The interest in epigenetics – the heritable changes of gene expression – has led to new findings about the relationship between epigenetic marks and a host of disorders including mental, immune and neuropsychiatric, and various cancers. In the recent years, there have been rapid advances in the understanding of epigenetic mechanisms. The most implicated is post-transcriptional modification of histones via phosphorylation (detailed in Lifetime Ser/Thr protein kinase assay note), ubiquitination (detailed in Lifetime protease & DUB assay note), acetylation, methylation and de-imination. Proteins engaged in the addition and removal of such histone marks (“epi-enzymes”) together with the proteins which specifically recognize them (“epi-readers”) have emerged as novel drug targets.
benefits of FLEXYTE lifetime assays
FLEXYTE® lifetime epigenetic assays utilise a unique long lifetime fluorophore FLEX17 site-specifically coupled to modified histone peptides or epi-reader proteins to provide a homogeneous, reliable and cost effective screening solution.
Utilisation of a long lifetime FLEX17 fluorophore enables discrimination of substrate and product from (or bound and free protein) discrimination from fluorescent interfering compounds whose lifetimes are commonly below 2 nanoseconds and also provides a stable, defined assay window upon which assays can be built in 384-or 1536-well formats.
The FLEXYTE lifetime epi-enzyme assay platform has been developed through the design and synthesis of FLEX17 labelled histone peptide substrates incorporating an aromatic residue within the sequence which modulates the lifetime of the fluorophore. Modification of the histone peptide by the epi-enzyme renders the peptide either susceptible or resistant to protease-based cleavage leading to a change in lifetime with respect to the substrate.
Assays have been configured for the following epi-enzyme targets:
- histone methyltransferases (HMT, both lysine and arginine)
- histone acetyltransferases (HAT)
- protein arginine deiminases (PAD)
- histone deacetylases (HDAC)
The FLEXYTE lifetime epi-reader assay platform comprises FLEX17 labelled epi-reader protein and Delta™ lifetime modulator-labelled histone peptide. When the histone peptide binds to the epi-reader protein, the Delta lifetime modulator is brought into close proximity to the FLEX17 fluorophore, reducing its lifetime. Compounds that disrupt this protein-peptide interaction are detected by an increase in lifetime (find more detail in Lifetime epi-reader assay note).
ameon lifetime reader
TTP Labtech’s ameon lifetime reader meets the challenge of accelerating the drug discovery process by integrating easily into screening workflows and offering a reliable fluorescence lifetime readout. The instrument applies a proprietary data recording technology to deliver precise lifetime measurement at high throughput.
✓ optimised for FLEXYTE lifetime reagents
✓ robust lifetime readout
✓ 1536-well compatible
specific enzyme list
The FLEXYTE lifetime platform provides appropriately modified histone peptides labelled with the long lifetime FLEX17 fluorophore for assaying a range of epi-enzyme targets as shown in the table below.
In addition, recombinant epi-reader proteins site-specifically labelled with the FLEX17 fluorophore together with modified histone peptides incorporating the Delta™ lifetime modulator are provided for assaying the therapeutically important epi-reader proteins, BRD4(1) and L3MBTL1.
Assays can be configured and reagents provided for other epi-enzymes or epi-readers upon request.