Development of a High Throughput Screening Assay for Inhibitors of Fibroblast Growth Factor-Receptor-Heparin Interactions

Abstract

High throughput screening (HTS) of large compound libraries for inhibitors of growth factors raises the requirement for simple yet reliable assays. Fibroblast growth factors (FGFs) play a pivotal role in the multistep pathway of malignant transformation, tumor progression, metastasis, and angiogenesis. FGF-2 (basic FGF) requires a cooperative interaction with heparin or heparan sulfate proteoglycans in order to form functional growth factor-receptor complexes that are essential for receptor binding and activation. We have developed a simple screening system, devised to identify molecules that modulate heparin-FGF-receptor interactions. The system is composed of a heparin matrix, FGF-2, and a FGF receptor-1 protein engineered by genetically fusing the extracellular domain of FGF receptor-1 to alkaline phosphatase (FRAP). The screen is conducted using 96-well plates to which heparin has been covalently attached. FGF-2 is then bound to the plates through heparin-FGF interactions, followed by the addition of FRAP and compounds to be screened for modulation of heparin-FGF, receptor-heparin, and receptor-FGF interactions. The endpoint of the assay is measured enzymatically using the alkaline phosphatase (AP)-catalyzed formation of a chromogenic product, which is directly proportional to the amount of FRAP present on the plates as a heparin-FGF-FRAP ternary complex. Reduced AP values relative to control, as measured by spectrophotometry, indicate inhibition of the formation of an active FGF-receptor-heparin complex. The simple and versatile nature of the assay makes it an attractive HTS system. The screen has identified several potent inhibitors of FGF-2 receptor binding and activation. Furthermore, secondary screening of the HTS-recognized compounds identified several compounds that have the capacity to block growth factor-mediated tumor progression and angiogenesis in vivo.