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CDithem
Responsable : Robin FAHRAEUS • Responsable Tech. : Agnes LEON
Adresse : Inserm U716
IGM, 27 rue Juliette Dodu
75010 Paris

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Certifications

Pas de certification ISO

Site web

http://www.cdithem.fr

Presentation de la plate-forme

Key words:
In vitro screening, cell pharmacology, medicinal chemistry, molecular modelling, ADMEtox, X Ray crystallography.

Operational specificity:
CDithem (consortium for drug discovery and therapeutic innovation) is the first academic, vertically integrated drug discovery platform in France.
This integration formalizes the collaboration between biologists, chemists, structural biologists, computational scientists and analysts. It allows forward planning from the early stages of discovery (target identification), an accurate focus of resources and maximizes the delivery of our therapeutic objectives. To support project management, we have built a strong data management system that allows a secure repository of all the data generated and easy data mining and manipulation over the internet.

Technical Specificity : protein-protein interactions.
Protein-protein interactions represent an emerging area of drug discovery. Whereas many protein-protein interactions are functionally valid drug targets, the techniques and methods needed to analyse and screen/drug them still needs development and are less straightforward compared to screening for compounds that interact with an enzyme catalytic site. Hence, one of the aims of the platform is to develop techniques for drug development based on targeting protein-protein interactions that can be shared within academia and also benefit the industrial sector. Protein Interfaces are not well understood, nor are the energetic of association/dissociation, and as such, new technologies will have to be developed along the process to insure success, from molecular and cellular biology, chemistry to computational methods.

CDithem aims to develop such techniques and to provide services to:
• Search for, evaluate and validate therapeutic targets.
• Identify and optimize hits using existing as well as cutting edge technologies and skills developed by the consortium.
• Optimize further selected compounds to produce drug candidates (developable compounds, displaying the expected effect in a recognized animal model).

Services :
--> General data management
Bibliographic searches (Beilstein, Scifinder, MDL databases): establishment of prior art in drug discovery concerning incoming targets. Freedom to operate, Patentability.

-->Target evaluation, functional assessment in cell models. (within U716)
The combination of in vitro characterized targets and delivery of target modifying molecules into cells carrying specific genetic backgrounds serves to rapidly evaluate the potential effects of modifying a specific intracellular protein interaction. Previous work carried out by members of U716 has demonstrated how in vitro characterised small peptides delivered into cells can be used to specifically and rapidly validate the cell biological effects of targeting protein interaction.

- In vitro target characterization: Yeast two hybrid, data mining, proteomics. Molecular targets are identified/selected using molecular biology, genetics and structural information. Target proteins and genetically modified variants are produced in prokaryotic and eukaryotic systems and biochemical properties are characterized. Protein-protein interactions are evaluated by biochemical as well as functional assays. Peptide display libraries and synthetic peptide libraries are used to identify protein interacting peptides. Scaffolding proteins and synthetic peptides are used to mimic/disrupt protein-protein interactions and to study the biochemical effects of targeting specific protein interactions.

- Evaluation in cell systems: BRET, Peptide delivery, siRNA, genetically modified cell lines, yeast-based assays. Scaffolding proteins or cell deliverable peptides are used to bring in vitro characterized peptides into cells. Cells carrying specific genetic endogenous/exogenous modifications and siRNA are used to evaluate specific cell biological effects of modifying protein-protein interactions. BRET and standard protein biochemistry assays are used in small well format to determine modifications of interactions in cells based on peptides or chemical compounds.

--> Screening, Medicinal Chemistry, Compound library and management, eADMEt (within U761)

CDithem has a library of 45.000 compounds (no string attached) formatted for screening in 96 well plates (SBS standards). Compounds have been selected from commercial vendors or prepared by our chemists using state-of-the-art selection and design criteria, in terms of diversity and “drug / lead likeness”.

Fast and robust assays allow medium to high throughput screening.
Screening parameter is evaluated to optimize the performance.
Sample analysis based on fluorescence (FRET, HTRF, fluorescence anisotropy, fluorescence intensity), luminescence, absorbance or thermal shift technologies are applied to study enzymatic reactions, ELISA, protein-protein interactions, DNA-protein interaction.

Thermal Shift Assays are used to measure the strength of the ligand/protein interaction at high throughput.

For developing structure-based drug design, the medicinal chemistry team works together with the molecular biology / X-Ray diffraction team, fully equipped for molecular biology, protein expression and purification and crystallization using a robotic platform.

Noteworthy, a coordination committee between CEA and Inserm has been created to avoid duplication of technologies and redundant development work or equipments.

Library design & compound enumeration is performed using Pipeline pilot, from 4000+ reagents.

Library synthesis is performed with various specialized devices such as a Tecan genesis, a custom-made 96-well multi-reflux oven, a microwave workstation, carrousels, Genevac HCl-resistance evaporator, lyophilisator.

To support the compound optimisation and to optimise the management and the interpretation of early in vivo experiments, the laboratory has developed efficient eADME tools :
- Solubility, pKa, logD; Metabolic stability (plasma half-life, microsomal clearance, cyp phenoyping);

- Pharmacokinetics (AUC in rodents, IV, IP and PO routes); Cytotoxicity (MTT assay on cultured fibroblasts and hepatocytes) Specificity on a case by case basis.

Integration of early ADME models is a differentiating asset of CDithem from the scientific, economical and ethical standpoints. It increases the relevance of in vivo data and reduces the number of animals used for validation. Indeed, only the compounds displaying the required PK properties will be administered to animals.

--> MTi - Molecular modeling, Virtual screening and Drug Design:

In the drug discovery process, a series of important tasks can be performed through computer calculations, such as ADME/tox filtering to obtain a chemical library with a specific physico-chemical profile, the virtual screening of chemical libraries using either ligand-based and/or structure-based approaches, the homology modeling of unknown target 3D structure, the protein-protein docking to get insight into the nature of a protein-protein interaction at the structural level, and finally, but non-exhaustively, hit to lead optimization through binding affinity comparison.

• Homology modeling
• Protein-protein docking
• Biostatistics simulations
• ADME/Tox profiling, in silico generation of compound collections (diversity set, similarity, ADME/Tox filtered, target specific)
• Structure-based in silico screening
• Ligand-based in silico screening
• Protein structure analysis
• in silico hit-to-lead optimization
• Experimental design based on structural analysis
• Development of mathematical models and use of machine learning approaches

Moyens et equipements

Capacité de calcul: cluster de 30 CPU
Equipement de criblage principaux:
LC/MS/MS Varian 1200
LCPrepa/MS Waters
Automate de dilution Cibio
Chimiothèque: > 45 000 composés

Galerie

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