitory activity against TPase, whereas KIN59 is markedly more anti-angiogenic than TP65 in the CAM assay. It may be postulated that the (noncompetitive) interaction of KIN59 with the new allosteric interaction site on TPase blocks the angiogenic activity of TPase better than the (competitive) interaction of TP65 with the substrate-binding sites, because of the lack of endogenous competitive (i.e. dThd and phosphate) substrate for KIN59. Alternatively, binding of KIN59 to TPase may affect the interaction of TPase with other proteins that are important for the angiogenic process. Also, the interaction of KIN59 with any other of the several angiogenic factors that may be present at suboptimal concentrations in the CAM cannot be discarded.

In conclusion, our data on the potent anti-angiogenic activity of the noncompetitive TPase inhibitor KIN59 indicate that the biological (i.e. angiogenic) activity of TPase might not only be confined to its functional nucleoside and phosphate-binding sites. Identification of other potential regulatory (i.e. allosteric) binding sites in TPase might shed new light on the mechanisms of TPase-triggered angiogenesis stimulation and apoptosis inhibition, which is important to obtain a better insight into the molecular role of TPase in the progression of cancer and angiogenic diseases.


   FOOTNOTES
 
* This work was supported by grants from the Belgische Federatie Tegen Kanker (to S. L.) and the Geconcerteerde Onderzoeksactie-Vlaanderen Nr 00/12 (to J. B., E. D. C.), European Commission Grant QLRT-2001-01004 (to J. B., M.-J. P.-P., and M.-J. C.), Spanish Ministerio de Educacion y Ciencia SAF2003-07219-C02-01, and Italian Ministry for Education, University and Research-Interuniversity Funds for Basic Research (to D. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Predoctoral fellow of the Ministerio de Ciencia y Tecnología. Back

§ Post-doctoral research assistant from the Onderzoeksfonds-K.U. Leuven. To whom correspondence should be addressed: Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium. Tel.: 32-16-33-73-55; Fax: 32-16-33-73-40; E-mail: sandra. liekens{at}rega.kuleuven.ac.be.

1 The abbreviations used are: TPase, thymidine phosphorylase; CAM, chick chorioallantoic membrane; dThd, thymidine; 7DX, 7-deazaxanthine; MBEC, mouse brain endothelial cell; Thy, thymine; HPLC, high pressure liquid chromatography. Back

2 S. Liekens, E. De Clercq, and J. Balzarini, unpublished results. Back


   ACKNOWLEDGMENTS
 
We are grateful to Ria Van Berwaer for excellent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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S. Liekens, A. Bronckaers, A.-I. Hernandez, E.-M. Priego, E. Casanova, M.-J. Camarasa, M.-J. Perez-Perez, and J. Balzarini
5'-O-Tritylated Nucleoside Derivatives: Inhibition of Thymidine Phosphorylase and Angiogenesis
Mol. Pharmacol., August 1, 2006; 70(2): 501 - 509.
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