Cell-Based Protein Stabilization Assays for the Detection of Interactions between Small-Molecule Inhibitors and BRD4

Abstract

Bromodomain protein 4 (BRD4), a member of the bromodomain and extra-terminal (BET) protein family, acts as a central element in transcriptional elongation and plays essential roles in cell proliferation. Inhibition of BRD4 binding to acetylated histone tails via its two bromodomains, BD1 and BD2, with small-molecule inhibitors has been shown to be a valid strategy to prevent cancer growth. We have evaluated and established two novel assays that quantify the interaction of transfected BRD4 BD1 with chemical inhibitors inside cultured cells. Both methods are based on the principle of ligand-induced protein stabilization by which the binding of a small-molecule inhibitor stabilizes intracellular BRD4 BD1 and protects it from proteolytic degradation. We demonstrate the universal character of this principle by using two orthogonal, highly sensitive detection technologies for the quantification of BRD4 BD1 levels in cellular lysates: enzyme fragment complementation and time-resolved fluorescence resonance energy transfer (TR-FRET). Upon optimization of both assays to a miniaturized high-throughput format, the methods were validated by testing a set of small-molecule BET inhibitors and comparing the results with those from a cell-free binding assay and a biophysical thermal shift assay. In addition, point mutations were introduced into BRD4 BD1, and the corresponding mutants were characterized in the TR-FRET stabilization assay.

Targeting BET Bromodomains for Cancer Treatment

“…Biophysical characterizations of the interactions of BET inhibitors with their targets using thermal shift assay/differential scanning fluorimetry, isothermal titration calorimetry, surface plasmon resonance, biolayer interferometry, nuclear magnetic resonance and x-ray crystallography have been reported [10,20,30,98,99]. More recently, assays enabling the verification of target engagement by compounds in living cells such as fluorescence recovery after photobleaching (FRAP) and cell-based protein stabilization assays have expanded the spectrum of technologies available for the discovery and characterization of BET inhibitors [10,100].…”

Implementation and Use of State-of-the-Art, Cell-Based In Vitro Assays

“…7 The term frozen cell describes the use of freshly resuscitated cells shortly after their recovery from liquid nitrogen storage and should not to be mistaken for the use of division-arrested cells, as exemplified/described by Digan et al (2005). screens 8 and cell-based follow-up activities 9 (Schulze et al 2015). However, the initial production of frozen cells for assay development and assay validation is still done in-house and on a small lab scale -something that can easily be recapitulated in an academic setting as well -based on scientifically sound, technical solutions that combine existing knowledge on the field of cryopreservation and resuscitation of cells while keeping the conditions of the functional assay to be performed in mind.…”

BET bromodomain proteins and epigenetic regulation of inflammation: implications for type 2 diabetes and breast cancer

“…Most efforts to develop selective inhibitors have been focused on BRD4 due to the discovery of a gene rearrangement that links BRD4 to aggressive carcinoma [169]. Additional efforts include the development of cell-based screening assays in BRD4-dependent cells [170], as well as designing small molecules that will target bromodomain 1 (BD1) of BRD4 [171, 172]. Taking the common approach to modify existing BET family inhibitors, I-BET and JQ1, Baud et al developed an approach to introduce selectivity to BET bromodomain inhibitors [173], which involves Proteolysis Targeted Chimeras (PROTACs).…”