Project: Industrialisation of Chemical Yeast 3-hybrid technology: applications to drug discovery and development and toxicoproteomics

_x000D__x000D_Finding the protein-binding profile of a small molecule for identifying new protein Ps is of great interest in many aspects of life sciences._x000D__x000D_- For drug safety evaluation: in terms of drug development, for identifying potential proteins connected to the molecule (“off target” effects). _x000D_- Identifying a new target for a known drug or ex-clinical candidate: drug repositioning._x000D_- Identifying the target of active molecule identified from “high content” screening techniques (phenotypic assays, whole animal ex. Zebra fish)._x000D_- For health and environment toxicology without animal experimentation: since the REACH directive of the EU, the safety of molecules needs to be addressed by all industries using large amount of particular of molecules such as the cosmetic, agro-food industry or agrochemical._x000D__x000D_We believe than Yeast Chemical Hybrid technology (CY3H) will be a very efficient new weapon in the chemical proteomic arsenal to address those problematic. For that purpose, we propose, based on our combine expertise in Yeast Hybrid technology and Chemistry, to develop a service to explore binding capacity of bioactive molecules from customers using chemical genetics technology in yeast._x000D__x000D_Chemical Yeast three hybrid._x000D_This complementary chemical proteomics technique, originally described by Schreiber and Crabtree (1) is an extension of the Yeast two Hybrid developed by Stanley Fields (2). It allows the identification of proteins Ps of a particular molecule._x000D_In the two-hybrid assay, the interactions of a particular bait-protein with its prey are detected as reconstitution of a transcriptional activator from its DNA binding (BD) and activation doCOs (AD). In the CY3H approach, a small molecule-protein interaction is detected by the dimerization of two receptor proteins via a bridged heterodimeric ligand. One ligand-receptor pair serves as an anchor (BD), while the other ligand-receptor pair (AD) is the small molecule-protein interaction of interest. The CY3H method was extended by Licitra and Liu who reported the first application of this CY3H method to small molecule target identification. (3)_x000D__x000D_During the ten past years, several groups as well as Biotech companies used this technology illustrating its relative efficacy. However despite very interesting results obtained, the use of the technique reCOed limited and no industrial service was offered for the following reasons:_x000D_- Weakness of the two hybrid screen quality and low capability to perform the screens_x000D_- Yeast permeability _x000D_- Case to case synthesis of fusion molecule_x000D_- Low number of anchor proteins available_x000D__x000D_The first point is address by Hybrigenics’ screening platform -ISO9001-2008 certified- which use an optimized version of the yeast two-hybrid screening technology (named: ULTImate Y2H™) that tackles most of the false positives and false negatives issues arising from the original technology. Indeed, unlike classical two-hybrid screening performed by sequential transformation of bait and then the library, ULTImate Y2H™ is derived from a unique and patented cell-to-cell mating process that allows the testing of 97 million interactions at once on average. (WO9942612)_x000D__x000D_The platform which has accommodated more than 5000 screens will greatly increase the output of the technique allowing the switch from a very nice one shot technique to a service dedicated to pharmaceutics, cosmetics and agro industry._x000D__x000D_Our project is thus based on a concept already validated but which failed to really reveal its potentiality and meet the market needs. The developments we proposed in this proposal are key innovations relative to this chemical protomic tool: _x000D__x000D_- A multi anchor and multi linker approach with a selection process based on yeast competition assays._x000D_- The use of antibodies systems as anchors._x000D_- Addressing chemical synthesis by “ready to use” multi building blocks_x000D_- Increasing results quality using cell-to-cell mating process_x000D_- Developing additional validating tools_x000D__x000D_The optimizations planned during this project, will shortened the timings for the customer, and the parallel approach will allow the launching of a high technology product. The real industrialization into a screening platform will also in mid term reduce the price of the service and by this way extend the interest of Academics, Biotechs and Pharma companies._x000D__x000D_This project proposes to develop a new service offer for research labs covering potentially the whole life science community._x000D__x000D__x000D__x000D_(1) Spencer, D.; Wandless, T.; Schreiber, S.; Crabtree, G. Science 1993, 262, 1019-1024._x000D_(2) Fields, S., and Song, O. (1989). Nature 340, 245–246. Fields, S. Proteomics. 2009, 9, 5209-13_x000D_(3) Licitra, E.J., and Liu, J. (1996). Proc. Natl. Acad. Sci. USA 93, 12817–12821._x000D_

Acronym	HybrY3Chem (Reference Number: 5646)
Duration	01/07/2010 - 30/06/2012
Project Topic	The project is dealing with the development of a new service based on yeast 3-hybrid technology for the detection of protein-ligand interactions in vivo. The development of such product combining chemistry and genetic, will address several very important aspects of drug discovery and development.
Project Results (after finalisation)	The CO result is the launch of a new service based on Chemical Yeast Three-Hybrid technology (CY3H). This technique allows the identification of all the binding Ps of a small bioactive molecule (e.g. on and off-targets of a Drug). it's a direct and unbiased approach. _x000D_The industrialization of this technology was posssible thanks to the heritage of Hybrigenics' ULTImate Y2H platform regarding the availability of highly complex cDNA libraies of preys, the patented mating screening procedure and the sophisticated bioinformatic analysis. _x000D_In parallel to these solid foundations, specific tools were developed :_x000D_- the permeability of the screening system (yeast cerevesiae) to chemical compounds : it was improved by deleting 5 genes involved in drug transport. _x000D_- selection of the most suitable anchorage sytems_x000D_- compound derivatization strategy and related chemistry in collaboration with our P Charnwood Molecular Ltd_x000D_- we started to screen with a conventional co-transformation protocol and moved, on optimization, to a more robust mating procedure_x000D_Early 2013, this new CY3H technique was transfered from development to labproduction (staff, procedure, ...)._x000D_
Network	Eurostars
Call	Eurostars Cut-Off 4

Project partner

Number	Name	Role	Country
2	Charnwood Molecular ltd	Partner	United Kingdom
2	HYBRIGENICS SA	Coordinator	France