Non-peptidic thrombospondin-1 mimics as fibroblast growth factor-2 inhibitors: an integrated strategy for the development of new antiangiogenic compounds

Abstract

Endogenous inhibitors of angiogenesis, such as thrombospondin-1 (TSP-1), are promising sources of therapeutic agents to treat angiogenesis-driven diseases, including cancer. TSP-1 regulates angiogenesis through different mechanisms, including binding and sequestration of the angiogenic factor fibroblast growth factor-2 (FGF-2), through a site located in the calcium binding type III repeats. We hypothesized that the FGF-2 binding sequence of TSP-1 might serve as a template for the development of inhibitors of angiogenesis. Using a peptide array approach followed by binding assays with synthetic peptides and recombinant proteins, we identified a FGF-2 binding sequence of TSP-1 in the 15-mer sequence DDDDDNDKIPDDRDN. Molecular dynamics simulations, taking the full flexibility of the ligand and receptor into account, and nuclear magnetic resonance identified the relevant residues and conformational determinants for the peptide-FGF interaction. This information was translated into a pharmacophore model used to screen the NCI2003 small molecule databases, leading to the identification of three small molecules that bound FGF-2 with affinity in the submicromolar range. The lead compounds inhibited FGF-2-induced endothelial cell proliferation in vitro and affected angiogenesis induced by FGF-2 in the chicken chorioallantoic membrane assay. These small molecules, therefore, represent promising leads for the development of antiangiogenic agents. Altogether, this study demonstrates that new biological insights obtained by integrated multidisciplinary approaches can be used to develop small molecule mimics of endogenous proteins as therapeutic agents.

FIGURE 1.

Identification of the FGF-2 interacting sequence of TSP-1. a, shown is peptide array analysis. Binding of biotin-labeled FGF-2 to the 237 peptides was analyzed and expressed as described under “Experimental Procedures.” Shown below the graph is a schematic representation of the structure of the type III repeats, organized in 13 C- or N-type motifs, and of the recombinant fragments Tr1–4 used in panel d. b, shown is binding of biotin-labeled peptides DD15, AQ9, AQ19, DIDG6, and DIDG15 to immobilized FGF-2. Bound peptide is expressed as the absorbance (Abs, mean and S.D. of triplicate values). c, shown is SPR analysis of the binding of DD15 to FGF-2. The figure shows representative sensorgrams obtained by simultaneous injection of three concentrations of DD15 (50, 12.5, or 6.25 μm) for 3 min over sensor chip surfaces immobilizing FGF-2. The curves could be globally analyzed by the Langmuir equation, modeling a simple bimolecular interaction, and the corresponding fittings are shown in white. Data are expressed as resonance units versus time. d, shown is binding of biotin-labeled FGF-2 to the recombinant Tr fragments. Data are the amounts of bound FGF-2, as absorbance (mean and S.D. of triplicates).

FIGURE 2.

Structural characterization of the interactions between DD15 and FGF-2. a, shown is a central structure of the most populated cluster from the simulation of the DD15·FGF-2 complex. The surface of FGF-2 is colored according to the electrostatic potential, with blue corresponding to positive charge density, red to negative charge density, and white hydrophobic surface. The residues highlighted for DD15 are the ones used as templates for pharmacophore design. b, the central structure of the most populated cluster from the simulation of the DD15·FGF-2 complex suggests that DD15 and heparin compete for the same binding site.

FIGURE 3.

a, STD NMR spectra are shown of DD15 peptide in the presence of FGF-2. Upper spectrum, shown is a reference ¹H NMR spectrum of 0.7 mm DD15 peptide in the presence of 40 μm FGF-2 in 30 mm buffer phosphate (95% D₂O, 5% H₂O), 50 mm NaCl, and pH 7.0 recorded at 7 °C on a 500 MHz Bruker spectrometer. Lower spectrum, shown is a ¹H NMR STD spectrum of DD15 peptide in the same experimental conditions as the reference spectrum (18:1 peptide:FGF-2 ratio) with 4.5 s irradiation time in the aromatic region. The assignment of the STD signals is reported, an asterisk marks a signal due to an impurity, and the Ac label indicates the acetyl N-terminal group (see supplemental Methods). b, shown is projection on the central structure of the most populated cluster of DD15 residues (cyan) in contact with FGF-2 residues (blue) is shown. Cyan spheres represent the peptide segment Lys-746-Asp-750, involved in the FGF-2 interaction as determined in STD-NMR experiments showing consistency between the model of the complex from simulations and NMR data. c, the pharmacophore model projected on the bound conformation of DD15 is shown.

FIGURE 4.

a, screening small molecules for their ability to prevent the binding of type III repeats to FGF-2 is shown. Labeled type III repeats were incubated with immobilized FGF-2 in the presence of 50 μm concentrations of the 19 small molecules selected by the pharmacophoric search of the NCI2003 data base of molecules. Binding is expressed as a percentage of control, mean, and S.E. from three experiments (p < 0.05(*) and p < 0.001 (**), ANOVA followed by Dunnett's post test). b, shown is the chemical structure of the most active molecules, sm8, sm10, and sm27. c, shown is SPR analysis of the binding of sm8 and sm27 to FGF-2. The figure shows representative sensorgrams obtained by simultaneous injection of two concentrations of the molecules (10 and 3 μm) for 3 min over sensor chip surfaces immobilizing FGF-2. The curves were analyzed by the Langmuir equation, modeling a simple bimolecular interaction, and the corresponding fittings are shown in white. Data are expressed as resonance units versus time.

FIGURE 5.

Antiangiogenic activity of small molecules mimetic of the FGF-2-binding sequence of TSP-1. a, shown is binding of FGF-2 to endothelial cells. BAEC were incubated with labeled FGF-2 in the presence of the small molecule (1–100 μm). The cell-bound FGF-2 is expressed as a percentage of control (in the absence of small molecules). b, shown is endothelial cell proliferation. BAEC were exposed to 5 ng/ml FGF-2 with the small molecule (6–100 μm) and incubated for 3 days. Proliferation is expressed as a percentage of control (in the absence of small molecules). c, shown is FGF-2-induced angiogenesis in the chorioallantoic membrane assay. FGF-2 (200 ng) was administered in the absence or presence of sm27 (0.5 μg) on day 8. A representative picture taken 4 days later is shown. Original magnification, 50×.