Resource Center

The Y2H technique is based on the reconstitution of a functional transcription factor (TF) followed by the expression of a reporter gene. This takes place in genetically modified yeast strains, in which the transcription of a reporter gene leads to a specific phenotype, usually growth on a selective medium or change in the color of the yeast colonies. Upon direct binding of the sequences of interest (bait) to protein fragments of the library (prey), the DNA Binding Domain (DBD) of the TF is brought closer to its Activation Domain (AD). The most popular fusions use the DBD and AD of the yeast TF Gal4. The bacterial protein LexA is also frequently used as a DBD in combination with Gal4 AD.
Reconstitution of the TF activates HIS3 reporter gene transcription, allowing the yeast cells to grow on a selective medium lacking for example histidine. The plasmid DNA of the positive clones is sequenced to identify the protein partners.

The development and gradual improvements of the yeast two-hybrid system (Y2H) since the early 90’s revolutionized the way protein interactions could be detected (1).

As a genetic technique, a yeast two-hybrid screen offers a sensitive and cost-effective means to test the direct interaction between two targeted proteins, or to use one’s favorite protein as a bait to screen libraries of proteins fragments prepared from the desired cell types, tissues or entire organisms. The identity of the interacting partners is then obtained by sequencing the corresponding plasmids in the selected yeast colonies. Collections of full-length proteins (‘ORFeomes’) are also becoming available for several species, but they do not cover the entire proteome yet.

The yeast two-hybrid system was rapidly adopted by the scientific community and screens in various species and research fields already led to dozens of thousands of publications. It remains the method of choice when it comes to discover novel protein interactions, as reflected by the recently published literature.

Variations of Y2H were developed to conduct screens in the presence of a co-factor, or an enzyme required for a given post-translational modification of the protein partners (2).

Other versions allow to screen integral membrane proteins (3) and the technique was adapted to detect protein-protein interactions in mammalian cells (4). Finally, yeast n-hybrid protocols were also devised to screen for novel DNA-protein5, RNA-protein6 and small molecule-protein interactions (7).

Contact our team of scientists for advice on your project.

References

1. Fields, S. and Song, O. A novel genetic system to detect protein-protein interactions (1989) Nature 340, 245-246
2. Naba, A., Reverdy, C., Louvard, D. and Arpin, M. Spatial recruitment and activation of the Fes kinase by ezrin promotes HGF-induced cell scattering (2008) EMBO J. 27(1), 38-50
3. Stagljar, I., Korostensky, C., Johnsson, N. and te Heesen, S. A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo (1998) PNAS 95(9), 5187-5192
4. Eyckerman, S. , Verhee, A., der Heyden, J.V., Lemmens, I., Ostade, X.V., Vandekerckhove, J. and Tavernier, J. Design and application of a cytokine-receptor-based interaction trap (2001) Nat Cell Biol. 3(12), 1114-1119
5. Li, J.J. and Herskowitz, I. Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system (1993) Science 262(5141), 1870-1874
6. Putz, U., Skehel, P. and Kuhl, D. A tri-hybrid system for the analysis and detection of RNA--protein interactions (1996) Nucleic Acids Res 24, 4838-4840
7. Licitra, E.J. and Liu, J.O. A three-hybrid system for detecting small ligand-protein
receptor interactions (1996) PNAS 93(23), 12817-12821

PRINCIPLE OF CHEMICAL YEAST THREE-HYBRID FOR YOUR DRUG PROFILING PROJECT

Because the mean duration for the development of a drug is around 10 years and the associated costs are rising, it has become crucial to streamline drug discovery and development and optimize your drug target profile from the start.
The understanding of small bioactive molecules' 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 is an unbiased compound profiling technique derived from the Yeast Two-Hybrid (Y2H) technology(1).

An optimized technology for drug target identification
In this adapted Y2H-based version, the small molecule of interest or drug is used as a bait to screen protein libraries prepared from any tissue, cell type or organism. The drug-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:

- A "Tagged Drug" (Tag – Linker – Drug)
- A hybrid protein containing a DBD fused to a hook protein binding the tag (Hook)
- A hybrid protein containing a transcriptional AD fused to the "target" protein fragment from the library.

Yeast Chemical Hybrid Principle for drug’s target deconvolution
Different linkers and tags can be chosen. The Tag is a molecule attached to the linker which binds to the chosen ABD.

The ""Tagged Drug"" is tested for its interaction with protein targets by screening a library in yeast. When a ""Drug-Target"" interaction takes place, the ""Tagged Drug"" bridges the gap between the DNA Binding Domain and the Activation Domain thanks to the Tag-Hook 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 submitted to a dependency assay, and then analyzed by sequencing, to identify the protein partners.

Several successful applications of the chemical yeast three-hybrid technique in drug profiling projects were published in the past few years (2,3,4).

Compared to other techniques for drug target identification like global proteomics, activity-based methods or affinity purification coupled with mass spectrometry technologies (5,6), the chemical yeast three-hybrid offers a sensitive and cost-effective means to test the direct interaction between a small molecule and its target proteins. Protein fragments are prepared from the desired cell types, tissues or entire organisms as cDNA libraries and 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.

Accelerate your drug profiling project
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 ULTImate Y2H technology. Your drug profiling project will benefit from our expertise of 25 years with yeast two-hybrid screens and our scientific assistance.

Of note, the YChemH process takes advantage of the use of a modified yeast strain that avoids drug efflux as well as the exhaustive library screening process "ULTImate", thereby considerably lowering the false negative rate.

References

1. Licitra, E.J. and Liu, J.O. (1996) PNAS, 93, 12817–12821

2. Becker, F. et al. A (2004), Chemistry & Biology, 11, 211-223

3. Chidley, C. et al.  (2011), Nature Chemical Biology, 7, 375-383

4. Shepard, A.R. et al. ACS Chemical Biology, 8, 549-558. Hybrigenics acquired Dualsystems Biotech yeast activities in July 2013.

5. Das, R.K. et al. (2011), Interdisciplinary Bio Central, 3:3, 1-18

6. Lee, J. and Bogyo, M. (2013) Current Opinion in Chemical Biology, 17, 118–126

Single domain antibodies isolated from camelids, so-called “VHHs” and more generally referred to as "nanobodies", correspond to the variable region of a heavy chain of a camelid antibody. They present many advantages such as a very small size of around 15 kDa, a high stability and solubility and still retain a high affinity for their antigens.
Like every variable domain, our VHHs contain 3 hypervariable regions called CDR (Complementarity Determining Regions) responsible for antigen recognition and thus antibody diversity. These CDRs are spaced with scaffold regions. A deep analysis of stable VHH's sequences and CDRs composition allowed to define possible CDRs length and amino acids composition for our synthetic nanobody library.

The applications and advantages of Nanobodies

Monoclonal antibodies are the most widely used reagents for specific detection and quantification of proteins. However, their production is long, time-consuming and requires animal immunization. In addition, some proteins are not immunogenic or reveal toxic to the animals and cannot lead to efficient antibody selection. Finally, monoclonal antibodies are large molecules of about 150 kDa and it sometimes limits their use in assays with several reagents competing for close epitopes recognition. Because of these limitations, the use of smaller antibody-derived molecules has emerged.

Single-chain variable-fragment (scFv) antibodies have been commonly used as alternatives. ScFv consist of only the light chain and heavy chain variable regions of immunoglobulins connected by a peptide linker. Their average molecular weight is about 27 kDa. ScFv contain the antigen-binding site and are as specific and affine as intact antibodies. In addition, they can be easily expressed in yeast or in E. coli with a high production yield.

The advantage of these antibody-derived molecules is their small size which enables their binding to hidden epitopes not accessible to whole antibodies. In the context of therapeutic applications, a small molecular weight also means an efficient penetration and fast clearance. A VHH nanobody has a higher probability of adopting identical intra- and extracellular folding. Therefore, it is a qualified candidate for intracellular probing (Intrabody).

Being a single-domain antibody molecule, a VHH nanobody is expressed in cells without the need for a supramolecular assembly in contrast to a full immunoglobulin made of 4 chains, 2 light chains and 2 heavy chains. A VHH nanobody is more stable and robust than a whole antibody.

Both scFv and VHH nanobodies can be linked to the Fc fragment of the desired species and keep their specificity and binding properties (Minibody).

Phage Display is a technique of reference for in vitro antibody selection. It consists in the expression of an antibody sequence in fusion with the gIIIp envelope protein of the M13 phage thanks to a phagemide vector. For phage display antibody selection, phages presenting antibodies are put in contact with the antigen of interest on beads in a homogenous format.
After a washing step, selected antibodies recognizing antigen are eluted and then transformed in bacteria for amplification. Another cycle of phage display antibody selection can start. 3 to 4 rounds of selection are necessary to obtain specific antibodies.

Hybrigenics' Phage Display Antibody selection features a unique process resulting in the enrichment of antigen-specific clones. Importantly, native full–length antigen is not adsorbed on plate and therefore preserved during multiple rounds of selection. Several antibodies recognizing different epitopes of an antigen can be identified. As a result, our antibodies are more likely to recognize endogenous antigens for in vitro and in vivo applications, such as immunofluorescence, live cell imaging, video microscopy or proteasome addressing. This also presents the great advantage of selecting putative conformational antibodies.

Since the mid 20th century, antibodies have become major components of the scientific toolkit to analyze fundamental biological mechanisms or develop diagnostics. In the last 25 years, antibodies have even turned into an inexhaustible source of new drugs. An antibody is a large molecule secreted by B lymphocytes. This large molecule is difficult to use and to monitor. To be able to modify intracellular targets, antibodies need to be injected inside the cells, which is not an easy task (1).

Molecular manipulations of antibodies now allow the expression of different parts of antibodies inside the cells from a cDNA clone (2). The most classical format is the scFv, a molecule in which the VH and the VL parts of an antibody have been fused by a small peptidic linker. A new family of antibodies, discovered in the camelid in 1993 (3), displays the unique feature of specifically recognizing the antigen with a single VH domain. They are called VHH or nanobodies®.

Thanks to their small size, the VHH or nanobodies are very good candidates for intrabody expression.
However, the folding in the secretion pathway and in the intracellular environment could be different between different VHH's and the intrabody capacity of a fragment has to be tested by screening inside mammalian cells or using the Yeast Two-Hybrid technology (4-6).

In basic research, such intracellular antibodies have been successfully used to visualize and understand the molecular dynamics of biological processes. For example, uncoupling of dynamin polymerization and GTPase activity has been recently revealed by a conformation-specific nanobody selected from our synthetic library [7] (see also [4, 8, 9])

Intrabodies can be used as

• Blocking molecules - a powerful tool for target engagement studies (10, 11)
• Be fused to an F-box or a PEST signal to mediate selective target degradation in cell (11-13)
• Block virus replication (14, 15)
  Inhibit protein/protein interactions.
  
  References
  
  1. Kreis, T.E., Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. EMBO J, 1986. 5(5): p. 931-41.
  2. Biocca, S., M.S. Neuberger, and A. Cattaneo,Expression and targeting of intracellular antibodies in mammalian cells.EMBO J, 1990. 9(1): p. 101-8.
  3. Hamers-Casterman, C., et al., Naturally occurring antibodies devoid of light chains. Nature, 1993. 363(6428): p. 446-8.
  4. Nizak, C., et al., Recombinant antibodies to the small GTPase Rab6 as conformation sensors. Science, 2003. 300(5621): p. 984-7.
  5. Tanaka, T. and T.H. Rabbitts, Intrabodies based on intracellular capture frameworks that bind the RAS protein with high affinity and impair oncogenic transformation. EMBO J, 2003. 22(5): p. 1025-35.
  6. Rothbauer, U., et al., Targeting and tracing antigens in live cells with fluorescent nanobodies.Nat Methods, 2006. 3(11): p. 887-9.
  7. Galli, V., et al., Uncoupling of dynamin polymerization and GTPase activity revealed by the conformation-specific nanobody dynab. Elife, 2017. 6.
  8. Fukata, Y., et al., Local palmitoylation cycles define activity-regulated postsynaptic subdomains. J Cell Biol, 2013. 202(1): p. 145-61.
  9. Dimitrov, A., et al.,Detection of GTP-tubulin conformation in vivo reveals a role for GTP remnants in microtubule rescues. Science, 2008. 322(5906): p. 1353-6.
  10. Tanaka, T., R.L. Williams, and T.H. Rabbitts, Tumour prevention by a single antibody domain targeting the interaction of signal transduction proteins with RAS. EMBO J, 2007. 26(13): p. 3250-9.
  11. Moutel, S., et al., NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. Elife, 2016. 5.
  12. Butler, D.C. and A. Messer, Bifunctional anti-huntingtin proteasome-directed intrabodies mediate efficient degradation of mutant huntingtin exon 1 protein fragments. PLoS One, 2011. 6(12): p. e29199.
  13. Caussinus, E. and M. Affolter, deGradFP: A System to Knockdown GFP-Tagged Proteins. Methods Mol Biol, 2016. 1478: p. 177-187.
  14. Gal-Tanamy, M., et al., Inhibition of protease-inhibitor-resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies. Antiviral Res, 2010. 88(1): p. 95-106.
  15. Kaku, Y., et al., Inhibition of rabies virus propagation in mouse neuroblastoma cells by an intrabody against the viral phosphoprotein. Antiviral Res, 2011. 91(1): p. 64-71.

Intrabody Enrichment by Yeast Two-Hybrid: how does it work?
Pre-selected hs2dAb (VHH) are transferred from phages to a Y2H prey vector by gap repair. This vector allows the fusion of the Activation Domain (AD) of a transcription factor to the single-domain antibody sequence. This Y2H Antibody library (made of VHH nanobodies) is then transformed into a genetically modified yeast strain lacking the HIS3 gene necessary for the synthesis of histidine. The antigen of interest is fused to the DNA Binding Domain (DBD) of a transcription factor in a Y2H bait vector and then transformed into a second yeast strain of opposite sexual type compared to the prey yeast strain.

Put together, the two yeast strains mate and form diploids expressing both the antigen and a VHH nanobody from the library. If the antibody recognizes the antigen, it allows for the reconstitution of the functional transcription factor. It activates the transcription of the HIS3 reporter gene and allows yeast cells to grow on a selective medium lacking histidine. The DNA of the positive clones is then sequenced and analyzed to identify the selected intrabodies.


In addition to the screening of the hs2dAb library for intrabody selection, Hybrigenics constructs its own cDNA libraries and offers to screen them to identify the binding partners of your favorite protein, DNA, RNA or Small Molecule. We have over 130 libraries from 35 different species and will construct dedicated libraries for your project upon request. They are the most complex and diverse Y2H libraries available on the market.

How can YChemH help you with polypharmacology challenges?

The last few years have witnessed a paradigm shift of the scientific community regarding the ""one drug, one target, one disease"" philosophy that have researchers focused on one specific biological target with high affinity and selectivity towards a molecule of interest, hoping to maximize efficacy and minimize side-effects1. Nowadays, it is generally acknowledged that one drug can have multiple targets that will affect various biological processes in different ways. Multi-target drug discovery is part of a concept called polypharmacology. When unintended, polypharmacology can be very dangerous because of unpredictable side-effects. Thus, a lot of drugs are regularly withdrawn from the market, mostly due to their toxicities.

On the other hand, anticipating these potential side-effects and managing polypharmacology can lead to successful outcomes. Many papers published these past 10 years suggest that more effective and safer drugs can be developed with polypharmacology approaches.(1,4,5)


That’s why polypharmacology has a major importance today in the drug discovery and development process and represents a real challenge for the pharmaceutical industry.

There are different approaches in polypharmacology; it goes from drug combination with two or more drugs that independently have only one specific target, to multi-target drugs where one molecule acting on two or more targets is administered. The risk with combination therapies is that the use of multiple drugs introduces problems with pharmacokinetics, drug-drug interaction, toxicity and patient compliance. Multi-target drugs usually present a higher safety profile.

Different strategies to identify or design multi-target drugs can be used. Among them:

 The identification of the relevant targets for a given disease followed by screening strategies to fish the molecules with the expected protein target profile

- or the identification of the targets of the most promising molecules following a disease-relevant screening campaign.

The objective is to bring information to elucidate the molecule's mechanism of action in relation to complex protein networks and to understand how it can impact one or several disease molecular pathways.

Hybrigenics ULTImate YChemH target deconvolution platform is set up to deliver this kind of information with a focus on the direct interactants. This is a good starting point to move further in the investigation instead of using a list of hundreds potential target hits.

Multi-target drug discovery is a complex approach but the investment is worth it, and lot of great opportunities can arise through polypharmacology.

Furthermore, it is generally accepted that such complex therapeutic approaches are particularly adapted for intricate multifactorial pathologies, such as cancers, metabolic diseases, cardiovascular diseases, and neurological diseases (4, 6). Indeed, these diseases generally involve complex networks of proteins that can be addressed to improve therapies.

As mentioned before, identifying the on and off-targets of a molecule of interest is a key step for any polypharmacology approaches. Traditionally, studies focus on a list of safety-relevant and validated targets to test, but it’s not exhaustive and doesn’t allow to explore all the possibilities or new targets. Many polypharmacological drugs are discovered only by chance when the molecule is already on the market and some great therapeutic avenues may never be uncovered because the drug’s targets remain unknown or only partially understood.

Hybrigenics’ ULTImate YChemH  is a drug target deconvolution method that is direct, in vivo and highly sensitive. It allows us to find all the protein partners of your molecule of interest, using it as a bait to screen one of our highly complex protein domain libraries.

Over 350 cDNA libraries available.

These unique libraries are prepared from a given tissue, primary cultures or a cell line of interest, which enables whole proteome screening in an unbiased manner. We can test more than 80 million interactions at the same time ensuring an exhaustive screening of the library. Furthermore, each interaction is tested individually. That means we can find all the targets of a molecule of interest for a given sample also unexpected targets whether it’s a strong or weak interaction. In addition, we can identify precisely the interacting domain of the target protein with the molecule which provides very useful information to optimize further analysis. Some drug discovery approaches can also take advantage of such finding. These key advantages put ULTImate YChemH as one of the best tool for drug target deconvolution on the market and a strong ally for those who want to design an efficient polypharmacology strategy.

It is most likely that the interest for polypharmacology will increase in the coming years to bring optimized drugs to the market, more efficient and less prone to toxicity. However, designing a successful polypharmacology strategy remains a challenge and several strategies are being developed (15, 16). Those who will take the time to properly identify their molecule’s targets will certainly gain a major advantage to uncover the pathways that lead to success.

References

1. Reddy, A.S.; Zhang, S. Expert Rev Clin Pharmacol. 2013, 6 (1), 41-47.

2. Manautou, J. E., Campion, S. N., Aleksunes, L. M. Comprehensive Toxicology, 2010, 175–220.

3. Segawa M., Sekine S., Sato T., Ito K., , J Toxicol Sci. 2018, 43(5), 339-351.

4. Peters, J.-U., P J Med Chem., 2013, 56(22), 8955-8971.

5. Anighoro, A., Bajorath, J and Giulio Rastelli, G., , J Med Chem., 2014, 57, 7874−7887.

6. Multi-Target Drugs: PLoS One. 2012; 7(6): e40262 Jin-Jian Lu, Wei Pan, Yuan-Jia Hu, and Yi-Tao Wang.

7. Wollin, L., Wex, E., Pautsch, A., Schnapp, G., Hostettler, K.E., Stowasser, S., Kolb, M.  Eur Respir J, 2015; 45, 1434–1445.

8. Roth, G.J., Binder, R., Colbatzky, F., Dallinger, C., Schlenker-Herceg, R., Hilberg, F., Wollin, S.-L., Kaiser, R.:  J. Med. Chem. 2015, 58, 1053−1063.

9. Liu, X., Zhu, F., X. Ma, H., Shi, Z., Yang, S. Y., Wei, Y. Q., Chen., Y. Z.  Curr. Med. Chem., 2013, 20, 1646-1661.

10. MTalevi, A. Expert Rev. Precision Med. Drug Dev., 2018, 3(1), 49-61.

11. Nishimura, Y., Hara H., Editorial:Front. Pharmacol., 2018, 9,1068.

12. Bordessa, A., Colin-Cassin, C., Grillier-Vuissoz, I., Kuntz, S., Mazerbourg, S., Husson, G., Myriam Vo, Flament, S., Martin, H., Chapleur, Y., Boisbrun, M., Eur. J. Med. Chem., 2014, 83, 129-140

13. De Souza, R.M., Schapira, A., Expert Opin. Pharmacotherapy, 2017, 18(9), 937-943.

14. Moya-García, A., Adeyelu, T., Kruger, F.A., Dawson, N.L., G. Lees, J.P., Overington, J.P., Orengo, C., Ranea, J.A.G.. Scientific Reports, 2017, 7, 10102.

15. Zhang, W., Bai, Y., Wang, Y., Xiao, W. , 2016, 22 (21), 3171-81.

16. Hopkins, A.L.. Nat. Chem. Biol , 2008, 4 (11), 682-690.