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Direct and allosteric inhibition of the FGF2/HSPGs/FGFR1 ternary complex formation by an antiangiogenic, thrombospondin-1-mimic small molecule.

Pagano K, Torella R, Foglieni C, Bugatti A, Tomaselli S, Zetta L, Presta M, Rusnati M, Taraboletti G, Colombo G, Ragona L - PLoS ONE (2012)

Bottom Line: The formation of a ternary complex with the transmembrane tyrosine kinase receptors (FGFRs), and heparan sulphate proteoglycans (HSPGs) is required for FGF2 pro-angiogenic activity.NMR and MD data demonstrate that sm27 engages the heparin-binding site of FGF2 and induces long-range dynamics perturbations along FGF2/FGFR1 interface regions.We propose that sm27 antiangiogenic activity is based on a twofold-direct and allosteric-mechanism, inhibiting FGF2 binding to both its receptors.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio NMR, Istituto per lo Studio delle Macromolecole, Consiglio Nazionale delle Ricerche, Milano, Italy.

ABSTRACT
Fibroblast growth factors (FGFs) are recognized targets for the development of therapies against angiogenesis-driven diseases, including cancer. The formation of a ternary complex with the transmembrane tyrosine kinase receptors (FGFRs), and heparan sulphate proteoglycans (HSPGs) is required for FGF2 pro-angiogenic activity. Here by using a combination of techniques including Nuclear Magnetic Resonance, Molecular Dynamics, Surface Plasmon Resonance and cell-based binding assays we clarify the molecular mechanism of inhibition of an angiostatic small molecule, sm27, mimicking the endogenous inhibitor of angiogenesis, thrombospondin-1. NMR and MD data demonstrate that sm27 engages the heparin-binding site of FGF2 and induces long-range dynamics perturbations along FGF2/FGFR1 interface regions. The functional consequence of the inhibitor binding is an impaired FGF2 interaction with both its receptors, as demonstrated by SPR and cell-based binding assays. We propose that sm27 antiangiogenic activity is based on a twofold-direct and allosteric-mechanism, inhibiting FGF2 binding to both its receptors.

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Effect of sm27 on the interaction of FGF2 with heparin/HSPGs and FGFR1.A) FGF2 (150 nM) was injected over heparin or FGFR1 immobilized to a SPR sensorchip in the absence or in the presence of increasing concentrations of sm27 and the amount of FGF2 bound to the surfaces in the different experimental conditions was then measured. Each point is the mean ± SEM of 3 separate experiments. B) Binding of Eu-FGF2 to CHO cells expressing either HSPGs or FGFR1 in the presence of sm27. C) Binding of Eu-FGF2 to endothelial cells (BAEC). D) Effect of sm27 on the formation of the HSPGs/FGF2/FGFR1 complex. FGFR1+/HSPG− A745 CHO flg-1A cells were added to a monolayer of FGFR1−/HSPG+ CHO-K1 cells in serum-free medium with FGF2 (1.66 nM) in the presence of increasing concentrations of sm27. After 2 h of incubation at 298K, the cells bound to the monolayer were counted under an inverted microscope. Each point is the mean ± SEM of 3 separate experiments performed in triplicate. In B and C, black and white arrows indicate the inhibitions obtained with heparin (0.1 µg/ml) and unlabeled FGF2 (1.5 µg/ml) used as reference competitor of HSPGs and FGFR1 binding, respectively
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pone-0036990-g005: Effect of sm27 on the interaction of FGF2 with heparin/HSPGs and FGFR1.A) FGF2 (150 nM) was injected over heparin or FGFR1 immobilized to a SPR sensorchip in the absence or in the presence of increasing concentrations of sm27 and the amount of FGF2 bound to the surfaces in the different experimental conditions was then measured. Each point is the mean ± SEM of 3 separate experiments. B) Binding of Eu-FGF2 to CHO cells expressing either HSPGs or FGFR1 in the presence of sm27. C) Binding of Eu-FGF2 to endothelial cells (BAEC). D) Effect of sm27 on the formation of the HSPGs/FGF2/FGFR1 complex. FGFR1+/HSPG− A745 CHO flg-1A cells were added to a monolayer of FGFR1−/HSPG+ CHO-K1 cells in serum-free medium with FGF2 (1.66 nM) in the presence of increasing concentrations of sm27. After 2 h of incubation at 298K, the cells bound to the monolayer were counted under an inverted microscope. Each point is the mean ± SEM of 3 separate experiments performed in triplicate. In B and C, black and white arrows indicate the inhibitions obtained with heparin (0.1 µg/ml) and unlabeled FGF2 (1.5 µg/ml) used as reference competitor of HSPGs and FGFR1 binding, respectively

Mentions: The combined NMR and MD characterization of sm27 binding to FGF2 revealed that the inhibitor targets a well defined region extending over the FGF2 heparin-binding site. We thus exploited SPR analysis to evaluate the ability of sm27 to bind FGF2 and consequently inhibit its interaction with surface-immobilized heparin, an experimental model that resembles the binding of heparin binding growth factors to cell-associated HSPGs [30]. As shown in Figure 5A, sm27 effectively prevents FGF2/heparin interaction in a dose-dependent way with an IC50 equal to 3.5 µM, suggesting that the same inhibitory effect can be exerted by sm27 also on the FGF2/HSPGs interaction at the cell surface.


Direct and allosteric inhibition of the FGF2/HSPGs/FGFR1 ternary complex formation by an antiangiogenic, thrombospondin-1-mimic small molecule.

Pagano K, Torella R, Foglieni C, Bugatti A, Tomaselli S, Zetta L, Presta M, Rusnati M, Taraboletti G, Colombo G, Ragona L - PLoS ONE (2012)

Effect of sm27 on the interaction of FGF2 with heparin/HSPGs and FGFR1.A) FGF2 (150 nM) was injected over heparin or FGFR1 immobilized to a SPR sensorchip in the absence or in the presence of increasing concentrations of sm27 and the amount of FGF2 bound to the surfaces in the different experimental conditions was then measured. Each point is the mean ± SEM of 3 separate experiments. B) Binding of Eu-FGF2 to CHO cells expressing either HSPGs or FGFR1 in the presence of sm27. C) Binding of Eu-FGF2 to endothelial cells (BAEC). D) Effect of sm27 on the formation of the HSPGs/FGF2/FGFR1 complex. FGFR1+/HSPG− A745 CHO flg-1A cells were added to a monolayer of FGFR1−/HSPG+ CHO-K1 cells in serum-free medium with FGF2 (1.66 nM) in the presence of increasing concentrations of sm27. After 2 h of incubation at 298K, the cells bound to the monolayer were counted under an inverted microscope. Each point is the mean ± SEM of 3 separate experiments performed in triplicate. In B and C, black and white arrows indicate the inhibitions obtained with heparin (0.1 µg/ml) and unlabeled FGF2 (1.5 µg/ml) used as reference competitor of HSPGs and FGFR1 binding, respectively
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351436&req=5

pone-0036990-g005: Effect of sm27 on the interaction of FGF2 with heparin/HSPGs and FGFR1.A) FGF2 (150 nM) was injected over heparin or FGFR1 immobilized to a SPR sensorchip in the absence or in the presence of increasing concentrations of sm27 and the amount of FGF2 bound to the surfaces in the different experimental conditions was then measured. Each point is the mean ± SEM of 3 separate experiments. B) Binding of Eu-FGF2 to CHO cells expressing either HSPGs or FGFR1 in the presence of sm27. C) Binding of Eu-FGF2 to endothelial cells (BAEC). D) Effect of sm27 on the formation of the HSPGs/FGF2/FGFR1 complex. FGFR1+/HSPG− A745 CHO flg-1A cells were added to a monolayer of FGFR1−/HSPG+ CHO-K1 cells in serum-free medium with FGF2 (1.66 nM) in the presence of increasing concentrations of sm27. After 2 h of incubation at 298K, the cells bound to the monolayer were counted under an inverted microscope. Each point is the mean ± SEM of 3 separate experiments performed in triplicate. In B and C, black and white arrows indicate the inhibitions obtained with heparin (0.1 µg/ml) and unlabeled FGF2 (1.5 µg/ml) used as reference competitor of HSPGs and FGFR1 binding, respectively
Mentions: The combined NMR and MD characterization of sm27 binding to FGF2 revealed that the inhibitor targets a well defined region extending over the FGF2 heparin-binding site. We thus exploited SPR analysis to evaluate the ability of sm27 to bind FGF2 and consequently inhibit its interaction with surface-immobilized heparin, an experimental model that resembles the binding of heparin binding growth factors to cell-associated HSPGs [30]. As shown in Figure 5A, sm27 effectively prevents FGF2/heparin interaction in a dose-dependent way with an IC50 equal to 3.5 µM, suggesting that the same inhibitory effect can be exerted by sm27 also on the FGF2/HSPGs interaction at the cell surface.

Bottom Line: The formation of a ternary complex with the transmembrane tyrosine kinase receptors (FGFRs), and heparan sulphate proteoglycans (HSPGs) is required for FGF2 pro-angiogenic activity.NMR and MD data demonstrate that sm27 engages the heparin-binding site of FGF2 and induces long-range dynamics perturbations along FGF2/FGFR1 interface regions.We propose that sm27 antiangiogenic activity is based on a twofold-direct and allosteric-mechanism, inhibiting FGF2 binding to both its receptors.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio NMR, Istituto per lo Studio delle Macromolecole, Consiglio Nazionale delle Ricerche, Milano, Italy.

ABSTRACT
Fibroblast growth factors (FGFs) are recognized targets for the development of therapies against angiogenesis-driven diseases, including cancer. The formation of a ternary complex with the transmembrane tyrosine kinase receptors (FGFRs), and heparan sulphate proteoglycans (HSPGs) is required for FGF2 pro-angiogenic activity. Here by using a combination of techniques including Nuclear Magnetic Resonance, Molecular Dynamics, Surface Plasmon Resonance and cell-based binding assays we clarify the molecular mechanism of inhibition of an angiostatic small molecule, sm27, mimicking the endogenous inhibitor of angiogenesis, thrombospondin-1. NMR and MD data demonstrate that sm27 engages the heparin-binding site of FGF2 and induces long-range dynamics perturbations along FGF2/FGFR1 interface regions. The functional consequence of the inhibitor binding is an impaired FGF2 interaction with both its receptors, as demonstrated by SPR and cell-based binding assays. We propose that sm27 antiangiogenic activity is based on a twofold-direct and allosteric-mechanism, inhibiting FGF2 binding to both its receptors.

Show MeSH
Related in: MedlinePlus