HTS AND ADME-PKSCREENING LAB Inserm U761
Institut Pasteur de Lille
Université Lille Nord de France Responsable Benoît DEPREZ
00 33 3 20 96 40 24
Group members DECAVEL Jean-Pierre | DEPREZ Benoît | LEROUX Florence
Mission of the platform
The Inserm U761, headed by Pr Benoît Déprez, includes a screening facility, set up at the Pasteur Institute in Lille. The mission of the group is to operate and constantly update a technology platform supporting project team activities in drug discovery. It covers the whole drug discovery and selection process : library management, assay development, medium to high throughput screening, hit-to-lead lead optimisation and early ADME-PK profiling. The platform contributes to the CDithem (Consortium for Drug Discovery and Therapeutic Innovation) network, that was recently selected by the Gis IBiSA.
The platform is lead by a research engineer and operated by 3 biologists (1 post-doctoral research fellow and 2 technicians).
Description of the platform Chemical Library
During the past 4 years, our lab has assembled a library of 45.000 compounds formatted for screening in 96-well plates (SBS standards). Compounds have been selected from commercial vendors or prepared by our chemists (10% of the library) using state-of-the-art selection and design criteria, in terms of diversity and "drug/lead-likeness".
A chemical library of about 30.000 extra compounds will be purchased in 2010 in order to address current "diversity holes" in our library. The financing come from an ANR grant (PIRIbio project : development and validation of a protein-protein interaction inhibitor focused library via in silico & in vitro approaches) and a selection procedure has already been defined in coordination with the Inserm U973 (Bruno Villoutreix, Paris 7). The chemical library management system avoids repeated freeze thaw cycles and ensures the longest possible lifetime for all the samples. Sample tracking is seamless throughout the sample’s life (from ordering to testing). To manage compounds and associated results, a high-throughput data analysis and mining system for drug discovery has been implemented in the Unit, using Access / Isis databases and the pipeline pilot™ software.
The Main equipments are : (1) a 1420 multilabel multi-mode fluorescence / luminescence reader Victor™3V (Perkin Elmer), (2) a lightcycler 480 real-time detection system (Roche), used to evaluate protein ligand binding in thermal shift assays, (3) a pipettor device CyBIWell (CyBio) to dilute and aliquot the chemical libraries and (3) a Genesis RSP instrument (Tecan) for serial dilutions. To improve the screening capacity, the platform will be equipped in 2010 with a second reader, keeping the current Victor as a back-up and assay development system. A cell culture unit was created at the beginning of 2008 with the purchase of a Thermo Scientific Heraeus HERAsafe KS safety cabinet, a Nikon inverted microscope Eclipse TS100 and a Thermo Scientific Steri-cycle CO2 incubator.
Compound Screening, Hit-to-Lead and Lead Optimisation.
The laboratory has a recognized expertise in the development of miniaturized, fast and robust assays for medium to high throughput screening. All critical screening parameters are optimized in terms of reagent cost, required manpower and time, as well as its discriminating power, as measured by the Z’ and Z factors. Fluorescence data (FRET, HTRF, fluorescence anisotropy, fluorescence intensity) or absorbance data are used to study enzymatic reactions, protein binding, proteinprotein interactions, DNA-protein interactions.
Except a few immuno-assays, most of the tests are homogeneous screening assays, highly convenient to perform HTS. One cell-based assay was developed using Mycobacterium smegmatis transformed with a reporter gene.
A cytometry-based method was used to evaluate the compound inhibition of a protein-protein interaction.
Compound profiling, beyond "on-target" activity.
To enable the generation of relevant in vivo data, we have developed a procedure to predict and eventually measure the exposure of an animal model to a given compound. This profile comprises data on physical chemical properties known to govern bioavailability (logD7.4, aqueous solubility, microsomal stability…). Only compounds displaying the desired ADME properties, beyond their activity on target are administered to animal. This saves animal lives and researcher’s time. Importantly the knowledge generated by these studies is applicable to virtually any chemical series, independently of the mode of action. Thus all the data are stored safely in a database that can be interrogated by any researcher in the lab and serve as learning sets for molecular modelling. In collaboration with Juergen Siepmann, professor of pharmaceutical sciences in Lille, we have obtained a grant from Région Nord-Pas-de-Calais, to set up a small platform for early formulation & early pharmacokinetics. By e-formulation we mean rapid and miniaturized formulation, adapted to molecules not yet fully optimized and to an administration of minute quantities to small animals (mice in particular). The animal experimentation takes place in the animal facilities of the Pasteur Institute of Lille. Sample preparation and bioanalysis are performed using our LC-MSMS platform.