Johana Tournois: Associate engineer (CECS)
Benjamin Brinon: Qualified research technician (CECS)
Aims and background:
The High Throughput Screening (HTS) activity is part of the drug discovery process, and consists in selecting among thousands of molecules the ones that could have a pharmaceutical use. To do that, large compounds libraries are tested on a biological model showing a specific therapeutic target. The HTS at I-Stem focuses on relevant biological models of genetic diseases: the human stem cells and their progenies. The self-renewable capacity of these cells, their ability to differentiate into several tissue progenitors (neural, mesenchymal stem cells…), and the possibility to work with mutated cell lines define human stem cells as a good basis for screening compounds libraries in order to discover new potential drugs for monogenic diseases.
Once a disease has been explored with genomic, proteomic and cell biology tools, relevant targets are identified by I-Stem research teams. Then, the first objective of the HTS platform is to develop a robust cell based assay in microplates (96- 384- wells) with a specific read out. When the assay has been set up, the 12 000 I-Stem compounds are tested during the primary screening campaign. The screening is then followed by the retest, counter-test and EC50 evaluation steps. Finally, the lead optimization is done in collaboration with our chemist partners.
To be able to develop reproducible and reliable tests in sterile conditions, the HTS platform was set up with robots, readers, and a good data mining system.
Strategy, means and methods:
The HTS platform is equipped with several robotic systems:
- The Bravo-Benchcel (Agilent) is a compact pippeting station connected to a plate storage system. This platform treats 5 to 15 plates and is mainly used for the assay development step, cherry picking and serial dilution protocols
- The Biocel 1800 (Agilent) allows to handle in sterile conditions nearly 200 plates (96- 384- wells plates) for compound management tasks and primary screenings
- An automated cell fixation and staining platform (Thermo, Biotek) is used for High Content Screening campaigns (HCS)
- Two readers are available: the AnalystGT (Molecular Device) is a multimode detection system (luminescence, fluorescence, TR-FRET) and the Arrayscan (Cellomics) is an automated fluorescence microscope
Results and future prospects:
Two main screening campaigns were performed lately on the HTS platform:
A high throughput screening was performed on a target identified in the Huntington disease, the REST protein. Neural stem cells transfected with a luciferase reporter gene construction were treated with 12 000 compounds.
7000 compounds were tested on human mesenchymal stem cells carrying Type 1 myotonic dystrophy. These cells show cytoplamic aggregates that can be detected and quantified with the Arrayscan.
A collaboration with the Roche pharmaceutical company led to the screening of 200 000 Roche compounds on human neural stem cells in order to identify proliferative drugs.
The main perspective is to use Induced Pluripotent Stem (iPS) cells as a biological model for the next HTS screenings.
Discngine, Romainville, France (database management systems (Hiscreen) and Multi-parameter visualization tools (Spotfire))
Chem-X infinity, Romainville, France (libraries of small organic molecules)
Institut Curie, Orsay, France (libraries of small organic molecules)