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Photoaffinity labelling displacement assay using multiple recombinant protein domains.

David J Fallon1,2, Alex Phillipou3, Christopher J Schofield4

  • 1Department of Chemical Biology, GSK R&D, Gunnels Wood Road, Stevenage SG1 2NY, U.K.

The Biochemical Journal
|July 4, 2023
PubMed
Summary

A novel photoaffinity labeling (PAL) displacement assay was developed to efficiently measure compound binding affinities to bromodomain and extra-terminal domain (BET) proteins. This accessible platform shows strong correlation with existing methods, offering a valuable tool for drug discovery.

Keywords:
assaybromodomainsphotoaffinity

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Area of Science:

  • Biochemistry
  • Chemical Biology
  • Drug Discovery

Background:

  • Photoaffinity labeling (PAL) is a powerful technique for studying molecular interactions.
  • Bromodomain and extra-terminal domain (BET) proteins are crucial epigenetic regulators and therapeutic targets.
  • Accurate measurement of compound binding affinity is essential for drug development.

Purpose of the Study:

  • To develop and optimize a photoaffinity labeling (PAL) displacement assay.
  • To assess the binding affinities of compounds to specific protein binding sites.
  • To benchmark the assay using known BET inhibitors.

Main Methods:

  • Utilized a highly efficient PAL probe for displacement assays.
  • Employed N- and C-terminal bromodomains of BRD4 as target proteins.
  • Tested 264 compounds with known BET family activity from ChEMBL.
  • Correlated assay results with orthogonal time-resolved Förster resonance energy transfer (TR-FRET) data.

Main Results:

  • The developed PAL displacement assay successfully reported relative binding affinities.
  • Assay performance was benchmarked using a diverse set of 264 BET-targeting compounds.
  • Obtained pIC50 values showed strong correlation with orthogonal TR-FRET data.
  • Demonstrated the assay's capability to analyze multiple protein domains in tandem.

Conclusions:

  • The optimized PAL displacement assay is a robust and accessible biochemical screening platform.
  • This method provides reliable binding affinity data for drug discovery efforts targeting BET proteins.
  • The assay's high throughput potential makes it valuable for screening compound libraries.