Testing of potential glycan-based heparanase inhibitors in a fluorescence activity assay using either bacterial heparinase II or human heparanase

• A simple fluorimetric heparanase activity assay using the synthetic sulfated pentasaccharide fondaparinux as substrate.
• The assay was used for testing potential heparanase inhibitors.
• The inhibitory activity of sulfated glycans showed to be strongly dependent on their degree of sulfation as well as their basic glycan structure, but independent of their molecular mass.
• The semisynthetic β-1,3-glucan sulfate PS3 showed the strongest inhibitory effect with an IC50 being 160 times lower than that of suramin.

Heparanase, an endo-β-glucuronidase cleaving heparan sulfate (HS) chains at cell surfaces and in the extracellular matrix (ECM), is involved in angiogenesis, tumor progression and metastasis as well as in inflammation and kidney dysfunction. Therefore, heparanase is considered a promising therapeutic target and diagnostic marker. Recently, we have developed a simple, rapid, fully automatable fluorimetric activity assay using the synthetic sulfated pentasaccharide fondaparinux as substrate and bacterial heparinase II (HEP-II) instead of human heparanase (hHEP). The aim of this study was to evaluate this assay for inhibitor testing as well as to check whether the assay principle is applicable to measure the activity and inhibition, respectively, of the actual target enzyme hHEP.Besides the known hHEP inhibitor suramin and the antiinflammatory and antimetastatic PS3, two series of β-1,3-glucan sulfates differing in their chain length and degree of sulfation, further semisynthetic sulfated glycans, and two sulfated polysaccharides isolated from algae were included to examine structure–activity relationships.The inhibitory activity of sulfated glycans showed to be greatly dependent on both their degree of sulfation and their basic glycan structure, but independent of their molecular size. The β-1,3-glucan sulfates were superior to suramin as well as to the other glycans with similar degree of sulfations. The most active inhibitor was found to be the β-1,3-glucan sulfate PS3 (IC50 = 0.017 μM). By using hHEP instead of HEP-II comparable results were obtained. With an IC50 being about 160 times lower than that of suramin, PS3 exhibited again the strongest inhibitory effects. Inhibition of hHEP may therefore contribute to the potent antiinflammatory and antimetastatic activities of PS3 in vivo. In conclusion, the fluorimetric hHEP activity assay proved to be a simple, fully automatable tool for testing potential inhibitors. In case of HS mimetic inhibitors, the assay variant with HEP-II may provide a fast and inexpensive option for initial screening purposes.

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