Lysine acetylation as the best-studied histone post-translational modification (PTM) to date is a hallmark of genes undergoing active transcription and is selectively recognized by a small protein motif called a bromodomain, which is composed of a left-handed bundle of four α-helices linked by loop domains of varying length. Bromodomains selectively recognize acetyl-lysine through a hydrogen bond at an asparagine located inside the hydrophobic binding pocket. Once bound with acetyl-lysine, these bromodomains recruit protein complexes involved in epigenetic regulation of chromatin structure and further gene expression. Bromodomains work in parallel with other proteins to give a high level of specificity.

Figure 1. Bromodomain regulates transcription (Ayoub, A. M.; et al. 2017)

With the identification of several potent, selective and cellularly active bromodomains in the biomedical community, aberrations in bromodomains are found to possess striking physiological effects and the appreciation of functional importance and therapeutic potential of this family has been increased. Numerous researches in the field over the past decades have demonstrated the druggability of epigenetic bromodomains as attractive targets. Indeed, small molecule inhibitors have showed successful disruption of interactions between bromodomain and histone, holding promise as potential therapeutics.

Creative BioMart offers a collection of validated bromodomain assays that allow researchers to quickly and accurately profile putative inhibitors against either a panel of bromodomains, or to screen a selected bromodomain for novel inhibitor chemotypes. The novel industry leading platform is based on TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) and AlphaScreen technology, which is available in a variety of flexible screening and profiling formats and virtually eliminates assay development bottlenecks. At Creative BioMart, we are working towards the complete coverage of the bromodomain family by providing purified proteins, assay kits, and inhibitors & activators, which can be applied to high-throughput screening (HTS), selectivity profiling and quantitative affinity determinations, thus accelerating screening, drug discovery and validation of hits.

Bromodomain Screening Assay at Creative BioMart

a. TR-FRET Assay

TR-FRET assay at Creative BioMart boasts a big performance boost from the specially made Europium (Eu³⁺) chelate that is directly labeled onto the bromodomain, called ‘donor’ fluorophore. A biotinylated peptide containing target acetylated histone acts as a ligand for bromodomain. Allophycocyanin (APC)-labeled avidin binds to the peptide substrate with high affinity via the biotin moiety and functions as the ‘acceptor’ fluorophore. Inhibition of the interaction by an inhibitor results in a loss of TR-FRET signal. One less binding event in this assay avoids the need for expensive antibodies and keeps Europium and APC closer together for stronger FRET signals to maximally reserve sensitivity.

Figure 2. Procedure of TR-FRET based bromodomain screening assay

b. AlphaScreen Assay

AlphaScreen assay is also designed to measure the inhibition of bromodomain from binding to its substrate. The key to this assay is the highly specific binding of the bromodomain to the acetylated histone substrate. Three simple steps on a microtiter plate are required. First, a sample containing bromodomain and an inhibitor to be determined is incubated with the biotinylated substrate for thirty minutes. Next, acceptor beads and then donor beads are added sequentially. The last step is to read the Alpha-counts.

Advantages of Our Services

1. A panel of assays to screen inhibitors or targets
2. Simple, mix-and-read assay format that’s stable for several hours
3. Scalable to higher density plate formats
4. Compatible with standard laboratory automation
5. IC₅₀ (half maximal inhibitory concentration) determination or single point concentration determination
6. Fast turnaround time
7. Get project guidance in a time-efficient manner

With years of professional experience in bromodomain field, Creative BioMart dedicates providing you superior methodologies and technologies to identify your compounds with high sensitivity, high reproducibility and quick turnaround time. Creative BioMart is a reliable partner that can work with you to accelerate your research proceeding. For more detailed information, please feel free to contact us or directly sent us an inquiry.

Reference
1. Ayoub, A. M.; et al. BET Bromodomain inhibitors with one-step synthesis discovered from virtual screen. J Med Chem. 2017, 60(12): 4805-4817.