Fibroblast growth factor 2‐antagonist activity of a long‐pentraxin 3‐derived anti‐angiogenic pentapeptide

Abstract

Fibroblast growth factor-2 (FGF2) plays a major role in angiogenesis. The pattern recognition receptor long-pentraxin 3 (PTX3) inhibits the angiogenic activity of FGF2. To identify novel FGF2-antagonistic peptide(s), four acetylated (Ac) synthetic peptides overlapping the FGF2-binding region PTX3-(97–110) were assessed for their FGF2-binding capacity. Among them, the shortest pentapeptide Ac-ARPCA-NH2 (PTX3-[100–104]) inhibits the interaction of FGF2 with PTX3 immobilized to a BIAcore sensorchip and suppresses FGF2-dependent proliferation in endothelial cells, without affecting the activity of unrelated mitogens. Also, Ac-ARPCA-NH2 inhibits angiogenesis triggered by FGF2 or by tumorigenic FGF2-overexpressing murine endothelial cells in chick and zebrafish embryos, respectively. Accordingly, the peptide hampers the binding of FGF2 to Chinese Hamster ovary cells overexpressing the tyrosine-kinase FGF receptor-1 (FGFR1) and to recombinant FGFR1 immobilized to a BIAcore sensorchip without affecting heparin interaction. In all the assays the mutated Ac-ARPSA-NH2 peptide was ineffective. In keeping with the observation that hydrophobic interactions dominate the interface between FGF2 and the FGF-binding domain of the Ig-like loop D2 of FGFR1, amino acid substitutions in Ac-ARPCA-NH2 and saturation transfer difference-nuclear magnetic resonance analysis of its mode of interaction with FGF2 implicate the hydrophobic methyl groups of the pentapeptide in FGF2 binding. These results will provide the basis for the design of novel PTX3-derived anti-angiogenic FGF2 antagonists.

Long Pentraxin-3 Inhibits FGF8b-Dependent Angiogenesis and Growth of Steroid Hormone–Regulated Tumors

“…Similar to FGF2, hydrophobic interactions are implicated in FGF8b binding to FGFRs (30), thus suggesting that this mode of interaction may also occur between Ac-ARPCA-NH 2 and FGF8b. Indeed, Ac-ARPCA-NH 2 inhibits the mitogenic activity exerted by FGF8b on endothelial cells (42) and S115 tumor cells (Leali D, unpublished observations). Experiments are in progress to translate the information about Ac-ARPCA-NH 2 /FGF8b interaction into a pharmacophore model to be used for the screening of small molecule databases (44), in the search for novel PTX3-derived FGF8b-binding low molecular weight antagonists suitable for in vivo therapeutic interventions.…”

“…Recently, N-terminal PTX3-derived low molecular weight FGF antagonists have been identified in our laboratory (10,42,43), including the acetylated (Ac) synthetic pentapeptide Ac-ARPCA-NH 2 (in single letter code), corresponding to the amino acid sequence 100 to 104 in PTX3 N-terminus (42). Ac-ARPCA-NH 2 binds FGF2 via hydrophobic interactions that may mimic the interaction of the growth factor with hydrophobic FGF2-binding domain(s) in FGFRs (42). Similar to FGF2, hydrophobic interactions are implicated in FGF8b binding to FGFRs (30), thus suggesting that this mode of interaction may also occur between Ac-ARPCA-NH 2 and FGF8b.…”

Long Pentraxin-3 Inhibits Epithelial–Mesenchymal Transition in Melanoma Cells

“…PTX3 and the PTX3-derived synthetic peptide ARPCA inhibit the mitogenic activity exerted by FGF2 on endothelial and tumor cells (19,21,22,24). Accordingly, recombinant PTX3 protein and the ARPCA pentapeptide inhibited the proliferation of B16-F10 cells following stimulation with FGF2 (Fig.…”

“…The specificity of the effect was confirmed by the lack of activity of the negative controls short pentraxin serum amyloid-P and scrambled PARAC pentapeptide (ref. 24; Fig. 1A).…”

“…PTX3 binds FGF2 (19,20) and other members of the FGF family (21,22) via its N-terminal extension, thus inhibiting FGF2-dependent endothelial cell proliferation in vitro and angiogenesis in vivo (18)(19)(20)23). Accordingly, the acetylated pentapeptide Ac-ARPCA-NH 2 (ARPCA), corresponding to the N-terminal PTX3-(100-104) sequence, represents the minimal FGF-binding peptide able to interfere with FGF/ FGFR interaction (24). Moreover, PTX3 and ARPCA inhibit the FGF-dependent mitogenic, angiogenic, and tumorigenic potential of different tumor cell lines, including breast carcinoma (21) and prostate cancer cells (22).…”