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Abstract: In contrast to many reports dealing with inhibitors of growth factor receptors like VEGF or FGFR, only few reports of low molecular weight inhibitors, which are directed against growth factors itself, are known. Here, foscarnet, an antiviral drug which inhibits several viral DNA polymerases by mimic pyrophosphate of nucleotides, was identified to interact with fibroblast growth factor 2 and stabilize the growth factor against tryptic digestion similar like the non-nitrogen containing bisphosphonates clodronate and etidronate that we have reported just recently as inhibitors of FGF-induced cell proliferation. Foscarnet competes with ATP against the binding on fibroblast growth factor 2 at the heparin/ATP-binding domain. This indicates binding of foscarnet at the heparin-binding domain of FGF2. This interaction of foscarnet with fibroblast growth factor 2 reduces FGF2-induced proliferation of human umbilical vein endothelial cells and intracellular signaling via ERK1/2 kinases in this cell line. Additionally, foscarnet reduces in a dose-dependent manner proliferation of CAL-62 cells that belong to anaplastic thyroid carcinoma, a rare but lethal type of thyroid cancer that expresses FGF2. [Copyright &y& Elsevier]

Abstract: We have shown previously that nerve growth factor (NGF) requires only low nanomolar ATP concentrations in the cell culture medium to protect cortical rat neurons (CRN) from cellular damage induced by staurosporine (STS). We have also demonstrated before that NGF and other growth factors form stable non-covalent complexes with ATP. Here we demonstrated that 8N1ATP–NGF, but not NGF, protected CRN against damage. The photo-reactive ATP derivative 8N3ATP was incubated with NGF and was trapped in its position by UV irradiation forming a covalent bond. The cross-link with a molar ratio of 1:1 (8N1ATP:NGF) was confirmed by mass spectrometry. Circular dichroism experiments revealed that 8N1ATP altered the secondary structure of NGF in the same way as ATP did. Covalently bound 8N1ATP–NGF was shown to be stable in the presence of the ATP-hydrolyzing enzyme alkaline phosphatase while the non-covalent ATP–NGF-complex dissociated with the removal of free ATP from the solution. 8N1ATP–NGF protected CRN against damage by STS independently of free ATP in the culture medium. These results suggest that the ATP–NGF-complex, but not NGF, is the active ligand. [Copyright &y& Elsevier]

Abstract: Basic fibroblast growth factor (bFGF) is a heparin-binding growth factor known to cause cell proliferation, angiogenesis and neuroprotection. We have performed site-directed mutagenesis to identify the amino acids that are essential for heparin/growth factor interaction and for neuroprotection. Binding to heparin-acrylic beads was markedly reduced when lysine in position 134 of bFGF was replaced by alanine. Wildtype (wt)-bFGF was shown to protect rat primary cultures of embryonic hippocampal neurons against damage caused by staurosporine and to reduce the infarct size in mice after focal cerebral ischemia. These neuroprotective effects of wt-bFGF could not be shown for the mutant bFGF(K134A). Furthermore, phosphorylation of Akt and ERK1/2 was significantly reduced in cultured neurons treated with bFGF(K134A) indicating diminished intracellular signaling compared to neurons treated with wt-bFGF. In conclusion, lysine at position 134 of bFGF is essential for bFGF to bind heparin, then to interact with its receptor and, subsequently, to protect neurons against damage. [Copyright &y& Elsevier]

Abstract: Growth factors and their mechanisms of action have been studied extensively. However, it remained widely unrecognized that binding of ATP to growth factors is a prerequisite for their bioactivity. Here we demonstrated the binding of ATP to nerve growth factor (NGF) as well as its relevance for neuroprotection. By using mass spectrometry-based methodology we identified one or two molecules of ATP as being bound to NGF. To test neuroprotective activity of NGF we used primary cultures of rat cortical neurons damaged by staurosporine. ATP was indispensable for the neuroprotective effect of NGF. When the ATP concentration in the culture medium was reduced below approximately 2nM by adding alkaline phosphatase (AP) or ATPase the neuroprotective activity of NGF was abolished. Site-directed mutagenesis within the heparin-binding domain (HBD) of NGF abolished ATP-binding and the neuroprotective effect. Thus, NGF has to bind ATP to be capable of protecting neurons. [Copyright &y& Elsevier]