From the
Departamento de Estructura y Función de Proteínas, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, CVelázquez 144, 28006 Madrid, Spain,
|| Departamento de Productos Naturales, Instituto de Química Orgánica General, CSIC, CJuan de la Cierva 3, 28006 Madrid, Spain,
** Servicio de Histología, Departamento de Investigación, Hospital Ramón y Cajal, 28034 Madrid, Spain
Received for publication, December 17, 2002
, and in revised form, March 20, 2003.
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ABSTRACT |
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INTRODUCTION |
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Acidic and basic fibroblast growth factors (aFGF and bFGF)1 are two very important angiogenesis-promoting polypeptides. They belong to a family of mitogens that to date includes 23 polypeptides (7, 8). The two members of the group that were first described are aFGF and bFGF. They have very similar biochemical and biological properties and have served as paradigms for the whole family (9, 10). FGFs are often detected in tumors (11, 12). Histological studies have shown that the growth of solid tumors is suppressed by monoclonal antibodies against bFGF and that this effect was due to the inhibition of bFGF-induced angiogenesis (13). In addition, it has been reported that antisense targeting of bFGF and FGF receptor-1 in human melanomas blocks intratumoral angiogenesis and tumor growth (14). Finally, angiogenesis promoted by another well characterized inducer of blood vessel development, the vascular endothelial growth factor, has been reported to require endogenous expression of bFGF by endothelial cells, and it is therefore blocked by neutralizing antibodies against bFGF (15, 16). Consequently, inhibition of FGF mitogenic activity seems a crucial target for the development of antiangiogenic cancer treatments. FGFs show a characteristically high affinity for the glycosaminoglycan heparin and the glycosidic moiety of heparan sulfate proteoglycan (9, 10, 17). It has been demonstrated that binding to either of these polysulfates is required for FGFs to recognize their specific tyrosine kinase receptor on the cell surface (1820). In the case of aFGF-driven mitogenesis, the presence of either heparin or some specific polyanions like myo-inositol hexasulfate is, in addition, a nearly absolute requirement (21, 22). Thus, disruption of the interaction of FGFs with heparin and heparan sulfates seems an obvious target for antiangiogenesis.
The polysulfonated binaphthyl ureas known as suramins are considered potential anti-cancer agents because of their anti-angiogenic activity (23). The antiangiogenic activity of suramins is based, at least in part, on their ability to disrupt the interaction of many growth factors with their membrane receptors, such as in the case of FGFs and their tyrosine kinase receptors (19, 2426). Because it has been shown that heparin disrupts aFGF·suramin complexes (27) and counteracts the antiangiogenic effect of these polysulfonated ureas (25, 26), suramins are considered to act by blocking the heparin-binding sites of FGFs (28). Another group of antiangiogenic and anti-tumoral compounds is comprised by suradistas, a type of non-cytotoxic synthetic binaphthalene sulfonic distamycin-A derivatives. These compounds tightly interact with FGFs, inhibit the binding of these polypeptides to the tyrosine kinase cell membrane receptors, and suppress FGF-induced angiogenesis and neovascularization in vivo (2931), probably by a mechanism similar to that of the suramins.
Despite the potential relevance of the suramins and the suradistas as inhibitors of the FGF angiogenic activity, no high resolution data of their complexes with these polypeptides are available so far. Knowledge of the molecular structure of these complexes could be used to improve their pharmacological properties and lead to the design of better angiogenesis inhibitors. A major reason for the lack of data is that these two inhibitors promote the appearance of heterogeneous aggregates of the complexes (23, 27). Of outstanding interest was the finding that 1,3,6-naphthalenetrisulfonate (NTS) constitutes a minimal model for the inhibition of aFGF mitogenic activity by suramins and suradistas (32). Moreover NTS has been tested with positive results both in vitro and in vivo as an inhibitor of aFGF-induced angiogenesis and glioma proliferation (3235). This suggests potential new avenues for the development of new antiangiogenic compounds. The three-dimensional structure of aFGF complexed with NTS obtained by 1H NMR showed that NTS binds weakly and quite heterogeneously to the heparin-binding site of aFGF, in agreement with its low binding constant (32). The studies of Lozano et al. (32) also showed that naphthalene derivatives containing a reduced number of sulfonate groups per aromatic ring seem to act as better inhibitors of aFGF mitogenic activity than NTS. However, this enhancement was accompanied by the appearance of a clear toxicity of the compounds against quiescent cells at concentrations where they inhibit aFGF mitogenic activity. Based on these preliminary data, we have explored a wide window of charge, size, and relative position of substituents of the naphthalene ring in an attempt to identify new naphthalene derivatives that combine the highest inhibitory activity with the lowest toxicity. In a further step, the most pharmacologically promising of those compounds, 5-amino-2-naphthalenesulfonate, was evaluated for its antiangiogenic activity in vivo with positive results, and the three-dimensional structure of its complex with aFGF was solved by x-ray crystallography to a resolution of 2.0 Å. All of these studies allow formulation of a set of stereochemical rules that may constitute the basis for the development of new antiangiogenesis treatments.
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EXPERIMENTAL PROCEDURES |
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Sodium SaltsSodium salts were prepared by adding the required amounts of 0.1 N sodium hydroxide to a suspension of the corresponding sulfonic acid in water. Evaporation of the solvents yielded the sodium salts.
Acetyl Amide Derivatives (C-4, C-10, C-15, and C-19)Acetyl amides were prepared by heating a suspension of the corresponding sodium sulfonate salts in acetic anhydride for 4 h at 90 °C. The solvent was evaporated, yielding the acetyl amide derivatives.
Benzoyl Amide Derivatives (C-5, C-11, and C-16)A slight excess of benzoyl chloride (1.2 equivalents) was added to a solution of the corresponding sulfonic acid in pyridine. The solvent was evaporated after 30 min, and the residue was chromatographed on a silica gel column (MeOH:CH2Cl2, 1:9 v/v). The purified product was transformed in its corresponding sodium salt (see above).
Compound C-2Concentrated H2SO4 (0.32 ml) was added to 2-methyl-naphtalene (2.8 mmol). The mixture was heated in an oil bath at 80 °C for 3 h. This solution was cooled, poured over ice, and made basic by the addition of 1 N NaOH. The product was separated in cold and isolated by filtration.
Compound C-8 Ion-exchange resin (Amberlite) (H+ form) was added to a solution of sodium 2-naphthalenesulfonate in methanol. The mixture was stirred overnight and filtered, and the solvent was evaporated. The residue was lyophilized and yields the acid.
Compound C-12Excess of phthalic anhydride (1.3 equivalents) was added to a solution of C-9 (0.6 mmol) in methanol. The mixture was stirred overnight and evaporated. The residue was chromatographed on silica gel column (MeOH:CH2Cl2, 3:7 v/v) to yield the required product.
Compound C-13Adipoyl chloride (0.5 equivalents) was added to a suspension of C-9 (1.0 mmol) in pyridine. Stirring was continued for 48 h, and the solvent was evaporated. The residue was digested with a small volume of methanol, and the solid that separated was collected and characterized.
Compound C-25Gramine (6.45 mmol) was dissolved in concentrated H2SO4 (10 ml) and stirred for 5 min at 0 °C. This solution was poured over ice and made basic by the addition of 1 N NaOH. This aqueous solution was extracted with dichloromethane, made neutral by addition of 1 N H2SO4, and evaporated. The residue was digested with hot ethanol, and the solids were separated by filtration. The ethanol solution was evaporated, and the residue was subjected to column chromatography (ethanol) to yield the desired compound.
Proliferation Assays
The effect of the compounds listed on Table I on aFGF-driven mitogenesis and quiescent cell viability was studied in vitro as described by Lozano et al. (32) using 100-µl cultures of plated fibroblasts Balb/c 3T3. The concentration of aFGF used in the assays (0.32 ng/ml) correspond to that eliciting a half-maximum mitogenic response at myo-inositol hexasulfate concentrations that induce a nearly full activation of the mitogen (20 µg/ml; 32). The inhibitors and, when pertinent, aFGF were added in 10 µl of a solution containing myo-inositol hexasulfate (200 µg/ml), bovine serum albumin (1 mg/ml) in Dulbecco's modified Eagle's medium, once the cultures have reached quiescence (15 h after being transferred to minimal culture medium).
Angiogenesis Assay
Pathogen-free C57/BI/6 mice (Charles River) weighing 25 ± 4 g were used. The animals were housed in plastic cages in temperature- and humidity-controlled conditions; food and water were available ad libitum, and a 12-h light/dark schedule was maintained. The animal welfare guidelines of the National Institutes of Health and the European Union were carefully followed.
Sterile gelatin sponge cubes of 10 mm3 (Curaspon Dental, Clinimed Holding, Zwanenburg, The Netherlands) were implanted subcutaneously in the backs of the mice after induction of intraperitoneal anesthesia as described (33). The animals were distributed as follows: Group A (n = 10) sponges loaded with 200 µl of phosphate-buffered saline containing 29 µg·ml-1 heparin; Group B (n = 40) sponges were embedded with the same solution containing 10 µg·ml-1 aFGF. After implantation of the sponge into the subcutaneous pouch, the skin was sutured. The mice of Group B were randomly divided in four groups (n = 10) that received 0.008, 0.08, 0.8, and 8 mg·kg-1 of 5-amino-2-NMS, respectively, in an intraperitoneal injection of 200 µl of phosphate-buffered saline, 24 h after surgery (25, 33). All procedures were performed under sterile conditions.
For angiogenesis evaluation, the mice were re-anesthetized as described, and the sponges were surgically extracted and treated for histological studies as described by Cuevas et al. (33) 7 days after the implants. Neovascularization was quantified with computerized morphometric software connected to a microscope. Ingrowths of neovessels into sponges was assessed by measuring surface area with erythrocyte content. Neovascularization was analyzed in four predetermined visual fields in three different sections at 10-fold magnification. The statistical analyses were performed by using Student's t test.
Protein Expression, Purification, and Crystallization
The protein was obtained using established protocols (22). The residues are numbered according to their positions in the primary structure of the 154-amino acid aFGF (36). Crystals of the complex between aFGF and either 5-amino-2-NMS or NTS were grown using the sitting drop vapor diffusion method at 295 K by mixing 0.75 mM protein, 1.5 mM of the inhibitor and 60% sodium/potassium tartrate buffered with 5 mM sodium phosphate (pH 7.85) over a well solution of 1.3 M Li2SO4. The typical crystal size achieved after 1 month was about 0.7 x 0.5 x 0.2 mm. To corroborate the presence of the inhibitor inside the crystals, several crystal specimens were washed using the crystallization buffer, solubilized with 1 N NaOH, and subjected to electrospray mass spectrometry. The mass spectra were recorded on a liquid chromatography coupled mass spectroscopy HP 1100 spectrometer using electrospray ionization.
Data Collection, Structure Determination, and Refinement
For the diffraction experiments, the crystals were cryo-cooled in a stream of nitrogen gas at 100 K using the crystallization solution supplemented with 20% glycerol as cryo-protectant. X-rays from a synchrotron source at Beamline X11-DESY (Hamburg, Germany) were employed to collect data on a Marccd detector. Diffraction was visible to 1.8 Å, but only data to 2.0 Å were suitable for subsequent processing with MOSFLM (37) and programs of the CCP4 suite (38). The crystals belong to the monoclinic system, space group P2 (a = 96.5 Å, b = 47.2 Å, c = 97.8 Å, = 107.0°), with six protein molecules/asymmetric unit corresponding to a 48% solvent content. The calculated VM value (39) is 2.4 Å3/Da. The structure was determined by molecular replacement using AMoRe (40). The searching model was the one derived from 2axm
[PDB]
(41), with one molecule searched six times at 3.5 Å resolution. The positions of the six molecules in the asymmetric unit were optimized using rigid body refinement, leading to an R value of 44.9 (Rfree = 45.2). Inspection of the initial sigma-A maps showed that certain side chains were quite disordered, although some electron density was obvious. This problem was overcome by use of noncrystallographic symmetry restraints. Several steps of simulated annealing and B factor refinement were carried out with CNS (42) until the Rwork and Rfree values dropped to 22.8 and 25.9%, respectively. Bulk solvent and anisotropic overall B factor corrections were applied through the refinement. The final model is of excellent geometry as shown in Table II.
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Coordinates
The coordinates for the final model have been deposited in the Protein Data Bank under the code 1hkn.
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RESULTS |
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The half-maximum inhibitory activity of C-9 (sodium 5-amino-2-naphthalenesulfonate or 5-amino-2-NMS) is 265 µM, more than 2 orders of magnitude lower than that of naphthalene trisulfonate (32). Fig. 1b shows that inhibition of cell proliferation by 5-amino-2-NMS was eliminated by increasing concentrations of heparin, as was observed in previous studies with suramins and NTS (25, 26, 32). However, a similar heparin concentration to that employed in the case of NTS could produce reversal of the antiangiogenic activity by 5-amino-2-NMS only when this compound is present at a concentration of ≤1 mM (not shown). This finding, which is in agreement with the lower half-maximum inhibitory activity of 5-amino-2-NMS relative to NTS, suggests that although both compounds compete with heparin for binding to aFGF, 5-amino-2-NMS does so with higher efficiency.
An evaluation of the in vivo relevance of 5-amino-2-NMS as an angiogenesis inhibitor was carried out using a standard mouse angiogenesis assay previously used with suramin and NTS (25, 33). Fig. 2 illustrates the inhibition of the neovascularization induced by aFGF in gelatin sponges subcutaneously implanted in mice treated with 5-amino-2-NMS. Computation of the number of neovessels/area unit at different dosages of the inhibitor showed that neovascularization inhibition is already appreciable in mice receiving between 0.008 and 0.08 mg·kg-1, drastic at 0.8 mg·kg-1 (results not shown), and almost total at 8 mg·kg-1, a condition at which only traces of neovascularization were seldom observed (Fig. 2). These data clearly reveal that 5-amino-2-NMS is a neovascularization inhibitor that is considerably more effective than suramin and NTS. In these two last cases concentrations of 200 mg·kg-1 appear to be required for substantial inhibition of neovascularization (25, 33). Equivalent results were obtained when aFGF was substituted with bFGF (not shown). The highest 5-amino-2-NMS doses assayed did not produce any toxic death, apparent disturbances in animal behavior, or changes in weight.
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Atomic Structure of aFGF Bound to 5-Amino-2-NMSAccording to Fig. 1a, stereochemistry seems to be a relevant factor in the inhibition of aFGF mitogenic activity by naphthalene sulfonates and related compounds. To gain insight into this stereochemistry, which may contribute to new pharmacological developments in the treatment of anomalous angiogenesis, aFGF was co-crystallized in the presence of 5-amino-2-NMS, the most active of the tested compounds (Table I) that did not show apparent toxic side effects within the range of its inhibitory activity. For comparative purposes, crystals were also grown in the presence of NTS (see "Discussion").
Monoclinic crystals of the complex between aFGF and 5-amino-2-NMS grew to their maximum size in about 2 weeks using sodium potassium-tartrate as the main precipitant (Fig. 3a). These crystals belong to space group P2, contain six molecules in the asymmetric unit, and diffract up to 2.0 Å under synchrotron radiation. The structure was solved by molecular replacement using the coordinates of aFGF in complex with heparin (Protein Data Bank code 2axm
[PDB]
) (41). 5-Amino-2-NMS molecules were only added after the first refinement cycle into clear peaks in both the 2Fo - Fc and the Fo - Fc electron density maps. In the final model, no backbone ,
torsions angles were located in the disallowed region of the Ramachandran plot, whereas more than 92% of the residues were located in the most favored regions, as defined by the program PROCHECK (44). The data collection, structure determination, and refinement statistics are summarized in Table II.
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Four of the six molecules present in the asymmetric unit do not bind 5-amino-2-NMS, probably because of steric hindrance or a different charge environment from the neighboring symmetry-related molecules. In the two remaining aFGF molecules a very substantial electron density of about 7 was observed, into which 5-amino-2-NMS could be easily modeled (solvent molecules occupy the same binding site in the other four cases). However, whereas the sulfonate group and the ring directly bound to it perfectly could be fitted to the electron density, some atoms of the ring that holds the amino group were outside, probably as a consequence of the different orientations of the compound across the asymmetric units of the crystal. Therefore, to check whether the compound was really inside the crystals, we proceeded to a mass spectrometry analysis of washed crystal specimens. As shown in Fig. 3, the distribution of masses observed in the spectrum of a pure sample of 5-amino-2-NMS (Fig. 3b) was also present in the crystal sample (Fig. 3c).
Inhibitor-binding Site5-Amino-2-NMS binds directly to a positively charged cavity present on the molecular surface of aFGF in a 1:1 stoichiometric ratio (Fig. 4a). This inhibitorbinding region corresponds to the heparin-binding site of aFGF (41, 45) as shown in Fig. 4b, where both the aFGF bound 5-amino-2-NMS and heparin appear superimposed. As this figure illustrates, a strong steric hindrance prevents both ligands from binding simultaneously to aFGF. The sulfonate substituent of 5-amino-2-NMS is located in a small pocket of the protein, which appears to be occupied by the sulfonate groups of the ligand in the heparin-bound aFGF (41, 45) and by a sulfate or phosphate anion in all of the reported crystallo-graphic structures of free aFGF (46, 47). Several amino acid residues of aFGF that interact with heparin in the crystal structures reported by DiGabriele et al. (41) and Pellegrini et al. (45) either establish contact with 5-amino-2-NMS or closely surround it. Five of those residues involved in the direct interaction with 5-amino-2-NMS have been described as essential for heparin binding: Asn32, Lys127, Lys132, Gln141, and Lys142 (the first three forming hydrogen bonds with the sulfonate group of 5-amino-2-NMS and the last two through hydrophobic interactions with the naphthalene ring; Fig. 4, a and c). In addition, the amino group of 5-amino-2-NMS forms a hydrogen bond with the carbonyl group of Gly140 (Fig. 4c).
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Effects of 5-Amino-2-NMS Binding on the Structure of aFGFAs illustrated in Fig. 5 (a and b), the aFGF patch to which heparin and 5-amino-2-NMS bind shows differences depending on the bound ligand. This probably arises because of the specific interactions that each of these ligands establishes with the protein. These differences arise from different conformations of the side chains of the amino acids contributing to the interaction (not shown) with the backbone topology remaining practically unaffected (Fig. 5c), except for the loop connecting -strands 8 and 9 (Glu104Tyr108;
8/
9 loop; Fig. 5c). In the heparin-bound protein this loop shows an open conformation, whereas in the 5-amino-2-NMS complex it closes toward
-strand 12, generating a more compact structure (Fig. 5c). Moreover, secondary structure analyses performed with the program DSSP (48) shows that the
8/
9 loop in the 5-amino-2-NMS complex forms a left-handed 310 helix that likely confers certain rigidity to this turn. In previous reports (49), the secondary structure of the
8/
9 loop is either a type I or type I' turn (this last case likely because of the crystal packing). The
8/
9 loop does not constitute a site of crystal contact either in the case of aFGF in the presence of heparin or 5-amino-2-NMS.
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The 8/
9 loop belongs to the main high affinity receptor recognition site of aFGF (45, 50, 51). Interaction with 5-amino-2-NMS produces additional rearrangements at this region of the protein (bottom face of the structures represented in Fig. 5, a and b); thus, this region appears considerably flatter in the heparin-bound protein than in the 5-amino-2-NMS bound one, especially at the left and right borders. In addition, this relative approximation of the left and right edges upon 5-amino-2-NMS binding appears to be accompanied by a certain alteration in the charge layout of the receptor-binding site (Fig. 5, a and b). All of these changes likely hamper the interaction of the protein with its high affinity receptor.
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DISCUSSION |
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Protein aggregation has been suggested to be a key process in the inhibition of FGFs mitogenic activity by suramins (27). In addition, these compounds have been proposed to block the interaction of aFGF with heparin, because it has been demonstrated that their anti-mitogenic activity can be reverted by this glycosaminoglycan (25, 26). This last effect is supported by the studies of aFGF complexed to naphthalene sulfonates (32), a common chemical function of suramins and the analogous inhibitor family of suradistas, and by the structural data presented here. Because heparin binding is an essential requirement for the assembly of the FGF-cell membrane receptor mitogenesis-triggering complex (1821), the obstruction of the aFGF/heparin interaction is a likely mechanism for the inhibition of aFGF mitogenic activity by 5-amino-2-NMS (Fig. 4b). Moreover, an additional conformational change is triggered upon 5-amino-2-NMS binding at the aFGF face that is recognized by the cell surface FGF receptor (Fig. 5). One of the alterations of this face involves the development of some rigidity at the 8/
9 loop. Because proteins often undergo conformational changes when they bind to other molecules or macromolecular structures, the decrease in flexibility of the
8/
9 loop of aFGF upon 5-amino-2-NMS binding should probably be considered an additional unfavorable contribution of 5-amino-2-NMS to the conformational fitting of aFGF to its receptor binding site. Slight changes on this loop have been shown to deeply affect the specific mitogenic activity of aFGF (52). Hindered heparin binding and poor fitting onto its receptor may both account for the low mitogenesis inducing capacity of aFGF complexed with 5-amino-2-NMS.
A set of stereochemical leads for improving the potential pharmacological applicability of naphthalene sulfonates in antiangiogenesis can be drawn out from a comparative analysis of the whole set of inhibition experiments reported here (supplemental data and Table I). First, the sulfonate group should preferentially be located at position 2 rather than 1 of the planar aromatic ring of naphthalene, because all 2-NMS derivatives are more potent inhibitors than are those of 1-NMS. This can be explained on the basis of the structural data reported here showing that the sulfonate group (the primary anchoring functional group of the inhibitor to the heparin-binding site of the protein) should pack better within its docking pocket when it is at position 2 of the naphthalene ring than at position 1. Second, among those 2-NMS derivatives containing one amino group (which is likely to be positively charged under physiological conditions), those with this group at positions 5 or 6 are better inhibitors of aFGF mitogenic activity (C9 and C17 versus C14 and C18, respectively), probably because of the hydrogen bonding of the amino group to residues surrounding the anchoring site (Fig. 4c). Third, the size of the functional group at position 5 seems very relevant, because although the substitution of the amino group at that position by a small amide does not substantially alter the inhibitory activity (C9 versus C10), a significant impairment was evident when a bulky amide was introduced (C9 versus C11 and C13). Fourth, although naphthalene seems to constitute an adequate template for the core of the inhibitor, it can be substituted by quinoline without appreciable decrease of the inhibitory activity, so long as the positions of the amino and sulfonate groups relative to 5-amino-2-NMS (C9 versus C23) are maintained. As a final remark, we should point out that there is one exception to the rule that the most optimal position for the sulfonate group is 2 in the set of compounds we studied: 4-amino-3-hydroxy-1-NMS, which showed the best inhibitory activity (C-7; Fig. 1a). The real significance of this exception is difficult to ascertain at this time. The cell division promoting activity of this compound suggests that it could be affecting the mitogenesis-signaling cascade at different levels, at concentrations close to those at which it was also observed to inhibit aFGF-driven mitogenesis. Thus, the assay summarized in Fig. 1a and in the supplemental data of C-7 might be the result of inhibitory processes in addition to the mere inactivation of aFGF. This does not seem to be the case with 5-amino-2-NMS, because the inhibition can be reverted by heparin as expected from the structural data reported here.
Additional leads can also be derived from the three-dimensional structure of the complex 5-amino-2-NMS·aFGF. The poor definition of the electron density corresponding to the external ring of 5-amino-2-NMS bound to aFGF probably points toward a structural locus of instability within the complex. Consequently, the affinity of the ligand for the protein could perhaps be improved by stabilizing that part of the naphthalene ring that lies outside the electron density, possibly by adding polar groups prone to form hydrogen bonds with neighboring residues of the protein, in a fashion similar to the amino group of 5-amino-2-NMS. In conclusion, the structural data reported here might help in the development of new antiangiogenic treatments against cancer and other pathophysiological situations in which inhibition of endothelial cell proliferation would be a desirable therapeutic goal.
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FOOTNOTES |
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* This work was supported in part by Grant 2FD97-0519 from the Ministerio de Ciencia y Tecnología (Spain), a research contract from the Programa de Grupos Estratégicos of the Comunidad Autónoma de Madrid, and a CSIC-Glaxo Wellcome S.A. agreement. 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.
The on-line version of this article (available at http://www.jbc.org) contains supplemental data.
Both authors contributed equally to this work.
¶ Supported by a Ph.D. fellowship from Ministerio de Ciencia y Tecnología and by a grant from the Residencia de Estudiantes.
To whom correspondence should be addressed: Centro de Investigaciones Biológicas, CSIC, C/Velázquez 144, 28006 Madrid, Spain. Tel.: 34-91-5649065; Fax: 34-91-5649065; E-mail: gimenez_gallego{at}cib.csic.es.
1 The abbreviations used are: aFGF, acidic FGF; bFGF, basic FGF; FGF, fibroblast growth factor; NTS, 1,3,6-naphthalenetrisulfonate; NMS, naphthalene monosulfonate.
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ACKNOWLEDGMENTS |
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REFERENCES |
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