Epigenetics


cycle inhibitor profiling studies, the Phospho- Histone H3 Ser10 and Cyclin B1 Assay facilitates distinction among G2, M and G0/G1/S phases of the cell cycle. For laboratories wanting to analyse spe- cific DNA-protein interactions without performing electrophoretic mobility shift assays, they offer EZ- TFA transcription factor assays. These 96-well plate-based DNA binding activity assays are avail- able as universal kits for virtually any target of inter- est or as a pre-configured target-specific assay. As epigenetics solution provider, EMD Millipore also offers products for chromatin immunoprecipitation (ChIP), RNA binding protein immunoprecipitation (RIP), and DNA methylation analysis (Figure 17).


Figure 17: EMD Millipore’s MILLIPLEX MAP detection of changes in phosphorylation of histone H2A.X (Ser139) in Jurkat cells stimulated with or without 25mM anisomycin. The Median Fluorescent Intensity (MFI) was measured using the Luminex instrument


Epigentek (www.epigentek.com) was the first to pioneer a drug discovery service programme in epi- genetics. Utilising its proprietary Epigenase™ screening platform, a fluorescence-coupled enzyme amplification technology, both DNA/histone mod- ifying enzyme inhibitors and histone modification pattern modulators can be screened in a high throughput format (using 96, 384, or 1536-well microplates) with both single dose and EC50 pro- filing. The DNA/histone modifying enzyme panels include all 11 HDACs and five SIRTs, five HATs, 18 histone methyltransferases, 12 histone demethylases, four DNA methyltransferases, and 12 histone kinases. Histone modification pattern panels include DNA methylation, histone acetyla- tion, histone methylation, histone phosphorylation and histone sumoylation (Figure 18).


Figure 18: The principle of Epigentek’s Epigenase™ for screening of inhibitors targeting epigenetic enzymes. In the absence of inhibitors, the enzyme and substrate coated on microplate wells interact with each other to form an enzyme-converted end product, which is then bound with a fluorescence antibody or ligand. The fluorescence intensity of the bound antibody/ligand can be detected with a fluorescence microplate reader. In the presence of an inhibitor, the compound may bind the enzyme to block the formation of the enzyme- converted end product. Therefore, if there is no fluorescence antibody/ligand binding to the wells, this would result in a much lower fluorescence intensity than the uninhibited control. In this mechanism, the inhibitors can be identified accordingly


For assessing global epigenetic modifications Invitrogen (www.invitrogen.com) is expanding its tools to investigate not only phosphorylation and ubiquitination, but also acetylation and methyla- tion. Its new biochemical assay for identifying HDAC inhibitors uses a LanthaScreen® Eu-anti tag antibody with a fluorescently labelled tracer to measure the binding affinity of compounds rather than enzyme activity. This approach does not require a substrate, therefore avoiding issues relat- ed to the specificity of existing substrates. In addi- tion, the signal is unaffected by the presence of residual endogenous HDACs from the host expres- sion system because it is dependent on the presence of an epitope tag on the HDAC of interest. On the cellular side, Invitrogen has increased its LanthaScreen® cellular assay offering to include the analysis of histone H3 site-specific acetylation, methylation and phosphorylation in a wide range of cell backgrounds, including primary cells. These assays utilise cells expressing GFP-Histone H3 transiently delivered via BacMam and terbium


50 Drug Discovery World Spring 2011


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