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Contenido archivado el 2024-04-30

Recombinantly tailored reporter cell lines and 3D structure evaluations for improved drug development

Objetivo

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 exposure
To 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 antagonists
To 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.

Convocatoria de propuestas

Data not available

Régimen de financiación

CSC - Cost-sharing contracts

Coordinador

Centre National de la Recherche Scientifique
Aportación de la UE
Sin datos
Dirección
1,LAURENT FRIES
67404 ILLKIRCH
Francia

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Coste total
Sin datos

Participantes (7)