CHARACTERIZATION OF GONADOTROPIN-RELEASING-HORMONE ANALOGS BASED ON ASENSITIVE CELLULAR LUCIFERASE REPORTER GENE ASSAY

A novel cellular assay for the functional characterization of agonisti c and antagonistic analogs of gonadotropin-releasing hormone (GnRH) wa s developed, This assay is based on a fusion of the c-fos immediate-ea rly gene promoter to Photinus pyralis luciferase (Luc) as a reporter g ene, stably transfected in a recombinant cell line expressing the huma n 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 rep orter gene was therefore used to monitor agonist-induced signaling via the human GnRH receptor, Whereas Luc activity was induced in a dose-d ependent manner by GnRH or [D-Trp(6)] GnRH, different antagonistic pep tides completely inhibited this stimulation, The antagonistic potency (IC50) of various peptides with Cetrorelix and Antarelix as lead compo unds in general correlated well with the binding affinity (K-D) as det ermined from ligand binding experiments, The specificity of an inhibit ory effect was confirmed by GnRH receptor-independent stimulation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate or basic fibro blast growth factor, Since this new reporter gene assay is sensitive a nd simple and can be performed in a microtiter plate, it will signific antly facilitate screening and functional characterization of GnRH ana logs. (C) 1997 Academic Press.