Setting Up a Bioluminescence Resonance Energy Transfer High throughput Screening Assay to Search for Protein/Protein Interaction Inhibitors in Mammalian Cells

ABSTRACT The binding of sigma factors to core RNA polymerase is essential for the specific initiation of transcription in eubacteria and is thus critical for cell growth. Since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic RNA polymerases, sigma factor binding is a promising target for drug discovery. A homogeneous assay for sigma binding to RNA polymerase (Escherichia coli) based on luminescence resonance energy transfer (LRET) was developed by using a europium-labeled σ70 and an IC5-labeled fragment of the β′ subunit of RNA polymerase (amino acid residues 100 through 309). Inhibition of sigma binding was measured by the loss of LRET through a decrease in IC5 emission. The technical advances offered by LRET resulted in a very robust assay suitable for high-throughput screening, and LRET was successfully used to screen a crude natural-product library. We illustrate this method as a powerful tool to investigate any essential protein-protein interaction for basic research and drug discovery.

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High-throughput screening of TRPV1 ligands in the light of the Bioluminescence Resonance Energy Transfer technique

Molecular Pharmacology ◽

10.1124/molpharm.121.000271 ◽

2021 ◽

pp. MOLPHARM-AR-2021-000271

Author(s):

Yann Chappe ◽

Pauline Michel ◽

Alexandre Joushomme ◽

Solène Barbeau ◽

Sandra Pierredon ◽

...

Keyword(s):

Energy Transfer ◽

High Throughput ◽

High Throughput Screening ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Bioluminescence Resonance Energy Transfer

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High-Throughput Screening of G Protein-Coupled Receptor Antagonists Using a Bioluminescence Resonance Energy Transfer 1-Based β-Arrestin2 Recruitment Assay

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057105275344 ◽

2005 ◽

Vol 10 (5) ◽

pp. 463-475 ◽

Cited By ~ 129

Author(s):

Fadi F. Hamdan ◽

Martin Audet ◽

Philippe Garneau ◽

Jerry Pelletier ◽

Michel Bouvier

Keyword(s):

Energy Transfer ◽

G Protein ◽

Cell Lines ◽

High Throughput ◽

High Throughput Screening ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Receptor Activation ◽

Bioluminescence Resonance Energy Transfer ◽

G Protein Coupled

In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amounts of β-arrestin2-Rluc and VENUS fusions of G protein-coupled receptors (GPCRs) belonging to both class A and class B receptors, which include receptors that interact transiently or stably with β-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1- β-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and β-arrestin2- Renillaluciferase was then used for high-throughput screening (HTS) for antagonists of the chemokine receptor CCR5, the primary co-receptor for HIV. Atotal of 26,000 compounds were screened for inhibition of the agonist-promoted β-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced β-arrestin2 recruitment to CCR5. Three of the potential hits were further tested using other functional assays, and their abilities to inhibit CCR5 agonist-promoted signaling were confirmed. This is the 1st study describing a BRET1- ßarrestin recruitment assay in stablemammalian cells and its successful application in HTS for GPCRs antagonists.

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Homogeneous TR-FRET High-Throughput Screening Assay for Calcium-Dependent Multimerization of Sorcin

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057107303973 ◽

2007 ◽

Vol 12 (6) ◽

pp. 842-848 ◽

Cited By ~ 9

Author(s):

Heidi Appelblom ◽

Jussi Nurmi ◽

Tero Soukka ◽

Michael Pasternack ◽

Kai E. Penttilä ◽

...

Keyword(s):

Energy Transfer ◽

Fluorescence Resonance Energy Transfer ◽

High Throughput ◽

High Throughput Screening ◽

Screening Method ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Calcium Dependent ◽

High Throughput Screening Assay ◽

Fluorescence Resonance

A homogeneous high-throughput screening method based on time-resolved fluorescence resonance energy transfer (TR-FRET) for the measurement of calcium-dependent multimerization of an EF-hand protein, sorcin, is described. The assay is based on a specific sorcin binding peptide conjugated either with an intrinsically highly fluorescent europium chelate (donor) or an Alexa Fluor 700 fluorophore (acceptor). Addition of calcium results in multimerization of sorcin, allowing several peptides to bind simultaneously to the epitopes of the multimeric protein complex, and the proximity of peptides labeled either with donor or acceptor label results in fluorescence resonance energy transfer between the 2 labels. When no calcium is present, the protein remains in a monomer form, and thus no FRET can take place. In the optimized assay construct, the assay was performed in 45 min, and a more than 20-fold signal-to-background ratio was achieved. The reversibility of sorcin multimerization was shown by chelating free calcium with ethylenediamine tetraacetic acid (EDTA). The developed homogeneous assay can be used in screening molecules that either inhibit or enhance multimerization of sorcin, and the assay format is applicable to various noncompetitive high-throughput screening assays detecting protein multimerization reactions. ( Journal of Biomolecular Screening 2007:842-848)

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Screening for Protein-Protein Interaction Inhibitors Using a Bioluminescence Resonance Energy Transfer (BRET)–Based Assay in Yeast

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555216689530 ◽

2017 ◽

Vol 22 (6) ◽

pp. 751-759 ◽

Cited By ~ 4

Author(s):

Caroline Corbel ◽

Sara Sartini ◽

Elisabetta Levati ◽

Pierre Colas ◽

Laurent Maillet ◽

...

Keyword(s):

Energy Transfer ◽

Conformational Changes ◽

Protein Interactions ◽

High Throughput Screening ◽

Mammalian Cells ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Technical Note ◽

Bioluminescence Resonance Energy Transfer ◽

Protein Protein Interactions

The bioluminescence resonance energy transfer (BRET) technology is a widely used live cell-based method for monitoring protein-protein interactions as well as conformational changes within proteins or molecular complexes. Considering the emergence of protein-protein interactions as a new promising class of therapeutic targets, we have adapted the BRET method in budding yeast. In this technical note, we describe the advantages of using this simple eukaryotic model rather than mammalian cells to perform high-throughput screening of chemical compound collections: genetic tractability, tolerance to solvent, rapidity, and no need of expensive robotic systems. Here, the HDM2/p53 interaction, related to cancer, is used to highlight the interest of this technology in yeast. Sharing the protocol of this BRET-based assay with the scientific community will extend its application to other protein-protein interactions, even though it is toxic for mammalian cells, in order to discover promising therapeutic candidates.

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