Epigenetic assays for chemical biology and drug discovery

Abstract

The implication of epigenetic abnormalities in many diseases and the approval of a number of compounds that modulate specific epigenetic targets in a therapeutically relevant manner in cancer specifically confirms that some of these targets are druggable by small molecules. Furthermore, a number of compounds are currently in clinical trials for other diseases including cardiovascular, neurological and metabolic disorders. Despite these advances, the approved treatments for cancer only extend progression-free survival for a relatively short time and being associated with significant side effects. The current clinical trials involving the next generation of epigenetic drugs may address the disadvantages of the currently approved epigenetic drugs. The identification of chemical starting points of many drugs often makes use of screening in vitro assays against libraries of synthetic or natural products. These assays can be biochemical (using purified protein) or cell-based (using for example, genetically modified, cancer cell lines or primary cells) and performed in microtiter plates, thus enabling a large number of samples to be tested. A considerable number of such assays are available to monitor epigenetic target activity, and this review provides an overview of drug discovery and chemical biology and describes assays that monitor activities of histone deacetylase, lysine-specific demethylase, histone methyltransferase, histone acetyltransferase and bromodomain. It is of critical importance that an appropriate assay is developed and comprehensively validated for a given drug target prior to screening in order to improve the probability of the compound progressing in the drug discovery value chain.

Keywords: Assay development; Bromodomain; Chemical biology; Chemical probe; Demethylase; Drug discovery; High throughput screening; Histone acetyltransferase; Histone deacetylase; Histone methyltransferase.

Fig. 1

AlphaLISA® histone deacetylate assay that detects Histone H3-K9(Ac) or Histone H3-K27(Ac). The acetylated histones are detected using a biotinylated anti-H3 antibody and AlphaLISA®-acceptor beads conjugated specific to the acetylated lysine. Streptavidin-donor beads then capture the biotinylated antibody, bringing the acceptor and donor beads into proximity. Upon laser irradiation of the donor beads at 680 nm, short-lived singlet oxygen molecules produced by the donor beads can reach the acceptor beads in proximity to generate an amplified chemiluminescent signal at 615 nm

Fig. 2

a Colorimetric coupled histone deacetylate assay that makes use of a chromogenic peptide substrate (proprietary Color de Lys® Substrate) containing a ε-acetylated lysine residue. When an HDAC enzyme acts upon the substrate and the sidechain of a ε-acetylated lysine residue is deacetylated, it becomes susceptible to further degradation by an enzyme in the developer reagent (proprietary Color de Lys® Developer). The action of the enzyme within the developer reagent results in the release of a chromophore detected by measuring the absorbance of the reaction at 405 nm. b Fluorometric coupled histone deacetylate assay that makes use of a fluorogenic peptide substrate (proprietary Fluor de Lys® Substrate) containing a ε-acetylated lysine residue. When an HDAC enzyme acts upon the substrate and the sidechain of a ε-acetylated lysine residue is deacetylated, it becomes susceptible to further degradation by an enzyme in the developer reagent (proprietary Fluor de Lys® Developer) resulting in the release of 7-amino-4-methylcoumarin fluorophore which undergoes excitation at 360 nm and emits at 460 nm

Fig. 3

Luminescence coupled histone deacetylate assay that makes use of specific amino-luciferin labelled ε-acetylated lysine peptide substrates for HDAC Class I/II enzymes. When the substrate undergoes deacetylation by the HDAC enzyme, the product becomes susceptible to the Developer reagent and results in the release of amino-luciferin. This amino-luciferin is the substrate for a luciferase enzyme (also in the Developer reagent) and yields a glow-type luminescence

Fig. 4

Time-resolved fluorescence resonance energy transfer histone deacetylase assay. A signal is generated when the deacetylated peptides are captured by the Europium-labelled antibody donor and streptavidin-ULight™-acceptor thus bringing the Europium-donor and ULight™-acceptor molecules into close proximity. Upon irradiation at 340 nm, the energy from the Europium-donor is transferred to the ULight™-acceptor, which, in turn, generates a signal at 665 nm