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-Trp6] 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-Trp6] 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 (KD) 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.