Photoaffinity labelling displacement assay using multiple recombinant protein domains

David J Fallon; Alex Phillipou; Christopher J Schofield; David House; Nicholas C O Tomkinson; Jacob T Bush

doi:10.1042/BCJ20230129

Photoaffinity labelling displacement assay using multiple recombinant protein domains

Biochem J. 2023 Aug 16;480(15):1183-1197. doi: 10.1042/BCJ20230129.

Authors

David J Fallon^{1

2}, Alex Phillipou³, Christopher J Schofield⁴, David House¹, Nicholas C O Tomkinson², Jacob T Bush¹

Affiliations

¹ Department of Chemical Biology, GSK R&D, Gunnels Wood Road, Stevenage SG1 2NY, U.K.
² Department of Pure and Applied Chemistry, University of Strathclyde, Thomas Graham Building, Glasgow G1 1XL, U.K.
³ Department of Screening, Profiling and Mechanistic Biology, GSK R&D, Gunnels Wood Road, Stevenage SG1 2NY, U.K.
⁴ Chemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute for Antimicrobial Research, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, U.K.

PMID: 37401534
DOI: 10.1042/BCJ20230129

Abstract

The development and optimisation of a photoaffinity labelling (PAL) displacement assay is presented, where a highly efficient PAL probe was used to report on the relative binding affinities of compounds to specific binding sites in multiple recombinant protein domains in tandem. The N- and C-terminal bromodomains of BRD4 were used as example target proteins. A test set of 264 compounds annotated with activity against the bromodomain and extra-terminal domain (BET) family in ChEMBL were used to benchmark the assay. The pIC50 values obtained from the assay correlated well with orthogonal TR-FRET data, highlighting the potential of this highly accessible PAL biochemical screening platform.

Keywords: assay; bromodomains; photoaffinity.