Epigenetics research has expanded rapidly over the last several years, as evidenced by the exponential increase in published literature in this field. Breakthroughs have been made in the elucidation of basic epigenetic mechanisms such as histone modification, and with these advances have come an understanding of the critical role epigenetic modifications play in the development and progression of cancer. For example, deregulation of specific histone H3 methylation sites has been associated with tumor suppessor down-regulation and the development of several types of cancer.¹ Not surprisingly, there has been an increased focus on the identification of clinical treatments for aberrant epigenetic states, and in particular on targeted inhibitors of the dozens of enzymes that carry out histone post-translational modifications. Histone modification enzymes can be classified as Writers, which add epigenetic marks such as methyl, acetyl or phospho- groups, and Erasers, which catalyze the removal of these marks. For example, tumor suppressor down-regulation has been correlated with the loss of H3K4me2 and H3K4me3 marks (di- and trimethylation at lysine 4 of histone  H3) and with loss of the H3K9ac mark through deacetylation.² Thus, a targeted inhibitor for one of these eraser enzymes could be a valuable drug candidate.

Challenges to epigenetic drug discovery

As clinical research identifies an increasing number of correlations between epigenetic marks and disease states, drug discovery efforts have focused on screening compound libraries for candidate molecules capable of targeted inhibition of epigenetic writer or eraser enzymes. These efforts have been hampered by limitations of available technologies as well as a lack of high quality reagents. Assays for epigenetic drug discovery must be capable of distinguishing between generalized and specific effects, since a lead compound with a narrowly targeted effect is less likely to have off-target effects in clinical application. Epigenetic assays should also be as biologically relevant as possible, ideally measuring epigenetic marks on endogenous histone proteins.

Screening for epigenetic lead candidates

One widely-used approach to screening for histone methylation changes in response to a candidate therapeutic employs in vitro assays with ³H S-adenosyl methionine (³H-SAM) as a radioactive substrate. This has proven to be the gold standard for detection of generalized methylation changes,³ but these assays are not site-specific and thus are only a first step in directing the process of lead identification. Screening for targeted epigenetic changes requires assays amenable to high throughput automation but also capable of detecting specific marks and differentiating between mono-, di-, and tri-methylation at a given residue. Immunoassays such as ELISA can offer the needed specificity, and several ELISA assays for histone modifications are commercially available. However, ELISA assays are labor-intensive and therefore of limited utility for high throughput screening. Mass spectrometry-based assays can distinguish differentially methylated histone peptides, but they are limited to biochemical assays, not cell extracts, and are not scalable to truly high throughput screening.

High throughput screening for compounds that alter epigenetic marks can be readily accomplished using LANCE and AlphaLISA immunoassays (PerkinElmer Life Sciences), which use proximity-based detection in a no-wash, mix-and-read (homogeneous) assay format. Homogeneous assays are easily performed in 384- or 1536-well format and are very amenable to automation and high throughput screening.^4-9 A suite of epigenetic assays and toolbox reagents have been developed for detection of specific epigenetic marks and of activities of targeted epigenetic writer or eraser enzymes. Antibody pairs have been developed for histone H3 marks spanning seven different residues (Figure1), enabling high throughput assays to be performed for 9 writer enzymes (histone acetyl-, methyl- and phosphotransferases) and 25 eraser enzymes (histone deacaetylases and demethylases).

in vitro assays for profiling inhibitors of histone modification enzymes

In addition to their usefulness for lead identification, in vitro biochemical assays are well suited for profiling the enzyme inhibitors identified through initial screening as lead candidates. Such profiling commonly includes analyses of each inhibitor’s selectivity, rank order of potency, and kinetics of inhibition. For this purpose, a matrix of experimental conditions is assembled in a multi-well plate and the levels of specific epigenetic marks are analyzed. To determine inhibitor selectivity, the matrix may include various histone peptide substrates bearing epigenetic marks at different locations or various histome modifying enzymes. Inhibitor titrations are used to determine rank order of potency. Microfluidic capillary electrophoresis in a 384-well format (LabChip EZ Reader, Caliper Life Sciences) has been widely used for profiling kinase inhibitors, and is particularly well-suited for evaluation of inhibitor kinetics. This platform has recently been shown to be highly effective for profiling of histone deacetylase inhibitors for both inhibitor kinetics and rank order of potency.¹⁰ The charge-to-mass ratio separation principle of capillary electrophoresis cannot directly measure methylation changes, which are charge-neutral. However, a modified protocol allows this convenient approach to be applied to histone methylases,¹¹

Cell-based assays for verification and profiling of epigenetic lead candidates

While in vitro biochemical assays are useful for initial lead discovery and characterization, they rely on purified histone modifying enzymes and histone peptide substrates in a synthetic system and thus cannot measure actual interactions occurring in the nucleus of living cells. A much more biologically relevant assessment of inhibitor effects on epigenetic marks is possible using AlphaLISA cellular assay technology. These bead-based proximity immunoassays are well-suited to directly measure changes to epigenetic marks on endogenous histones catalyzed by endogenous modification enzymes. In a typical assay, the candidate inhibitor compound is used to treat a relatively small number of cells seeded in a microplate (1,000 to 10,000 cells/well) overnight. A 30 min, 2-step reagent addition releases histones from cell nuclei, and a 90 min all-in-one-well AlphaLISA assay is then performed (Figure 2a). The histone modification of interest is detected by the addition of a biotinylated anti-Histone H3 (C-terminus) antibody and AlphaLISA Acceptor beads conjugated to an antibody (Ab) specific to the epigenetic mark. The biotinylated antibody is then captured by Streptavidin (SA) Donor beads, bringing the two 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 fluorescentsignal at 615 nm (Figure 2b).

In the example presented here, changes in the levels of H3K4me2 were measured after treatment of cells with sodium butyrate (NaB) and Trichostatin A (TSA), two non-selective histone deacetylase (HDAC) inhibitors. Characterization of NaB (Figure 3a) with the AlphaLISA cellular assay shows that NaB also has effects on histone methylation. Overnight treatment of cells with NaB led to significantly higher levels of the dimethyl mark on at lysine 4 than did untreated controls. A Western blot reveals the same pattern of NaB-specific modification, but requires much more time and effort to perform and is less amenable to quantification and high throughput. Inhibitor titration performed with the AlphaLISA assay (Figure 3b) allowed quantification of a 5,000-fold difference in potency for the two inhibitors at this epigenetic mark. 

Together, the assay technologies presented in this article constitute a toolbox with many benefits for epigenetics drug discovery aimed at cancer therapeutics. Homogeneous LANCE and AlphaLISA assay technologies are simple and scalable for high throughput screening or for lead profiling, with validated assays and reagents available for a wide range of specific histone targets. AlphaLISA cellular assays are of particular value in the lead discovery process, as they allow cellularanalysis of endogenous nuclear responses to candidate inhibitors.

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