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The understanding of small molecule mechanism of action through target identification is now essential to improve drug efficiency and prevent side effects. In this context, chemical biology and in particular chemical proteomics have developed and various methods have emerged.
Chemical Yeast Three-Hybrid (CY3H) is a compound profiling technique derived from the Yeast Two-Hybrid (Y2H) technology1.
In this adapted version, the small molecule of interest is used as a bait to screen protein libraries. The small molecule - protein interactions are detected thanks to the reconstitution of an active transcription factor from DNA Binding Domain (DBD) and Activation Domain (AD) moieties.
Three components are used:
Different linkers and anchors can be chosen. The linker generally consists in a polymer of Poly-Ethylene Glycol (PEGn). The anchor is a molecule attached to the linker which binds to the chosen ABD. The main Anchor /Anchor Binding Domain combinations are: Methotrexate (MTX) / Dihydrofolate Reductase (DHFR); Trimethoprim (TMP) / DHFR, Biotin / Streptavidin; Dexamethasone (DEX) /Glucocorticoïd Receptor (GR) and O6-benzylguanine derivatives (BG) / SNAP-Tag.
The bait derivative is tested for its interaction with protein targets by screening a library in yeast. When a "small molecule – prey protein" interaction takes place, the bait derivative bridges the gap between the DBD and the AD thanks to the anchor-ABD interaction. This enables the expression of a reporter gene. The most popular are HIS3 (allowing yeast growth on a selective medium lacking histidine) and LacZ (to screen yeast in a colorimetric assay). Positive clones are then analyzed by sequencing, to identify the protein partners.
Several successfull applications of the chemical yeast three-hybrid technique in drug profiling projects were published in the past few years2,3,4.
Compared to others techniques for drug target identification like global proteomics, activity-based methods or affinity purification coupled with mass spectrometry technologies5,6, the chemical yeast three-hybrid offers a sensitive and cost-effective mean to test the direct interaction between a small molecule and its targeted proteins. Protein fragments are prepared from the desired cell types, tissues or entire organisms as cDNA libraries, transformed in yeast. This makes the method unbiased thanks to the proteome-wide screening of the given cell line or tissue. In addition, all kind of proteins targets are identified, not only enzymes.
Another advantage is that no protein purification is required because of the positive selection of the yeast clones (only the clones containing a protein target of the molecule are growing on the selective medium).
No link to solid supports of the small molecule is necessary and the small molecule interacts with its targets in a cellular context.
This compound profiling method allows to decipher drug mechanism of action, thanks to target deconvolution further to a phenotypic screening. This facilitates hit to lead optimization and candidate selection.
Also, the identification of off-targets provides new avenues to anticipate drug side-effects and evaluate safety. As a consequence, chemical yeast-three hybrid helps pharmaceutical and biotech companies to obtain market authorization for new drugs. Finally, it is a powerful technique to support drug repositioning of approved drugs in new therapeutic areas.
Hybrigenics has developed its own Yeast Chemical-Hybrid screening process that benefits from all the optimizations and improvements of its Y2H technology. Your drug profiling project will benefit from our expertise of 15 years with yeast two-hybrid screens and our scientific assistance.
Of note, this includes exhaustive library screening process "ULTImate" lowering considerably the false negative rate and the use of a modified yeast strain that avoid drug efflux.
Learn more on our ULTImate YChemH service for drug target identification, drug profiling and target deconvolution.
1. Licitra, E.J. and Liu, J.O. A three-hybrid system for detecting small ligand–protein receptor interactions (1996) PNAS, 93, 12817–12821
2. Becker, F. et al. A Three-Hybrid Approach to Scanning the Proteome for Targets of Small Molecule Kinase Inhibitors (2004), Chemistry & Biology, 11, 211-223
3. Chidley, C. et al. A yeast-based screen reveals that sulfasalazine inhibits tetrahydrobiopterin biosynthesis (2011), Nature Chemical Biology, 7, 375-383
4. Shepard, A.R. et al. Identification of PDE6D as a Molecular Target of Anecortave Acetate via a Methotrexate-Anchored Yeast Three-Hybrid Screen (2013), ACS Chemical Biology, 8, 549-558. Hybrigenics acquired Dualsystems Biotech yeast activities in July 2013.
5. Das, R.K. et al. Target Identification: A Challenging Step in Forward Chemical Genetics (2011), Interdisciplinary Bio Central, 3:3, 1-18
6. Lee, J. and Bogyo, M. Target deconvolution techniques in modern phenotypic profiling (2013) Current Opinion in Chemical Biology, 17, 118–126