FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2039324166> ?p ?o ?g. }

• W2039324166 endingPage "29605" @default.
• W2039324166 startingPage "29598" @default.
• W2039324166 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 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. 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. Angiogenesis is the process by which new blood vessels arise from pre-existing vessels (1Liekens S. De Clercq E. Neyts J. Biochem. Pharmacol. 2001; 61: 253-270Crossref PubMed Scopus (645) Google Scholar). 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) 1The 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. (1Liekens S. De Clercq E. Neyts J. Biochem. Pharmacol. 2001; 61: 253-270Crossref PubMed Scopus (645) Google Scholar). 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 (2Desgranges C. Razaka G. Rabaud M. Bricaud H. Balzarini J. De Clercq E. Biochem. Pharmacol. 1983; 32: 3583-3590Crossref PubMed Scopus (143) Google Scholar). 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 (3Usuki K. Saras J. Waltenberger J. Miyazono K. Pierce G. Thomason A. Heldin C.H. Biochem. Biophys. Res. Commun. 1992; 184: 1311-1316Crossref PubMed Scopus (211) Google Scholar). Mutational analysis revealed that the enzymatic activity of TPase is essential for its angiogenic effect (4Miyadera K. Sumizawa T. Haraguchi M. Yoshida H. Konstanty W. Yamada Y. Akiyama S. Cancer Res. 1995; 55: 1687-1690PubMed Google Scholar). In vitro, TPase/platelet-derived endothelial cell growth factor stimulates endothelial cell migration and is therefore not an endothelial cell growth factor (5Ishikawa F. Miyazono K. Hellman U. Drexler H. Wernstedt C. Hagiwara K. Usuki K. Takaku F. Risau W. Heldin C.H. Nature. 1989; 338: 557-562Crossref PubMed Scopus (694) Google Scholar). 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 (6Brown N.S. Jones A. Fujiyama C. Harris A.L. Bicknell R. Cancer Res. 2000; 60: 6298-6302PubMed Google Scholar). Moreover, Hotchkiss et al. (7Hotchkiss K.A. Ashton A.W. Klein R.S. Lenzi M.L. Hui Zhu G. Schwartz E.L. Cancer Res. 2003; 63: 527-533PubMed Google Scholar) 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 (8Toi M. Hoshina S. Taniguchi T. Yamamoto Y. Ishitsuka H. Tominaga T. Int. J. Cancer. 1995; 64: 79-82Crossref PubMed Scopus (189) Google Scholar, 9Hata K. Nagami H. Iida K. Miyazaki K. Collins W.P. Obstet. Gynecol. 1998; 12: 201-206Google Scholar, 10Matsuura T. Kuratate I. Teramachi K. Osaki M. Fukuda Y. Ito H. Cancer Res. 1999; 59: 5037-5040PubMed Google Scholar, 11Seki N. Kodama J. Hongo A. Miyagi Y. Yoshinouchi M. Kudo T. Eur. J. Cancer. 2000; 36: 68-73Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 12Okamoto E. Osaki M. Kase S. Adachi H. Kaibara N. Ito H. Pathol. Int. 2001; 51: 158-164Crossref PubMed Scopus (25) Google Scholar), pointing to an important role for this enzyme in tumor vascularization. In addition, TPase has been shown to inhibit tumor cell apoptosis (12Okamoto E. Osaki M. Kase S. Adachi H. Kaibara N. Ito H. Pathol. Int. 2001; 51: 158-164Crossref PubMed Scopus (25) Google Scholar). 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 (13Matsushita S. Nitanda T. Furukawa T. Sumizawa T. Tani A. Nishimoto K. Akiba S. Miyadera K. Fukushima M. Yamada Y. Yoshida H. Kanzaki T. Akiyama S. Cancer Res. 1999; 59: 1911-1916PubMed Google Scholar). Based on the structure of Escherichia coli TPase (14Walter M.R. Cook W.J. Cole L.B. Short S.A. Koszalka G.W. Krenitsky T.A. Ealick S.E. J. Biol. Chem. 1990; 265: 14016-14022Abstract Full Text PDF PubMed Google Scholar), which shows 40% sequence identity with human TPase (15Norman R.A. Barry S.T. Bate M. Breed J. Colls J.G. Ernill R.J. Luke R.W. Minshull C.A. McAlister M.S. McCall E.J. McMiken H.H. Paterson D.S. Timms D. Tucker J.A. Pauptit R.A. Structure. 2004; 12: 75-84Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), we recently designed and synthesized the first purine derivative (7-deazaxanthine (7DX)) with inhibitory activity against E. coli TPase (16Balzarini J. Esteban-Gamboa A. Esnouf R. Liekens S. Neyts J. De Clercq E. Camarasa M.J. Pérez-Pérez M.J. FEBS Lett. 1998; 438: 91-95Crossref PubMed Scopus (78) Google Scholar). 7DX was then used as a lead compound to develop more potent purine-based multisubstrate inhibitors of TPase (17Esteban-Gamboa A. Balzarini J. Esnouf R. De Clercq E. Camarasa M.J. Pérez-Pérez M.J. J. Med. Chem. 2000; 43: 971-983Crossref PubMed Scopus (81) Google Scholar). 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 (18Balzarini J. Degrève B. Esteban-Gamboa A. Esnouf R. De Clercq E. Engelborghs Y. Camarasa M.J. Pérez-Pérez M.J. FEBS Lett. 2000; 483: 181-185Crossref PubMed Scopus (38) Google Scholar). In addition, TP65 inhibited TPase-induced angiogenesis in vitro and in the chick chorioallantoic membrane (CAM) assay (19Liekens S. Bilsen F. De Clercq E. Priego E.M. Camarasa M.J. Perez-Perez M.J. Balzarini J. FEBS Lett. 2002; 510: 83-88Crossref PubMed Scopus (40) Google Scholar). 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. 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 (20Muramatsu N. Takenishi T. J. Org. Chem. 1965; 30: 3211-3212Crossref Scopus (7) Google Scholar) 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, (20Muramatsu N. Takenishi T. J. Org. Chem. 1965; 30: 3211-3212Crossref Scopus (7) Google Scholar) 207-210 °C; mass spectrometry (electrospray, positive mode), m/z 511 (M +1)+; 1H NMR (Me2SO-d6) δ 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) δ 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. (17Esteban-Gamboa A. Balzarini J. Esnouf R. De Clercq E. Camarasa M.J. Pérez-Pérez M.J. J. Med. Chem. 2000; 43: 971-983Crossref PubMed Scopus (81) Google Scholar). 5′-O-(t-Butyldimethylsilyl)inosine was synthesized according to the published procedure (21Chu C.K. Bhadti V.S. Doboszewski B. Gu Z.P. Kosugi Y. Pullaiah K.C. Van Roey P. J. Org. Chem. 1989; 54: 2217-2225Crossref Scopus (150) Google Scholar). 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 (22Bastaki M. Nelli E.E. Dell'Era P. Rusnati M. Molinari-Tosatti M.P. Parolini S. Auerbach R. Ruco L.P. Possati L. Presta M. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 454-464Crossref PubMed Scopus (103) Google Scholar). Purification of Recombinant TPase—The E. coli TPase gene was expressed in E. coli as a glutathione S-transferase fusion protein, as described previously (19Liekens S. Bilsen F. De Clercq E. Priego E.M. Camarasa M.J. Perez-Perez M.J. Balzarini J. FEBS Lett. 2002; 510: 83-88Crossref PubMed Scopus (40) Google Scholar). 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 (19Liekens S. Bilsen F. De Clercq E. Priego E.M. Camarasa M.J. Perez-Perez M.J. Balzarini J. FEBS Lett. 2002; 510: 83-88Crossref PubMed Scopus (40) Google Scholar). 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). CAM Assay in Fertilized Chicken Eggs—The in vivo CAM angiogenesis model was performed as described (23Liekens S. Neyts J. De Clercq E. Verbeken E. Ribatti D. Presta M. Cancer Res. 2001; 61: 5057-5064PubMed Google Scholar). 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. (24Ribatti D. Gualandris A. Bastaki M. Vacca A. Iurlaro M. Roncali L. Presta M. J. Vasc. Res. 1997; 34: 455-463Crossref PubMed Scopus (198) Google Scholar). 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 50× 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 (24Ribatti D. Gualandris A. Bastaki M. Vacca A. Iurlaro M. Roncali L. Presta M. J. Vasc. Res. 1997; 34: 455-463Crossref PubMed Scopus (198) Google Scholar). 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).Fig. 3Lineweaver-Burk plots of E. coli TPase inhibition by KIN59, in the presence of variable concentrations of dThd (upper panel) and phosphate (lower panel).View Large Image Figure ViewerDownload (PPT) 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 (19Liekens S. Bilsen F. De Clercq E. Priego E.M. Camarasa M.J. Perez-Perez M.J. Balzarini J. FEBS Lett. 2002; 510: 83-88Crossref PubMed Scopus (40) Google Scholar). 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).Fig. 5Inhibition 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.View Large Image Figure ViewerDownload (PPT) 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 (19Liekens S. Bilsen F. De Clercq E. Priego E.M. Camarasa M.J. Perez-Perez M.J. Balzarini J. FEBS Lett. 2002; 510: 83-88Crossref PubMed Scopus (40) Google Scholar). 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 tho" @default.
• W2039324166 created "2016-06-24" @default.
• W2039324166 creator A5038319371 @default.
• W2039324166 creator A5039283798 @default.
• W2039324166 creator A5040662398 @default.
• W2039324166 creator A5041169941 @default.
• W2039324166 creator A5053397046 @default.
• W2039324166 creator A5054370212 @default.
• W2039324166 creator A5060088876 @default.
• W2039324166 date "2004-07-01" @default.
• W2039324166 modified "2023-10-17" @default.
• W2039324166 title "The Nucleoside Derivative 5′-O-Trityl-inosine (KIN59) Suppresses Thymidine Phosphorylase-triggered Angiogenesis via a Noncompetitive Mechanism of Action" @default.
• W2039324166 cites W1964417650 @default.
• W2039324166 cites W1965324898 @default.
• W2039324166 cites W1977972494 @default.
• W2039324166 cites W1978347107 @default.
• W2039324166 cites W1981295123 @default.
• W2039324166 cites W2000845874 @default.
• W2039324166 cites W2004296906 @default.
• W2039324166 cites W2012826954 @default.
• W2039324166 cites W2018562415 @default.
• W2039324166 cites W2021256565 @default.
• W2039324166 cites W2024361203 @default.
• W2039324166 cites W2027081836 @default.
• W2039324166 cites W2027928806 @default.
• W2039324166 cites W2033158086 @default.
• W2039324166 cites W2036797676 @default.
• W2039324166 cites W2043448793 @default.
• W2039324166 cites W2044179131 @default.
• W2039324166 cites W2051750498 @default.
• W2039324166 cites W2054303135 @default.
• W2039324166 cites W2055218751 @default.
• W2039324166 cites W2056002310 @default.
• W2039324166 cites W2060728660 @default.
• W2039324166 cites W2088982851 @default.
• W2039324166 cites W2094810573 @default.
• W2039324166 cites W2103348066 @default.
• W2039324166 cites W2108445535 @default.
• W2039324166 cites W2109197670 @default.
• W2039324166 cites W2129069064 @default.
• W2039324166 cites W2159699022 @default.
• W2039324166 cites W2165138879 @default.
• W2039324166 cites W2325931277 @default.
• W2039324166 cites W4238026009 @default.
• W2039324166 cites W60298408 @default.
• W2039324166 doi "https://doi.org/10.1074/jbc.m402602200" @default.
• W2039324166 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15123637" @default.
• W2039324166 hasPublicationYear "2004" @default.
• W2039324166 type Work @default.
• W2039324166 sameAs 2039324166 @default.
• W2039324166 citedByCount "44" @default.
• W2039324166 countsByYear W20393241662012 @default.
• W2039324166 countsByYear W20393241662013 @default.
• W2039324166 countsByYear W20393241662014 @default.
• W2039324166 countsByYear W20393241662015 @default.
• W2039324166 countsByYear W20393241662016 @default.
• W2039324166 countsByYear W20393241662018 @default.
• W2039324166 countsByYear W20393241662019 @default.
• W2039324166 countsByYear W20393241662020 @default.
• W2039324166 countsByYear W20393241662022 @default.
• W2039324166 countsByYear W20393241662023 @default.
• W2039324166 crossrefType "journal-article" @default.
• W2039324166 hasAuthorship W2039324166A5038319371 @default.
• W2039324166 hasAuthorship W2039324166A5039283798 @default.
• W2039324166 hasAuthorship W2039324166A5040662398 @default.
• W2039324166 hasAuthorship W2039324166A5041169941 @default.
• W2039324166 hasAuthorship W2039324166A5053397046 @default.
• W2039324166 hasAuthorship W2039324166A5054370212 @default.
• W2039324166 hasAuthorship W2039324166A5060088876 @default.
• W2039324166 hasBestOaLocation W20393241661 @default.
• W2039324166 hasConcept C106159729 @default.
• W2039324166 hasConcept C111472728 @default.
• W2039324166 hasConcept C111771559 @default.
• W2039324166 hasConcept C138885662 @default.
• W2039324166 hasConcept C162324750 @default.
• W2039324166 hasConcept C181199279 @default.
• W2039324166 hasConcept C185592680 @default.
• W2039324166 hasConcept C202751555 @default.
• W2039324166 hasConcept C2776120743 @default.
• W2039324166 hasConcept C2776476023 @default.
• W2039324166 hasConcept C2776543447 @default.
• W2039324166 hasConcept C2776735402 @default.
• W2039324166 hasConcept C2776991684 @default.
• W2039324166 hasConcept C2777108182 @default.
• W2039324166 hasConcept C2777610669 @default.
• W2039324166 hasConcept C552990157 @default.
• W2039324166 hasConcept C55493867 @default.
• W2039324166 hasConcept C71240020 @default.
• W2039324166 hasConcept C71615608 @default.
• W2039324166 hasConcept C89611455 @default.
• W2039324166 hasConceptScore W2039324166C106159729 @default.
• W2039324166 hasConceptScore W2039324166C111472728 @default.
• W2039324166 hasConceptScore W2039324166C111771559 @default.
• W2039324166 hasConceptScore W2039324166C138885662 @default.
• W2039324166 hasConceptScore W2039324166C162324750 @default.
• W2039324166 hasConceptScore W2039324166C181199279 @default.
• W2039324166 hasConceptScore W2039324166C185592680 @default.
• W2039324166 hasConceptScore W2039324166C202751555 @default.

• next