Epigenetics
The histone deacetylase enzyme family contains many attractive targets for drug discovery because these enzymes are among the most critical post- translational regulators of transcriptional processes and gene expression. To date, methods developed for interrogating HDAC class I and II and Sirtuin activities have been complicated by insufficient or labour-intensive throughput, detection platform interferences, and/or poor sensitivity owing to low catalytic activity. Promega (
www.promega.com) has developed two highly sensitive, single addition, ‘add-mix-measure’ assays (HDAC-Glo™ I/II Assay and Screening System and SIRT-Glo™ Assay and Screening System) which obviate these traditional limitations in screening environments. These pan- selective assays utilise novel luminogenic substrates that contain optimised peptide sequences culminat- ing in an acetyllysine conjugated to aminoluciferin. When combined in a homogeneous reagent with recombinant Ultra-Glo™ luciferase and a lysine- specific developer enzyme, this near simultaneous, coupled reaction chemistry produces a ‘glow-type’ luminescent signal that is stable, proportional and quantifiable using standard luminometry in plate- based formats. The assays produce large signal win- dows with low variation (Z’ > 0.8) making them suitable for miniaturisation into high density plate formats. Although both assay systems can be used with recombinant enzyme sources, the HDAC- Glo™ I/II Assay offers additional utility in lytic or non-lytic cell-based formats with primary and can- cer cell types. Furthermore, the HDAC-Glo™ I/II Assay can be multiplexed in same-well formats with spectrally distinct viability or cytotoxicity assays to assess the cellular consequences of prolonged HDAC inhibition; useful for evaluating both on- target efficacy and off-target safety. Lastly, conven- ient counter-screen chemistries are available for both assays which are useful for confirming deacetylase specific effects (Figure 21).
Reaction Biology (RB) (
www.reactionbiology.com) is a premier service provider for drug profiling, screening and early drug discovery collaborations. Its proprietary HotSpot™ technology is an innova- tive, high-throughput platform for screening and profiling small molecules against any biological tar- get which may be assayed by radioisotope detec- tion. For transferase enzymes in particular, such assays are recognised as the ‘gold standard’. RB has become the first and only service provider to pro- vide screening and profiling against all major class- es of epigenetic transferases via the radioisotope approach: histone lysine and arginine methyltrans- ferases (HMTs), DNA methyltransferases
Drug Discovery World Spring 2011
Figure 21: Serial dilutions of the HDAC inhibitor Mocetinostat were applied to K562 cells for 48hrs. Cell viability was determined using Promega’s CellTiter-Fluor™ in a same-well, multiplexed format prior to the addition of Promega’s HDAC-Glo™ I/II Assay Reagent to detect remaining HDAC activity
(DNMTs), Histone acetylases (HATs), and histone- modifying kinases (and all other kinase types). All HotSpot™ assays are performed in a miniaturised reaction format to reduce cost and waste, while
Figure 22: Reaction Biology takes a comprehensive approach to serving the epigenetic drug discovery industry, providing high quality proteins, robust and low cost assay development, library screening, drug profiling and validation of drug activity in cellular assays
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