Development of a high throughput screening assay for inhibitors of fibroblast growth factor-receptor-heparin interactions.
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.
Aviezer
D
D
ProChon Biotech, Rehovot, Israel. aviezer@prochon.co.il
Seddon
A P
AP
Wildey
M J
MJ
Böhlen
P
P
Yayon
A
A
D000818:Animals; D001011:Aorta; D001331:Automation; D016466:CHO Cells; D002417:Cattle; D002478:Cells, Cultured; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004727:Endothelium / Q000166:cytology / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D017468:Receptors, Fibroblast Growth Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015203:Reproducibility of Results
D017468:Receptors, Fibroblast Growth Factor; D016222:Fibroblast Growth Factor 2; D006493:Heparin