Direct and Allosteric Inhibition of the FGF2/HSPGs/FGFR1 Ternary Complex Formation by an Antiangiogenic, Thrombospondin-1-Mimic Small Molecule

Katiuscia Pagano,Laura Ragona,Antonella Bugatti,Chiara Foglieni,Marco Rusnati,Giorgio Colombo,Rubben Torella,Simona Tomaselli,Marco Presta,Giulia Taraboletti,Lucia Zetta,Paolo Carloni

doi:10.1371/journal.pone.0036990

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.

Highlights

Fibroblast growth factors (FGFs) and their receptors are emerging as promising therapeutic targets for a wide array of pathologies, including angiogenesis-driven diseases
The chemical shift perturbation (CSP) analysis of the FGF2 1H and 15N Nuclear Magnetic Resonance (NMR) resonances upon addition of sm27 clearly indicated no global tertiary structure modification of the FGF2 molecule upon sm27 binding
The separate analysis revealed a high variation of R129 15N chemical shift, which was interpreted as a change of K128 side chain conformation upon sm27 binding (Figure S1)

Summary

Introduction

Fibroblast growth factors (FGFs) and their receptors are emerging as promising therapeutic targets for a wide array of pathologies, including angiogenesis-driven diseases. Therapeutic strategies, aimed at interfering with the formation of the complex between FGF and its receptors (either FGFRs or HSPGs), are being developed and include small molecule inhibitors of FGFR tyrosine kinase activity, monoclonal antibodies targeting FGFRs, and a wide array of natural or synthetic molecules able to sequester FGFs preventing their interaction with FGFRs and HSPGs [3]. 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]. Since its stereochemical properties optimally match the design rules proposed to improve the pharmacological applicability of naphthalene sulfonates in antiangiogenesis [10,11], with an activity not suitable to make it an immediate drugcandidate, sm can be considered the prototype lead compound for the ongoing development of potent FGF2-targeting drugs

Methods

Results

Conclusion