Modulation of Fibroblast Growth Factor-2 Receptor Binding, Signaling, and Mitogenic Activity by Heparin-Mimicking Polysulfonated Compounds

Abstract

Basic fibroblast growth factor (FGF-2) interacts with high-affinity tyrosine-kinase fibroblast growth factor receptors (FGFRs) and low-affinity heparan sulfate proteoglycans (HSPGs) in target cells. Both interactions are required for FGF-2-mediated biological responses. Here we report the FGF-2 antagonist activity of novel synthetic sulfonic acid polymers with distinct chemical structures and molecular masses (MMs). PAMPS [poly(2-acrylamido-2-methyl-1-propanesulfonic acid)], (MM approximately 7,000-10,000), PAS [poly(anetholesulfonic acid)], (MM approximately 9,000-11,000), PSS [poly(4-styrenesulfonic acid)], (MM = 70,000), and poly(vinylsulfonic acid) (MM = 2,000), inhibited FGF-2 binding to HSPGs and FGFRs in fetal bovine aortic endothelial GM 7373 cells. They also abrogated the formation of the HSPG/FGF-2/FGFR ternary complex, as evidenced by their capacity to prevent FGF-2-mediated cell-cell attachment of FGFR-1-overexpressing, HSPG-deficient Chinese hamster ovary cells to wild-type HSPG-bearing cells. Direct interaction of the polysulfonates with FGF-2 was demonstrated by their ability to protect the growth factor from proteolytic cleavage. Accordingly, molecular modeling, based on the crystal structure of the interaction of FGF-2 with a heparin hexamer, showed the feasibility of docking PAMPS into the heparin-binding domain of FGF-2. In agreement with their FGF-2-binding capacity, PSS, PAS, and PAMPS inhibited FGF-2-induced cell proliferation in GM 7373 cells and murine brain microvascular endothelial cells. The antiproliferative activity of these compounds was associated with the abrogation of FGF-2-induced tyrosine phosphorylation of FGFR-1. Moreover, the polysulfonates PSS and PAS inhibited FGF-2-induced activation of mitogen-activated protein kinase-1/2, involved in FGF-2 signal transduction. In conclusion, sulfonic acid polymers bind FGF-2 by mimicking heparin interaction. These compounds may provide a tool to inhibit FGF-2-induced endothelial cell proliferation in angiogenesis and tumor growth.

The Thymidine Phosphorylase Inhibitor 5′-O-Tritylinosine (KIN59) Is an Antiangiogenic Multitarget Fibroblast Growth Factor-2 Antagonist

“…Thus, KIN59 seems to differ from various negatively charged FGF2 inhibitors identified so far, including polysulfonated and polysulfated molecules, able to bind the basic domain of FGF2, thereby preventing its interaction with HSPGs (15). Indeed, in contrast to these compounds (14), KIN59 is uncharged and does not protect the growth factor from proteolytic digestion (data not shown). Recently, we have shown that the pattern recognition receptor long pentraxin-3 (PTX3) and the PTX3-derived N-acetylated pentapeptide ARPCA (in single letter code) bind FGF2 with high affinity and specificity, thus preventing HSPG/FGF2/FGFR1 ternary complex formation and angiogenesis (18,47).…”

“…These cells are characterized by a sustained rate of growth in vitro and by the capacity to generate opportunistic vascular lesions in nude mice as a consequence of an autocrine loop of stimulation triggered by the release of the overexpressed growth factor (44). Accordingly, different FGF2 antagonists inhibit the growth of FGF2-T-MAE cells in vitro and in vivo (14,15,17,18). On this basis, as a tool for further in vivo experimentation, FGF2-T-MAE cells were stably transfected with firefly luciferase, thus generating F2T-luc2.9 cells.…”

“…The latter interaction prevents the proteolytic degradation of FGF2 in the extracellular environment and induces growth factor oligomerization, which facilitates FGFR dimerization and subsequent transactivation (13). As such, various compounds, such as soluble heparin, polysulfonated compounds, and FGF2-binding peptides, which abrogate the interaction of FGF2 with HSPGs and/or FGFRs, have been shown to inhibit the biologic activity of FGF2 and to possess antitumor activity in vivo (14)(15)(16)(17)(18).…”

Heparin-Like Drugs with Antiangiogenic Activity

“…Liekens et al (Bugatti et al, 2007;Liekens et al, 1999;Liekens et al, 1997) studied different sulfonic acid-bearing polymers and demonstrated their capacity to impair the binding of bFGF to HSPGs. In these studies different polymers (poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (polyAMPS), poly(anetholesulfonic acid) and poly(4-styrenesulfonic acid)) showed a inhibition of the aFGF-mediated mitogenic response.…”

“…A polysulfonated naphthylurea (Suramin) was investigated in the inhibition of different proangiogenic factors (Danesi et al, 1993;Gagliardi et al, 1992). Based on the structure of suramin, different polysulfated and polysulfonic acid polymers were developed with capacity to interact with proangiogenic factors in similar mode to heparin (Fernández-Tornero et al, 2003;Liekens et al, 1999;Liekens et al, 1997). Actually there are different polyanionic and nonpolysulfonated components under study with capacity to interact with proangiogenic factor in the same binding-site that HSPGs (Fernández et al, 2010).…”