Binding Constant Measurements for Inhibitors of Growth Factor Binding to Heparan Sulfate Proteoglycans

Abstract

A large number of proteins (>100) have been demonstrated to bind to heparan sulfate proteoglycans, and in many instances these interactions have important biological consequences (1). The best-studied example is the fibroblast growth factor (FGF) family of proteins. The FGFs regulate a wide range of cellular functions, including proliferation, differentiation, and migration. The best-characterized FGF family member, basic fibroblast growth factor (bFGF) or FGF-2, has been shown to require interaction with heparan sulfate on cell surfaces in order to induce maximal activity (2,3). However, interaction of bFGF with heparan sulfate within the extracellular matrix can limit bFGF diffusion and access to cell surfaces (4,5). Thus the interaction of bFGF with heparan sulfate has been targeted as a site for regulation of both endogenous and exogenously administered bFGF. For example, bFGF and many other heparin-binding proteins have been demonstrated to play pivotal roles in the growth of the new blood vessels (angiogenesis), a process that is essential for both efficient wound healing and the development of malignant tumors. Indeed, inhibition of endogenous bFGF activity has been suggested as a possible treatment to prevent tumor growth and metastasis, whereas enhancement of angiogenesis by pharmacological bFGF has been proposed to stimulate repair of damaged tissue (i.e., ischemic heart muscle) (6). In both instances, effective treatments might make use of small compounds, which inhibit bFGF binding to heparan sulfate proteoglycans. These compounds might have applications as inhibitors of bFGF binding and activity at cell surfaces, or in turn they might enhance the transport of added bFGF through connective tissue and allow cell stimulation distant from the administration site. Compounds that bind either to the growth factor or to heparan sulfate could block bFGF binding to heparan sulfate, yet might have very different effects biologically. To screen various potential inhibitors of growth factor/heparan sulfate binding effectively, a simple, rapid, and semi-quantitative assay is required.