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A Bead-Based Proximity Assay for BRD4 Ligand Discovery.

Justin M Roberts¹, James E Bradner^1,2

¹Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.

Current Protocols in Chemical Biology

|December 3, 2015

Summary

We developed a high-throughput AlphaScreen assay to discover BET bromodomain inhibitors. This method detects protein-protein interactions, enabling the identification of potential anti-cancer and anti-inflammatory drugs targeting BRD4.

Keywords:

AlphaScreen BRD4 JQ1 bromodomain high-throughput screening protein-protein interaction

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Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Published on: July 12, 2014

Area of Science:

Biochemistry
Drug Discovery
Molecular Biology

Background:

Bromodomain-containing proteins, particularly the BET family, are key targets for anti-cancer and anti-inflammatory drug development.
Developing selective inhibitors requires robust assays to detect interactions between bromodomains and acetylated histone peptides.

Purpose of the Study:

To optimize and report a bead-based AlphaScreen assay protocol for measuring inhibition of ligand binding to bromodomain-containing protein 4 (BRD4).
To enable high-throughput screening (HTS) for the discovery of selective BET bromodomain inhibitors.

Main Methods:

Utilized an AlphaScreen assay involving donor and acceptor beads to detect proximity between bromodomains and poly-acetylated histone peptides.
Optimized bead-based assays by determining appropriate concentrations for beads, probes, and BRD4 protein.
Developed methods for deriving protein-probe inhibition curves and implementing the assay for HTS.

Main Results:

Successfully optimized an AlphaScreen assay for detecting BRD4-ligand interactions and measuring inhibition.
Demonstrated the assay's suitability for secondary validation and specificity profiling in drug discovery.
Established a protocol for the discovery and optimization of potential BRD4 inhibitors.

Conclusions:

The optimized AlphaScreen assay is a powerful tool for high-throughput screening of potential BRD4 inhibitors.
This assay facilitates lead discovery and optimization for anti-neoplastic and anti-inflammatory drug development targeting BET bromodomains.
The reported protocol provides a reproducible method for assessing bromodomain-ligand interactions.