Receptor-Dependence of the Transcription Read-Out in a Small-Molecule Three-Hybrid System

Abida, Wassim; Carter, Brian T.; Althoff, Eric A.; Lin, Hening; Cornish, Virginia W.

doi:10.1002/1439-7633(20020902)3:9<887::aid-cbic887>3.0.co;2-f

Cited by 37 publications

(43 citation statements)

References 9 publications

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“…The first published example of a yeast three-hybrid system used the rat glucocorticoid receptor as bait scaffold and dexamethasone as the small molecule anchor part [19] and other groups have used this three-hybrid approach as well [20]. Other anchor-bait scaffold combinations include, e.g., methotrexate and dihydrofolate reductase (DHFR; [21][22][23][24] and benzylguanine and the SNAP-tag [25,26].…”

Section: Investigating Drug-protein Interactions: the Yeast Threehybrmentioning

confidence: 99%

Yeast “N”-hybrid systems for protein–protein and drug–protein interaction discovery

Rezwan¹,

Auerbach²

2012

Methods

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Section: Investigating Drug-protein Interactions: the Yeast Threehybrmentioning

confidence: 99%

Yeast “N”-hybrid systems for protein–protein and drug–protein interaction discovery

Rezwan¹,

Auerbach²

2012

Methods

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“…Here, the bait is a hybrid ligand in which an “anchor” moiety is covalently connected via a linker to a compound of interest (Figure 1). Each of these components can be varied, thus leaving head space for optimization and innovation (Licitra and Liu, 1996; Abida et al, 2002; Henthorn et al, 2002; Baker et al, 2003; Becker et al, 2004; de Felipe et al, 2004; Dirnberger et al, 2006; Gallagher et al, 2007). However, for effective and affordable chemical synthesis of functional baits a few criteria should be considered.…”

Section: Tool Box For a Fishing Tour: Implementing The Y3h Platformmentioning

confidence: 99%

“…Second, the linker not only determines the distance (spacing) between the two ligands, it may also affect the solubility of the resulting hybrid ligand. Previous, systematic studies revealed that the linker between the two functional groups should have a minimum length of 5 carbon atoms to afford dimerization of the receptors, but a spacer length of 12 atoms would be most favorable, whereas the chemical nature of the linker had little impact on the biological read-out (Abida et al, 2002). However, a polyethylene glycol (PEG) linker will improve solubility of the hybrid ligand in comparison to an aliphatic linker.…”

Section: Tool Box For a Fishing Tour: Implementing The Y3h Platformmentioning

confidence: 99%

“…The Mtx–Dex hybrid ligand as synthesized from the thiol derivatives of Mtx and Dex by Abida et al (2002) contains a 10-methylene chain linker. All other baits were synthesized with polyethylene glycol diamine linker and the functional group of the small molecule used for coupling to the Mtx–(PEG) 3 –NH 2 intermediate is highlighted in red, the hydroxy groups were converted to a succinyl ester HOOC–CH 2 –CH 2 –COO–R prior to coupling.…”

Section: Tool Box For a Fishing Tour: Implementing The Y3h Platformmentioning

confidence: 99%

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The yeast three-hybrid system as an experimental platform to identify proteins interacting with small signaling molecules in plant cells: Potential and limitations

Cottier

Mönig

Wang

et al. 2011

fpls

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Chemical genetics is a powerful scientific strategy that utilizes small bioactive molecules as experimental tools to unravel biological processes. Bioactive compounds occurring in nature represent an enormous diversity of structures that can be used to dissect functions of biological systems. Once the bioactivity of a natural or synthetic compound has been critically evaluated the challenge remains to identify its molecular target and mode of action, which usually is a time-consuming and labor-intensive process. To facilitate this task, we decided to implement the yeast three-hybrid (Y3H) technology as a general experimental platform to scan the whole Arabidopsis proteome for targets of small signaling molecules. The Y3H technology is based on the yeast two-hybrid system and allows direct cloning of proteins that interact in vivo with a synthetic hybrid ligand, which comprises the biologically active molecule of interest covalently linked to methotrexate (Mtx). In yeast nucleus the hybrid ligand connects two fusion proteins: the Mtx part binding to dihydrofolate reductase fused to a DNA-binding domain (encoded in the yeast strain), and the bioactive molecule part binding to its potential protein target fused to a DNA-activating domain (encoded on a cDNA expression vector). During cDNA library screening, the formation of this ternary, transcriptional activator complex leads to reporter gene activation in yeast cells, and thereby allows selection of the putative targets of small bioactive molecules of interest. Here we present the strategy and experimental details for construction and application of a Y3H platform, including chemical synthesis of different hybrid ligands, construction of suitable cDNA libraries, the choice of yeast strains, and appropriate screening conditions. Based on the results obtained and the current literature we discuss the perspectives and limitations of the Y3H approach for identifying targets of small bioactive molecules.

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“…These dimerization systems are all based, in part, on the chemical structure of rapamycin or FK506. In addition, other scaffolds have been explored (for some recent reviews see: [53,[65][66][67][68][69][70]), such as the coumermycin system [71,72], alkyltransferase [73], estradiol-biotin chimeras [74], acyl homoserine lactone-based methods [75] and methotrexatederived dimerizers [76]. Together, CIDs provide a versatile collection of synthetic reagents for controlling proteinprotein interactions.…”

Section: Inspirations From Nature: Chemical Indu-cers Of Dimerizationmentioning

confidence: 99%

Chemical Control Over Protein-Protein Interactions: Beyond Inhibitors

Gestwicki¹,

Marinec²

2007

CCHTS

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Protein-protein interactions have become attractive drug targets and recent studies suggest that these interfaces may be amenable to inhibition by small molecules. However, blocking specific interactions may not be the only way of manipulating the extensive network of interacting proteins. Recently, several approaches have emerged for promoting these interactions rather than inhibiting them. Typically, these strategies employ a bifunctional ligand to simultaneously bind two targets, forcing their juxtaposition. Chemically "riveting" specific protein contacts can reveal important aspects of regulation, such as the consequences of stable dimerization or the effects of prolonged dwell time. Moreover, in some cases, entirely new functions arise when two proteins, which normally do not interact, are brought into close proximity with one another. Together with inhibitors, bifunctional molecules are part of a growing toolbox of chemical probes that can be used to reversibly and selectively control the interact-ome. Using these reagents, new insights into the dynamics of protein-protein interactions and their importance in biology are beginning to emerge. Future hurdles in this area lie in the development of robust synthetic platforms for rapidly generating compounds to meet the challenges of diverse protein-protein interfaces.

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Receptor-Dependence of the Transcription Read-Out in a Small-Molecule Three-Hybrid System

Cited by 37 publications

References 9 publications

Yeast “N”-hybrid systems for protein–protein and drug–protein interaction discovery

Yeast “N”-hybrid systems for protein–protein and drug–protein interaction discovery

The yeast three-hybrid system as an experimental platform to identify proteins interacting with small signaling molecules in plant cells: Potential and limitations

Chemical Control Over Protein-Protein Interactions: Beyond Inhibitors

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