A simple assay for evaluating inhibitors of proteoglycan-ligand binding

Proteoglycans, once thought to primarily serve as structural components of extracellular matrix, are now being focused on for their role in tissue and cell regulation, particularly angiogenesis. Many growth factors, notably t he fibroblast growth family:(FGF) which now numbers 19 members, bind to hep arin and heparan sulfate proteoglycans and this binding has been shown to h ave a significant impact on the availability and activity of these growth f actors. Proteoglycans can serve as both temporal and spatial regulators and effective inhibitor design may depend on disruption of these interactions. We have developed a simple assay for evaluating small inhibitors of proteo glycan-ligand binding. The assay is based on cell-free incubation of the re actants and filtration across a cationic membrane. Conditions were establis hed that allow one to semiquantitatively determine binding constants for bo th direct proteogly can as well as soluble inhibitor affinity. The assay ha s been demonstrated using a model heparan sulfate proteoglycan preparation (perlecan from cultured bovine endothelial cells) and FGF-2. Protamine sulf ate, sucrose octasulfate, and heparin were analyzed as model inhibitor mole cules. This type of assay may have wide application as a fast and easy scre ening tool for small potential agonists and antagonists of proteoglycan-pro tein interactions. (C) 2000 Biomedical Engineering Society. S0090-6964(00)0 1301-1].