Gentisic Acid, a Compound Associated with Plant Defense and a Metabolite of Aspirin, Heads a New Class of in Vivo Fibroblast Growth Factor Inhibitors

Israel S Fernández,Pedro Cuevas,Javier Angulo,Pilar López-Navajas,Ángeles Canales-Mayordomo,Rocío González-Corrochano,Rosa M Lozano,Serafín Valverde,Jesús Jiménez-Barbero,Antonio Romero,Guillermo Giménez-Gallego

doi:10.1074/jbc.m109.064618

Abstract

Fibroblast growth factors are key proteins in many intercellular signaling networks. They normally remain attached to the extracellular matrix, which confers on them a considerable stability. The unrestrained accumulation of fibroblast growth factors in the extracellular milieu, either due to uncontrolled synthesis or enzymatic release, contributes to the pathology of many diseases. Consequently, the neutralization of improperly mobilized fibroblast growth factors is of clear therapeutic interest. In pursuing described rules to identify potential inhibitors of these proteins, gentisic acid, a plant pest-controlling compound, an aspirin and vegetarian diet common catabolite, and a component of many traditional liquors and herbal remedies, was singled out as a powerful inhibitor of fibroblast growth factors. Gentisic acid was used as a lead to identify additional compounds with better inhibitory characteristics generating a new chemical class of fibroblast growth factor inhibitors that includes the agent responsible for alkaptonuria. Through low and high resolution approaches, using representative members of the fibroblast growth factor family and their cell receptors, it was shown that this class of inhibitors may employ two different mechanisms to interfere with the assembly of the signaling complexes that trigger fibroblast growth factor-driven mitogenesis. In addition, we obtained evidence from in vivo disease models that this group of inhibitors may be of interest to treat cancer and angiogenesis-dependent diseases.

Highlights

FGFs in humans and mice that differ significantly in both size (17–20 kDa) and sequence, each contains a core homology region encompassing 120 –130 residues
FGFs, at times expressed at very high levels, normally remains trapped in the inhibitory activity; IDS-3, O2-sulfoglucuronic acid; GA, gentisic acid (2,5dihydroxybenzoic acid); HGA, homogentisic acid (2-(2,5-dihydroxyphenyl) acetic acid); SGN-4, N,O6-disulfoglucosamine; Saturation transfer difference (STD), saturation transfer difference NMR spectroscopy; HSQC, heteronuclear single quantum correlation; vascular endothelial cell growth factors (VEGFs), endothelial cell growth factor
We found that gentisic acid (GA; 2,5-dihydroxybenzoic acid), a widespread plant secondary metabolite involved in pest defense and a catabolite of aspirin, is a potential inhibitor of FGF [27,28,29,30,31,32]

Summary

EXPERIMENTAL PROCEDURES

Protein Expression Purification and Analysis—The cDNAs encoding FGF-1 and FGF-2 were cloned into the pRAT-4 plasmid, expressed in Escherichia coli BL21(DE3), and purified by heparin-Sepharose chromatography [33, 34]. Once the cells reached confluence, a linear wound across the diameter of the coverslip was drawn with a rubber cell scrapper, and the medium was changed to Ham’s F-12/Dulbecco’s modified Eagle’s medium (1:3) supplemented with a 1/100 (v/v) dilution of culture supplement ITSϩ (Collaborative Research), 2.5 mM L-histidine, 2 mM L-glutamine, 50 mM ethanolamine, 0.1 mg/ml myo-inositol hexasulfate, 103 units/ml penicillin, 12 mg/ml gentamicin, and the doses of FGF and inhibitor detailed under “Results.” The cultures were fixed 48 h afterward in a 1% solution of glutaraldehyde, and they were stained with crystal violet as described [36]. A STD experiment was carried out in which exdFGFR2IIIc was omitted from the solution

RESULTS

Refinement statistics

DISCUSSION