scholarly journals Luminescence Resonance Energy Transfer-Based High-Throughput Screening Assay for Inhibitors of Essential Protein-Protein Interactions in Bacterial RNA Polymerase

2003 ◽  
Vol 69 (3) ◽  
pp. 1492-1498 ◽  
Author(s):  
Veit Bergendahl ◽  
Tomasz Heyduk ◽  
Richard R. Burgess
Keyword(s):  
Energy Transfer ◽  
Drug Discovery ◽  
Rna Polymerase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Screening Assay ◽  
Essential Protein ◽  
High Throughput Screening Assay

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.

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

2007 ◽  
Vol 12 (6) ◽  
pp. 842-848 ◽  
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)

scholarly journals TR-FRET-Based High-Throughput Screening Assay for Identification of UBC13 Inhibitors

2011 ◽  
Vol 17 (2) ◽  
pp. 163-176 ◽  
Author(s):  
Charitha Madiraju ◽  
Kate Welsh ◽  
Michael P. Cuddy ◽  
Paulo H. Godoi ◽  
Ian Pass ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Inflammatory Diseases ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Chemical Library ◽  
Screening Assay ◽  
Time Resolved ◽  
High Throughput Screening Assay

UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63–linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)–conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)–conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z′ scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.

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

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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