Characterization of gonadotropin-releasing hormone analogs based on a sensitive cellular luciferase reporter gene assay

A novel cellular assay for the functional characterization of agonistic and antagonistic analogs of gonadotropin-releasing hormone (GnRH) was developed, This assay is based on a fusion of the c-fos immediate-early gene promoter to Photinus pyralis luciferase (Luc) as a reporter gene, stably transfected in a recombinant cell line expressing the human GnRH receptor, Transcription of endogenous c-fos and fos-Luc fusion gene are transiently induced quite similar by fetal calf serum or the superagonistic analog [D-Trp(6)] GnRH in a selected cell line, The reporter gene was therefore used to monitor agonist-induced signaling via the human GnRH receptor, Whereas Luc activity was induced in a dose-dependent manner by GnRH or [D-Trp(6)] GnRH, different antagonistic peptides completely inhibited this stimulation, The antagonistic potency (IC50) of various peptides with Cetrorelix and Antarelix as lead compounds in general correlated well with the binding affinity (K-D) as determined from ligand binding experiments, The specificity of an inhibitory effect was confirmed by GnRH receptor-independent stimulation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate or basic fibroblast growth factor, Since this new reporter gene assay is sensitive and simple and can be performed in a microtiter plate, it will significantly facilitate screening and functional characterization of GnRH analogs. (C) 1997 Academic Press.