Gentisic acid, a compound associated with plant defense and a metabolite of aspirin, heads a new class of in vivo fibroblast growth factor inhibitors

Abstract

Fibroblast growth factors are key proteins in many intercellular signaling networks. They normally remain attached to the extracellular matrix, which confers on them a considerable stability. The unrestrained accumulation of fibroblast growth factors in the extracellular milieu, either due to uncontrolled synthesis or enzymatic release, contributes to the pathology of many diseases. Consequently, the neutralization of improperly mobilized fibroblast growth factors is of clear therapeutic interest. In pursuing described rules to identify potential inhibitors of these proteins, gentisic acid, a plant pest-controlling compound, an aspirin and vegetarian diet common catabolite, and a component of many traditional liquors and herbal remedies, was singled out as a powerful inhibitor of fibroblast growth factors. Gentisic acid was used as a lead to identify additional compounds with better inhibitory characteristics generating a new chemical class of fibroblast growth factor inhibitors that includes the agent responsible for alkaptonuria. Through low and high resolution approaches, using representative members of the fibroblast growth factor family and their cell receptors, it was shown that this class of inhibitors may employ two different mechanisms to interfere with the assembly of the signaling complexes that trigger fibroblast growth factor-driven mitogenesis. In addition, we obtained evidence from in vivo disease models that this group of inhibitors may be of interest to treat cancer and angiogenesis-dependent diseases.

Figures

FIGURE 1.

Inhibition of the FGF-driven mitogenesis in cultures of Balb/c 3T3 fibroblasts by 2,5- dihydroxyphenyl acids. A, GA. B and D, 2,5DHPS. C, HGA. A–C, FGF-1. D, FGF-2. AU, absorbance units.

FIGURE 2.

Binding of GA and 2,5DHPS to FGF-1. Top row, stereo diagrams of the 2,5DHPS (left) and GA (right) FGF-1 binding sites show the electron density maps for the inhibitors and their adjacent side chains. Electron densities, corresponding to the final 2Fo − Fc maps are shown at the 1σ contour level. Molecular models of the side chains and the inhibitors are represented by solid sticks (white, carbon; red, oxygen; blue, nitrogen), and the protein backbone is shown as a transparent gray schematic. The figure was produced using the PyMOL program (DeLano Scientific LLC). Middle row, shown are molecular surface representations of FGF-1 complexed with heparin (A) (45), GA (B), and 2,5DHPS (C). The ligands are shown as solid sticks colored as above. The electrostatic potential of the protein surface mapped in blue (positive) and red (negative) was generated using the same parameters in each of three cases with the APBS program (89). Bottom row, shown are stereoviews of the three-dimensional structure of the FGF-1 binding site for 2,5DHPS (left) and GA (right). Both inhibitors, shown as stick-and-ball models (gray, carbon; red, oxygen; yellow, sulfur) appear inside the semitransparent representation of their van der Waals volume. The electrostatic potential of the protein surface is mapped in blue (positive) and red (negative), and it was generated with the Discovery Studio Visualizer program (2.0.1.7347; Accelrys Software Inc.) using its internal parameters to calculate the electrostatic potential.

FIGURE 3.

Binding of GA, 2,5DHPS, heparin, and 5A2NMS to FGF-1. Top row, left, shown is a superimposed three-dimensional structure of the FGF-1 backbone bound to 2,5DHPS (cyan) and GA (white) at the inhibitor binding site represented in Fig. 2; right, shown is a stereoview of the superimposed three-dimensional structure of the FGF-1 backbone bound to 2,5DHPS (cyan) and heparin (yellow) (45). For orientation purposes 2,5DHPS is represented at its binding site as a stick model, with the carbon, oxygen, and sulfur atoms colored white, red, and yellow, respectively. Representations were generated with the Discovery Studio Visualizer program in the first case and with the PyMOL program in the latter. In both cases the backbones of the superimposed molecules were reciprocally oriented to a minimal root mean square deviation with the program used to generate their representations. Displacement of the backbone at the level of the β3/β4 loop is not properly appreciated at the figure because the perspective of the drawing. Middle row, left, shown are superimposed three-dimensional structures of the amide backbone of FGF-1 bound to 2,5DHPS (cyan), GA (white), and 5A2NMS (green). Middle row, right, shown are superimposed three-dimensional structures of the amide backbone of FGF-1 bound to heparin (yellow) and incorporated into two different models of the FGF-1·FGFR2 complexes (orange, symmetric; red, asymmetric). The complex also includes heparin in the case of the asymmetric model (45, 51, 57). Superimposed molecules were reciprocally oriented to a minimal root mean square deviation and represented using PyMOL. Bottom row, shown is a network of non-covalent interactions between FGF-1 and 2,5DHPS (left) and GA (right). The ligands and the protein are shown as stick-and-ball models, respectively, with the atoms colored as above. Superimposed molecules were reciprocally oriented to a minimal root mean square deviation and represented using PyMOL.

FIGURE 4.

Surface defined by the amino acids of FGF-1 that interface with FGFR2, in the case of the symmetric model of the complex and of the asymmetric model, when bound to heparin and 2,5DHPS, respectively. Top row, the surface is defined by the amino acids in FGF-1 that interact with the D2 immunoglobulin domain. These residues are the same in the asymmetric and symmetric three-dimensional structures of the FGF-1·FGFR2 complex (51, 57). A, FGF-1 bound to heparin is shown. B, FGF-1 bound to 2,5DHPS is shown. In both cases the protein is oriented identically, as the backbone trace (yellow) shows. The following amino acids of FGF-1 interact with the D2 immunoglobulin domain: Tyr-29 (β strand 1); Arg-49 (β strand 2); Arg-51 (loop β strands 2/3); Glu-101, Leu-103 (β strand 8); Asn-106, His-107 (loop β strands 8/9); Tyr-108, Asn-109 (β strand 9); Leu-147, Pro148; Leu-149, Pro-150 (β strand 12). Bottom row, shown is a three-dimensional representation of the surface defined by the whole set of amino acids of FGF-1 that interact with the D3 immunoglobulin domain in the asymmetric and in the symmetric three-dimensional structures of the FGF-1·FGFR2 complex. C, FGF-1 bound to heparin is shown. D, FGF-1 bound to 2,5DHPS is shown. In both cases the protein is identically oriented, as the backbone trace (yellow) shows. Circled residues are common to the interfaces of the symmetric and the asymmetric models. The interface with the D3 domain includes the residues circled plus those on top of them in the case of the asymmetric model and those to their left in the case of the symmetric one. The following amino acids are specific to the interface with the D3 domain in the asymmetric model: Leu-60, Arg-102, Leu-103, Glu-105, Asn-106, His-107, and Ile-112. Those specific to the symmetric model are: Tyr-22, Lys-23, Pro-25, Lys-26, Leu-28, and Tyr-69. The following residues are common to the interface in both models (circled): Ser-61, Ala-62, Glu-63, Ser-64, Val-65, Gly-67, Val-68, Ile-70, and Glu-101. Arrows point toward the residues most displaced when FGF-1 binds 2,5DHPS instead of heparin. The figure was generated, and the surfaces are colored according the electrostatic potential (red, negative; blue, positive) using Discovery Studio Visualizer and its internal parameters.

FIGURE 5.

Stabilization of 2,5DHPS in solution by FGF-1. Absorbance spectra are shown of 2,5DHPS (100 μm) in the presence (2 and 4) and absence (1 and 3) of an equimolar concentration of FGF-1, recorded just after the preparation of the solution (spectra 3 and 4) and 48 h later (spectra 1 and 2). Solutions were at the pH (7.5) and ionic strength (20 mm sodium phosphate, 75 mm NaCl) of the mitogenesis assay cultures. Approximately 10% of 2,5DHPS is lost by oxidation during the 48 h of incubation, according to the 1H NMR spectra of the initial and final solutions. mAU, milliabsorbance units.

FIGURE 6.

Competition between heparin and 2,5DHPS. A, shown is the amount of FGF retained on a 1-ml HiTrapTM heparin HP column (GE Healthcare) when co-injected with either heparin (■) or 2,5DHPS (▴, ●). The amount of protein retained is represented by the height of the peak eluted with a 5-min linear gradient of 300 to 1.5 m NaCl in the chromatography buffer after post-injection column re-equilibration (20 mm sodium phosphate, 300 mm NaCl, pH 7.2). The flow rate was 1 ml/min. The protein (13 μm in 250 μl; ■, ▴, FGF-1; ●, FGF-2) was co-injected in the presence of different concentrations of the competitor, as plotted in the graph. mAU, milliabsorbance units; B, shown is reversion of the inhibition of the mitogenic activity of FGF-1 by 2,5DHPS at increasing heparin concentrations. The mitogenesis assay was carried out as described under “Experimental Procedures” using murine Balb/c 3T3 fibroblasts. C, FGF-1-driven mitogenesis of FR1c-11 cells preincubated as indicated in the figure and under “Experimental Procedures” (*, equivalent levels of mitogenesis were observed when 1 μg/ml of heparin was included in the pretreatment). D, shown is the amount of exdFGFR2IIIc retained in a HiTrapTM Heparin HP column (GE Healthcare) when co-injected with increasing concentrations of heparin (▴) or 2,5DHPS (●) using the chromatographic conditions described above.

FIGURE 7.

Characterization of recombinant exdFGFR2IIIc. A, circular dichroism spectra in the far-UV region of exdFGFR2IIIc are shown. Closed circles represent the spectrum reconstructed on the basis of the percentage of the secondary structure components of exdFGFR2IIIc obtained by deconvolution of the experimental spectrum (DICHROWEB server (68, 90). Upper inset, Coomassie Brilliant Blue-stained SDS/polyacrylamide electrophoresis gel (15%) (50) of recombinant exdFGFR2IIIc after purification and refolding (the horizontal lines to the right indicate the position of molecular mass markers of 250, 75, 50, 37, and 25 kDa). Lower insert, UV spectra (∼0.5 mg/ml; 20 mm Hepes, 150 mm NaCl (pH 7.5)) of exdFGFR2IIIc before (dotted line) and after refolding are shown. AU, absorbance units. B, shown is a characteristic 1H,15N HSQC spectrum of the backbone of 15N-labeled FGF-1 (150 μm in 20 mm sodium phosphate (pH 7.2), 300 mm NaCl) in the presence (lower panel) and absence (upper panel) of 50 μm exdFGFR2IIIc. Framed 1H,15N cross-peaks correspond to Gln-54 and Gln-91 Nδ and His-138 Nα (1), Asp-154 Nα (2), and Lys-23 and Lys-24 Nα (3). C, shown is an STD spectrum of a solution of 2,5DHPS and exdFGFR2IIIc in 20 mm sodium phosphate, 150 mm NaCl (pH 7.2) at a molar ratio of 1 to 0.05 (middle spectrum). The top record is a 1H NMR spectrum of the aromatic region of a 1 mm solution of 2,5DHPS in the same buffer. The bottom trace is a STD spectrum equivalent to that of the middle trace, except that the protein was omitted. Circular dichroism spectra in the far UV region were obtained using a Jasco 710 spectropolarimeter at 20 °C in a 1-mm path length cuvette. The protein (0.13 mg/ml) was in 50 mm sodium phosphate (pH 7.0), and the spectra were averaged by accumulating four scans. A four-scan averaged spectrum of the buffer was routinely subtracted from the protein spectra.

FIGURE 8.

Chromatography of C-LYT/aFGF·exdFGFR2IIIc complexes. The protein (∼3.2 nmol in each case) was injected in 250 μl of the chromatography buffer at the arrows labeled INJ. The buffer contained 150 mm choline from the mark (arrow labeled CHL) to the end of the chromatogram. A, C-LYT/aFGF is shown. B, exdFGFRIIIc is shown. C, an equimolar mixture of C-LYT/aFGF and exdFGFRIIIc is shown. D, an equimolar mixture of C-LYT/aFGF and exdFGFRIIIc is shown as in C, except that 1 ml of a 100 mm 2,5DHPS was injected onto the column (arrow) before raising the choline concentration of the buffer to 150 mm (in C and D, the peak eluted with choline has a long tail only partially shown in the figure). E, shown is the chromatography of an equimolar mixture (∼3.5 nmol) of C-LYT/aFGF and exdFGFRIIIc (containing 5 mm 3-kDa heparin, in the case of the dashed line) using a NaCl gradient (dotted line; except for this NaCl gradient, the other chromatographic conditions are those used in the remainder of the figure). mAU, milliabsorbance units. F, shown is Coomassie Brilliant Blue-stained SDS/PAGE (15%) of the fractions a, b, and c of the chromatograms C and D. The horizontal lines to the left indicate the migration of C-LYT/aFGF (30.8 kDa) and exdFGFRIIIc (38.7 kDa). Chromatography was carried out on 1-ml HiTrapTM DEAE FF columns (GE Healthcare) at a flow rate of 2 ml/min and a pH of 7.5 (Hepes 20 mm, 300 mm NaCl), conditions chosen to avoid any appreciable ionic interaction of FGF-1 (pI 7.9) and exdFGFR2IIIc (pI 5.8), respectively, with the solid phase of the column.

FIGURE 9.

Inhibition by 2,5DHPS of FGF-induced cell migration and of uncontrolled growth in vivo. A, top panel, the effect of different doses of 2,5DHPS on the migratory phenotype induced by either FGF-1 or FGF-2 in wounded confluent cultures of fibroblasts Balb/c 3T3 cells is shown. A, bottom panel, shown are representative photographs of the experiment (2,5DHPS, 200 μm). The black arrow points to a cell with a long filopodium, a characteristic of migrating cells colonizing the denuded area. Data are plotted as the mean ± S.E. of the number of migrating cells invading the wound made in the culture. ***, p < 0.001 versus column 1; †††, p < 0.001 versus columns 3 and 6 for the wells treated with FGF-1 and FGF-2, respectively, as assessed by one-factor analysis of variance followed by the Student-Newmann-Keuls test. B, left plot, shown is the effect of orally administered 2,5DHPS on blood vessel invasion of gelatin sponges soaked in FGF-1 and implanted subcutaneously in rats. B, right panel, shown are representative photographs of the experiment. The image in the second row is an amplification of the boxed area in the image above with the color hue manually modified to better appreciate of the blood vessels. Data are plotted as the mean ± S.E. ***, p < 0.001 versus vehicle by unpaired t test. C, left panel, shown are the effects of 2,5DHPS on the progress of subcutaneously implanted rat gliomas. C, right panel, shown are representative photographs of tumors with a size representing approximately the mean of the plot at the left. The data are expressed as the mean ± S.E. of the volume of excised gliomas at the end of the intraperitoneal treatment. *, p < 0.05 versus vehicle by unpaired t test.