title: A simple assay for evaluating inhibitors of proteoglycan-ligand binding.
abstract: 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 the fibroblast growth family (FGF) which now numbers 19 members, bind to heparin and heparan sulfate proteoglycans and this binding has been shown to have a significant impact on the availability and activity of these growth factors. 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 proteoglycan-ligand binding. The assay is based on cell-free incubation of the reactants and filtration across a cationic membrane. Conditions were established that allow one to semiquantitatively determine binding constants for both direct proteoglycan as well as soluble inhibitor affinity. The assay has been demonstrated using a model heparan sulfate proteoglycan preparation (perlecan from cultured bovine endothelial cells) and FGF-2. Protamine sulfate, sucrose octasulfate, and heparin were analyzed as model inhibitor molecules. This type of assay may have wide application as a fast and easy screening tool for small potential agonists and antagonists of proteoglycan-protein interactions.
authors.affiliation: Department of Chemical Engineering, Virginia Polytechnic Institute and State University, Blacksburg 24061-0211, USA. kforsten@vt.edu
authors.forename: K E
authors.initials: KE
authors.lastname: Forsten
authors.forename: N
authors.initials: N
authors.lastname: Wang
authors.forename: R M
authors.initials: RM
authors.lastname: Robinson
authors.forename: M A
authors.initials: MA
authors.lastname: Nugent
mesh_terms: D000818:Animals; D001698:Biomedical Engineering; D002417:Cattle; D002474:Cell-Free System; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004730:Endothelium, Vascular / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin / Q000494:pharmacology; D006497:Heparan Sulfate / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D011479:Protamines / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011509:Proteoglycans / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013395:Sucrose / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D017077:Culture Media, Conditioned; D016222:Fibroblast Growth Factor 2; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin; D006497:Heparan Sulfate; D007334:Insulin-Like Growth Factor I; D008024:Ligands; D011479:Protamines; D011509:Proteoglycans; D011994:Recombinant Proteins; D013395:Sucrose; C071927:perlecan; C053081:sucrose octasulfate