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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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Mapping of FGF2/sm27 interaction by NMR and docking simulations.A) Sm27 molecule: 4-hydroxy-6-[(8-hydroxy-6-sulfonaphthalen-2-yl)carbamoylamino] naphthalene-2-sulfonic acid. B) Superimposed 1H-15N HSQC spectra of free FGF2 (black), FGF2:sm27 in stoichiometric ratios 1∶1 (blue) and 1∶2 (red). Spectral regions showing R129 and K144 behaviors are zoomed. C) Graphical representation of the combined HN and N FGF2 chemical shift perturbation determined for the various residues, according to [53] (), following the addition of sm27 in 2∶1 stoichiometric ratio. Gray and black bars refer to backbone and Asn, Gln and Arg side chain variations, respectively. D) Bundle of the first 10 structures of cluster 1 obtained with HADDOCK. FGF2 in shown as a blue cartoon, sm27 is shown in yellow sticks. E) Summary of the conserved protein-inhibitor interactions. K128, R129, Q143, and K144 side chains are shown as sticks and are labelled. H-bonds are depicted with black dotted lines and atoms involved in hydrophobic interactions are showed as blurred spheres.
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pone-0036990-g001: Mapping of FGF2/sm27 interaction by NMR and docking simulations.A) Sm27 molecule: 4-hydroxy-6-[(8-hydroxy-6-sulfonaphthalen-2-yl)carbamoylamino] naphthalene-2-sulfonic acid. B) Superimposed 1H-15N HSQC spectra of free FGF2 (black), FGF2:sm27 in stoichiometric ratios 1∶1 (blue) and 1∶2 (red). Spectral regions showing R129 and K144 behaviors are zoomed. C) Graphical representation of the combined HN and N FGF2 chemical shift perturbation determined for the various residues, according to [53] (), following the addition of sm27 in 2∶1 stoichiometric ratio. Gray and black bars refer to backbone and Asn, Gln and Arg side chain variations, respectively. D) Bundle of the first 10 structures of cluster 1 obtained with HADDOCK. FGF2 in shown as a blue cartoon, sm27 is shown in yellow sticks. E) Summary of the conserved protein-inhibitor interactions. K128, R129, Q143, and K144 side chains are shown as sticks and are labelled. H-bonds are depicted with black dotted lines and atoms involved in hydrophobic interactions are showed as blurred spheres.

Mentions: One of the most potent endogenous inhibitors of angiogenesis is thrombospondin-1 (TSP-1) [4], [5]. It binds to FGF2 with an affinity similar to heparin [6], [7], inhibiting the FGF2-mediated angiogenic activation of endothelial cells. We have recently identified an antiangiogenic FGF2-binding site in the type III repeats of TSP-1, and demonstrated that binding of FGF2 to this site inhibits angiogenesis by sequestration of the growth factor [8]. Then, peptide array analysis, binding experiments and SPR analysis guided us to identify a linear amino acidic sequence of type III repeats of TSP-1 that bound FGF2 in the μM range. Using a pharmacophore-based approach, three non-peptidic small molecules, retaining the antiangiogenic activity of the entire TSP-1 and the type III repeats, were identified. The most active molecule, sm27 (IUPAC name: 4-hydroxy-6-((((8-hydroxy-6-sulfo-2-naphthyl) amino)carbonyl)amino)-2-naphthalenesulfonic acid) (Figure 1A), prevented the binding of FGF2 to endothelial cells, inhibited FGF2-induced endothelial cell proliferation and FGF2-induced angiogenesis in the chicken chorioallantoic membrane assay [9]. Since its stereochemical properties optimally match the design rules proposed to improve the pharmacological applicability of naphthalene sulfonates in antiangiogenesis [10], [11], although with an activity not suitable to make it an immediate drug-candidate, sm27 can be considered the prototype lead compound for the ongoing development of potent FGF2-targeting drugs.


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)

Mapping of FGF2/sm27 interaction by NMR and docking simulations.A) Sm27 molecule: 4-hydroxy-6-[(8-hydroxy-6-sulfonaphthalen-2-yl)carbamoylamino] naphthalene-2-sulfonic acid. B) Superimposed 1H-15N HSQC spectra of free FGF2 (black), FGF2:sm27 in stoichiometric ratios 1∶1 (blue) and 1∶2 (red). Spectral regions showing R129 and K144 behaviors are zoomed. C) Graphical representation of the combined HN and N FGF2 chemical shift perturbation determined for the various residues, according to [53] (), following the addition of sm27 in 2∶1 stoichiometric ratio. Gray and black bars refer to backbone and Asn, Gln and Arg side chain variations, respectively. D) Bundle of the first 10 structures of cluster 1 obtained with HADDOCK. FGF2 in shown as a blue cartoon, sm27 is shown in yellow sticks. E) Summary of the conserved protein-inhibitor interactions. K128, R129, Q143, and K144 side chains are shown as sticks and are labelled. H-bonds are depicted with black dotted lines and atoms involved in hydrophobic interactions are showed as blurred spheres.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0036990-g001: Mapping of FGF2/sm27 interaction by NMR and docking simulations.A) Sm27 molecule: 4-hydroxy-6-[(8-hydroxy-6-sulfonaphthalen-2-yl)carbamoylamino] naphthalene-2-sulfonic acid. B) Superimposed 1H-15N HSQC spectra of free FGF2 (black), FGF2:sm27 in stoichiometric ratios 1∶1 (blue) and 1∶2 (red). Spectral regions showing R129 and K144 behaviors are zoomed. C) Graphical representation of the combined HN and N FGF2 chemical shift perturbation determined for the various residues, according to [53] (), following the addition of sm27 in 2∶1 stoichiometric ratio. Gray and black bars refer to backbone and Asn, Gln and Arg side chain variations, respectively. D) Bundle of the first 10 structures of cluster 1 obtained with HADDOCK. FGF2 in shown as a blue cartoon, sm27 is shown in yellow sticks. E) Summary of the conserved protein-inhibitor interactions. K128, R129, Q143, and K144 side chains are shown as sticks and are labelled. H-bonds are depicted with black dotted lines and atoms involved in hydrophobic interactions are showed as blurred spheres.
Mentions: One of the most potent endogenous inhibitors of angiogenesis is thrombospondin-1 (TSP-1) [4], [5]. It binds to FGF2 with an affinity similar to heparin [6], [7], inhibiting the FGF2-mediated angiogenic activation of endothelial cells. We have recently identified an antiangiogenic FGF2-binding site in the type III repeats of TSP-1, and demonstrated that binding of FGF2 to this site inhibits angiogenesis by sequestration of the growth factor [8]. Then, peptide array analysis, binding experiments and SPR analysis guided us to identify a linear amino acidic sequence of type III repeats of TSP-1 that bound FGF2 in the μM range. Using a pharmacophore-based approach, three non-peptidic small molecules, retaining the antiangiogenic activity of the entire TSP-1 and the type III repeats, were identified. The most active molecule, sm27 (IUPAC name: 4-hydroxy-6-((((8-hydroxy-6-sulfo-2-naphthyl) amino)carbonyl)amino)-2-naphthalenesulfonic acid) (Figure 1A), prevented the binding of FGF2 to endothelial cells, inhibited FGF2-induced endothelial cell proliferation and FGF2-induced angiogenesis in the chicken chorioallantoic membrane assay [9]. Since its stereochemical properties optimally match the design rules proposed to improve the pharmacological applicability of naphthalene sulfonates in antiangiogenesis [10], [11], although with an activity not suitable to make it an immediate drug-candidate, sm27 can be considered the prototype lead compound for the ongoing development of potent FGF2-targeting drugs.

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