Obiettivo To establish reporter cell systems for the in vivo monitoring of the agonistic activity of nuclear receptor ligands and high throughput screening. To screen for tissue-specific progestins and bone-specific (anti)estrogens . To establish reporter cell systems for the screening of "dissociated" nuclear receptor ligands which selectively antagonize AP1 activity. To screen for "dissociated" glucocorticoids and establish their anti-inflammatory activity. To study the chromatin structure of reporters genes and the effect of ligand exposureTo optimize the efficiency of expression and intracellular compartmentalization of the reporter gene product. . To extend reporter gene technology to monitor intracellular Ca++.To extend reporter gene technology to monitor membrane receptor activity and screen for agonists and antagonistsTo crystallize nuclear receptor apo-and holo-ligand binding domains and model unknown ones.The aim of this proposal is to join the efforts of industrial and basic research laboratories to (i) develop technologies for the establishment of recombinantly tailored "reporter" cell lines and (ii) determine the structure of nuclear receptor ligand binding domains in order to identify, design and improve lead compounds. This study wants to pave the way towards the development of a new generation of high throughput screening systems in which the activity of a drug is monitored in the intact actual cellular target relative to its action in non-target cells. As model systems the nuclear receptor (steroid, thyroid, retinoid, vitamin D) signalling pathways will be used, with the screening strategy being based on ligand-induced (anti-ligand-inhibited) activation or repression of reporter gene transcription, including the transrepression of AP1 activity. Stable cell lines will be established in which anti-ligand binding promotes a transcriptional response that can be easily monitored and quantified. The study of the chromatin structure of the integrated reporter will reveal to what extent it influences hormonal inducibility. In parallel the 3D structure of nuclear receptors will be determined in the absence and presence of ligands with well-defined functional characteristics. To extend this approach stable cell lines expressing the targeted photoprotein aequorin and "reporting" alterations in Ca++ homeostasis in given cellular compartments will be established. The following technologies will be developed and/optimised: (I) Four reporter systems will be investigated : ligand-induced luciferase, aequorin GFP and DNA recombination-dependent production of bioluminescence. (2) Stable cell lines with heterologous or homologous activators will be compared. (3) Reporter cell lines will originate from the same cell type in which the drug supposed to exhibit its action (ex.: bone vs endometrial cells). (4) To assess the promoter specificity of the transcriptional response in stably transfected cell lines different reporter genes will be compared. Possible applications of this technology comprise the identification of novel (tissue-specific`) agonists/antagonists, such as the development of bone-specific estrogens for the treatment of osteoporosis. and anti-inflammatory glucocorticoids with anti-AP1 activity but no hepatic side effects. 3D structure information will be used to optimise lead compounds by molecular modelling. Collectively,. the above described projects will provide novel tools for pharmaceutical drug development and explore the possibility of "tailoring" screening systems according to the drug target. Such systems are not only limited to nuclear receptors, but can be applied also to other signalling pathways which couple to specific transcription factors. This possibility will be explored by monitoring membrane receptor-mediated transcriptional responses of suitable reporter gene constructs. Campo scientifico medical and health sciencesbasic medicinepharmacology and pharmacydrug discoverymedical and health sciencesbasic medicinepharmacology and pharmacypharmaceutical drugsnatural sciencesbiological sciencesgeneticsDNAmedical and health sciencesbasic medicinephysiologyhomeostasis Programma(i) FP4-BIOMED 2 - Specific research, technological development and demonstration programme in the field of biomedicine and health, 1994-1998 Argomento(i) 1.1 - Pharmacotoxicology Invito a presentare proposte Data not available Meccanismo di finanziamento CSC - Cost-sharing contracts Coordinatore Centre National de la Recherche Scientifique Contributo UE Nessun dato Indirizzo 1,LAURENT FRIES 67404 ILLKIRCH Francia Mostra sulla mappa Costo totale Nessun dato Partecipanti (7) Classifica in ordine alfabetico Classifica per Contributo UE Espandi tutto Riduci tutto Centre National de la Recherche Scientifique Francia Contributo UE Nessun dato Indirizzo 1,RUE LAURENT FRIES 67404 ILLKIRCH Mostra sulla mappa Costo totale Nessun dato Centre National de la Recherche Scientifique Francia Contributo UE Nessun dato Indirizzo 118,ROUTE DE NARBONNE 31062 TOULOUSE Mostra sulla mappa Costo totale Nessun dato EUROPEAN MOLECULAR BIOLOGY LABORATORY Germania Contributo UE Nessun dato Indirizzo Meyerhofstrasse 1 HEIDELBERG Mostra sulla mappa Costo totale Nessun dato Roussel Uclaf Francia Contributo UE Nessun dato Indirizzo 102,Route de Noisy 102 93235 Romainville Mostra sulla mappa Costo totale Nessun dato Schering AG Germania Contributo UE Nessun dato Indirizzo 178,MULLERSTR. 13342 BERLIN Mostra sulla mappa Costo totale Nessun dato Università degli Studi di Padova Italia Contributo UE Nessun dato Indirizzo Via 8 Febbraio 3 35121 Padova Mostra sulla mappa Costo totale Nessun dato Université de Montpellier 1 Francia Contributo UE Nessun dato Indirizzo 70,RUE DE NAVACELLES 34090 MONTPELLIER Mostra sulla mappa Costo totale Nessun dato