Johana Tournois: Associate engineer (CECS)
Benjamin Brinon: 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 30 000 I-Stem compounds are tested during the primary screening campaign. The screening is then followed by the retest, counter-test and EC/IC50 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 20 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)
- One HTS reader is available: the Clariostar (BMG Labtech) is a multimode detection system (luminescence, fluorescence, TR-FRET, Alphascreen, Alphalisa, HTRF…)
- Two HCS readers are available: The Arrayscan (Cellomics) and the Image Express (Molecular device) are automated fluorescence microscope. From the cell imaging platform .http://www.istem.eu/en/technological-platforms/cell-imaging/
Results and future prospects:
Recently several screening campaigns were performed on the HTS platform:
A drug screening on Hutchinson Gilford progeria pluripotent stem cells, to the identification of inhibitors of prelamin A farnesylation (published)
High throughput screening for inhibitors of REST in neural derivatives of human embryonic stem cells reveals a chemical compound that promotes expression of neuronal genes (published)
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.
Examples of screening campaigns’ analyses with Hiscreen and Spotfire tools (Hit selection, Z ’Factor calculation, EC50 curves)
Discngine, Paris, 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)