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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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Backbone dynamics of apo and holo FGF2 by NMR.A) 1H15N-HSQC cross peaks normalized intensity (I) upon sm27 binding vs residue number. Gray and black diamond refers to backbone amides, and glutamine, asparagine and arginine side chains, respectively. B) 1H15N-HSQC cross peaks intensity variations plotted on FGF2 molecule. Blue (filled) and red (dotted) spheres indicate residues showing an increased and reduced intensity upon sm27 binding, respectively. The sphere radius is set according to the size of the variation. Side chains affected by intensity variations are shown in sticks. R129 and K144 side chains are shown in yellow sticks for clarity reasons. C) Modelfree analysis order parameters (S2) for the apo- and holo-FGF2 are reported in black and gray, respectively, as a function of the residue number. The black and gray straight lines represent the S2 average value for the apo and holo FGF2, respectively. S2 values are mapped on FGF2 structure in D and E for the apo and holo form, respectively. Color code: red for , where  and are the average and the standard deviation for S2 values, respectively; orange for ; white for ; marine for ; blue for . Secondary structure elements are numbered for clarity purposes. F) and G) R2/R1rhovalues as a function of the residue number are reported for the apo and holo FGF2, respectively. The horizontal straight and dotted lines represent the average value and the average plus one standard deviation, respectively. H) Residues with R2/R1rho exceeding from one in the apo and holo FGF2 are mapped on the protein structure. Green spheres represent residues affected by conformational exchange in both the apo and holo form. Marine dotted spheres represent residues affected by conformational exchange only in the apo-FGF2 form. Red spheres represent residues affected by conformational exchange upon sm27 binding. Residues with R2/R1rho values affected by significant errors, either in the apo or in the holo form, are represented as dotted spheres.
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pone-0036990-g002: Backbone dynamics of apo and holo FGF2 by NMR.A) 1H15N-HSQC cross peaks normalized intensity (I) upon sm27 binding vs residue number. Gray and black diamond refers to backbone amides, and glutamine, asparagine and arginine side chains, respectively. B) 1H15N-HSQC cross peaks intensity variations plotted on FGF2 molecule. Blue (filled) and red (dotted) spheres indicate residues showing an increased and reduced intensity upon sm27 binding, respectively. The sphere radius is set according to the size of the variation. Side chains affected by intensity variations are shown in sticks. R129 and K144 side chains are shown in yellow sticks for clarity reasons. C) Modelfree analysis order parameters (S2) for the apo- and holo-FGF2 are reported in black and gray, respectively, as a function of the residue number. The black and gray straight lines represent the S2 average value for the apo and holo FGF2, respectively. S2 values are mapped on FGF2 structure in D and E for the apo and holo form, respectively. Color code: red for , where and are the average and the standard deviation for S2 values, respectively; orange for ; white for ; marine for ; blue for . Secondary structure elements are numbered for clarity purposes. F) and G) R2/R1rhovalues as a function of the residue number are reported for the apo and holo FGF2, respectively. The horizontal straight and dotted lines represent the average value and the average plus one standard deviation, respectively. H) Residues with R2/R1rho exceeding from one in the apo and holo FGF2 are mapped on the protein structure. Green spheres represent residues affected by conformational exchange in both the apo and holo form. Marine dotted spheres represent residues affected by conformational exchange only in the apo-FGF2 form. Red spheres represent residues affected by conformational exchange upon sm27 binding. Residues with R2/R1rho values affected by significant errors, either in the apo or in the holo form, are represented as dotted spheres.

Mentions: Peak intensity in HSQC spectra is a sensitive probe of exchange rates and relaxation rates affecting individual residues. The changes in the normalized peak intensities as a function of ligand additions thus provide useful dynamic and conformational information about the binding event. The peak intensity variations of each FGF2 residue on going from the apo to holo (2∶1 sm27:FGF2 ratio) form is reported in Figure 2A and are summarized on FGF2 structure in Figure 2B. The strongest effects were observed for R129, K144 and A145. Interestingly, these residues showed a very low intensity in the apo-FGF2 HSQC spectrum, probably due to conformational exchange, and their intensities grew up to the average value of the molecule, upon sm27 binding. The behavior of glutamines and asparagine side-chains could be analyzed from the same HSQC spectra, and significant changes were observed for residues N36, Q132 and Q143.


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)

Backbone dynamics of apo and holo FGF2 by NMR.A) 1H15N-HSQC cross peaks normalized intensity (I) upon sm27 binding vs residue number. Gray and black diamond refers to backbone amides, and glutamine, asparagine and arginine side chains, respectively. B) 1H15N-HSQC cross peaks intensity variations plotted on FGF2 molecule. Blue (filled) and red (dotted) spheres indicate residues showing an increased and reduced intensity upon sm27 binding, respectively. The sphere radius is set according to the size of the variation. Side chains affected by intensity variations are shown in sticks. R129 and K144 side chains are shown in yellow sticks for clarity reasons. C) Modelfree analysis order parameters (S2) for the apo- and holo-FGF2 are reported in black and gray, respectively, as a function of the residue number. The black and gray straight lines represent the S2 average value for the apo and holo FGF2, respectively. S2 values are mapped on FGF2 structure in D and E for the apo and holo form, respectively. Color code: red for , where  and are the average and the standard deviation for S2 values, respectively; orange for ; white for ; marine for ; blue for . Secondary structure elements are numbered for clarity purposes. F) and G) R2/R1rhovalues as a function of the residue number are reported for the apo and holo FGF2, respectively. The horizontal straight and dotted lines represent the average value and the average plus one standard deviation, respectively. H) Residues with R2/R1rho exceeding from one in the apo and holo FGF2 are mapped on the protein structure. Green spheres represent residues affected by conformational exchange in both the apo and holo form. Marine dotted spheres represent residues affected by conformational exchange only in the apo-FGF2 form. Red spheres represent residues affected by conformational exchange upon sm27 binding. Residues with R2/R1rho values affected by significant errors, either in the apo or in the holo form, are represented as dotted spheres.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0036990-g002: Backbone dynamics of apo and holo FGF2 by NMR.A) 1H15N-HSQC cross peaks normalized intensity (I) upon sm27 binding vs residue number. Gray and black diamond refers to backbone amides, and glutamine, asparagine and arginine side chains, respectively. B) 1H15N-HSQC cross peaks intensity variations plotted on FGF2 molecule. Blue (filled) and red (dotted) spheres indicate residues showing an increased and reduced intensity upon sm27 binding, respectively. The sphere radius is set according to the size of the variation. Side chains affected by intensity variations are shown in sticks. R129 and K144 side chains are shown in yellow sticks for clarity reasons. C) Modelfree analysis order parameters (S2) for the apo- and holo-FGF2 are reported in black and gray, respectively, as a function of the residue number. The black and gray straight lines represent the S2 average value for the apo and holo FGF2, respectively. S2 values are mapped on FGF2 structure in D and E for the apo and holo form, respectively. Color code: red for , where and are the average and the standard deviation for S2 values, respectively; orange for ; white for ; marine for ; blue for . Secondary structure elements are numbered for clarity purposes. F) and G) R2/R1rhovalues as a function of the residue number are reported for the apo and holo FGF2, respectively. The horizontal straight and dotted lines represent the average value and the average plus one standard deviation, respectively. H) Residues with R2/R1rho exceeding from one in the apo and holo FGF2 are mapped on the protein structure. Green spheres represent residues affected by conformational exchange in both the apo and holo form. Marine dotted spheres represent residues affected by conformational exchange only in the apo-FGF2 form. Red spheres represent residues affected by conformational exchange upon sm27 binding. Residues with R2/R1rho values affected by significant errors, either in the apo or in the holo form, are represented as dotted spheres.
Mentions: Peak intensity in HSQC spectra is a sensitive probe of exchange rates and relaxation rates affecting individual residues. The changes in the normalized peak intensities as a function of ligand additions thus provide useful dynamic and conformational information about the binding event. The peak intensity variations of each FGF2 residue on going from the apo to holo (2∶1 sm27:FGF2 ratio) form is reported in Figure 2A and are summarized on FGF2 structure in Figure 2B. The strongest effects were observed for R129, K144 and A145. Interestingly, these residues showed a very low intensity in the apo-FGF2 HSQC spectrum, probably due to conformational exchange, and their intensities grew up to the average value of the molecule, upon sm27 binding. The behavior of glutamines and asparagine side-chains could be analyzed from the same HSQC spectra, and significant changes were observed for residues N36, Q132 and Q143.

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