title: A Bead-Based Proximity Assay for BRD4 Ligand Discovery.
abstract: Bromodomain-containing proteins have emerged as desirable targets for anti-neoplastic and anti-inflammatory drug discovery. Toward the development of selective inhibitors of the BET family of bromodomains, we optimized bead-based assays to detect interactions between bromodomains and poly-acetylated histone peptides. Donor and acceptor beads bound to target and ligand are brought into proximity by this protein-protein interaction. After laser illumination, singlet oxygen evolved from donor beads travels to the spatially close acceptor beads, resulting in chemiluminesence. This AlphaScreen assay has proven amendable to high-throughput screening, secondary validation, and specificity profiling during lead discovery and optimization. Here we report our protocol for assay development to measure inhibition of ligand binding to bromodomain-containing protein 4 (BRD4). We discuss the discovery of an appropriate probe, optimization of bead, probe, and protein concentrations, and the derivation of protein-probe inhibition curves. Finally, we explore the implementation of this technology for high-throughput screening of potential BRD4 inhibitors.
authors.affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Roberts
authors.affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.|Department of Medicine, Harvard Medical School, Boston, Massachusetts.
authors.forename: James E
authors.initials: JE
authors.lastname: Bradner
mesh_terms: D001381:Azepines / Q000737:chemistry / Q000378:metabolism; D001710:Biotin / Q000737:chemistry; D018797:Cell Cycle Proteins; D057166:High-Throughput Screening Assays; D006801:Humans; D020128:Inhibitory Concentration 50; D008024:Ligands; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014230:Triazoles / Q000737:chemistry / Q000378:metabolism; D000092468:Bromodomain Containing Proteins
chemical_list: D001381:Azepines; D001710:Biotin; D018797:Cell Cycle Proteins; D008024:Ligands; D015335:Molecular Probes; D009687:Nuclear Proteins; D014157:Transcription Factors; D014230:Triazoles; C561695:(+)-JQ1 compound; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins
keywords: AlphaScreen; BRD4; JQ1; bromodomain; high-throughput screening; protein-protein interaction