Radioactivity-based assays to measure histone methyltransferase activity are sensitive, easy to configure, and applicable to a diverse range of enzymes. Filter binding assays measure transfer of tritiated methyl groups from [³H]-SAM to a substrate, followed by removal of unreacted [³H]-SAM and retention of radiolabeled-product by filtration and subsequent scintillation counting (Fig. 1A). The fungal mycotoxin metabolite chaetocin (3,6-epi-dithio-diketopiperazine) was one of the first methyltransferase inhibitors discovered by screening 3,000 compounds against the Drosophila melongaster SU(VAR)3-9 lysine methyltransferase using a radioactive histone H3 peptide filter binding assay (Table 1) [6Greiner D, Bonaldi T, Eskeland R, Roemer E, Imhof A. Identification of a specific inhibitor of the histone methyltransferase SU(VAR)3-9 Nat Chem Biol 2005; 1(3): 143-5.]. Filters assembled in 96- and 384-well vacuum plates have increased the throughput of this assay. Other plate-based techniques include immobilization of a biotinylated peptide substrate in an avidin/streptavidin-plate and removal of free [³H]-SAM with washing steps. The washing steps may be eliminated with the use of avidin/streptavidin-coated FlashPlates that contain a thin layer of scintillant bound to the walls of the microplate wells where the signal is based on isotope proximity [7Rathert P, Cheng X, Jeltsch A. Continuous enzymatic assay for histone lysine methyltransferases Biotechniques 2007; 43(5): 602-8.].

Fig. (1) Histone methyltransferases assay formats. Filter binding assays to measure histone methyltransferase activity utilize radiolabeled SAM to measure transfer of tritiated methyl groups to histone substrates (A). Unreacted 3H-SAM is removed from reaction solutions via filtration through a vacuum plate. Addition of scintillation fluid permits quantitation of radiolabeled products retained on the filter plate. (B) SPA methyltransferases assays utilize beads containing scintillation fluid that emit light when excited by β-particles released from radioactive decay of bead-bound substrates. (C) Coupled enzyme assays measure histone methyltransferase activity by transformation of SAH to products that can be measured by fluorescence or UV/Vis spectroscopy via Hcy or S-ribosyl-homocysteine intermediates. (D) Methylation of a biotinylated histone peptide by AlphaScreen is measured using streptavidin-coated donor beads and anti-immunoglobulin–conjugated acceptor beads in the presence of a specific antibody raised against the methylated lysine product. Transfer of singlet oxygen from laser-excited donor beads to acceptor beads in close proximity results in a chemiluminescent signal. (E) The LysC endoproteinase distinguishes between methyltransferase substrate and product peptides by selective proteolysis at unmodified lysine residues. Electrophoretic separation of tagged fluorescent peptides permits quantitation of enzymatic activity following proteolysis with LysC.

Table 1

Methods Used in the Discovery of Small Molecule Inhibitors of Histone Methyltransferase and Demethylase Enzymes

Transfer of tritiated methyl groups from [³H]-SAM to a bead-bound substrate provides a homogeneous, sensitive method for rapidly measuring methyltransferase activity (Fig. 1B). Scintillation proximity assays (SPA) utilize microscopic beads containing scintillant. The interaction of these beads with β-particles generated by the radioactive decay of tritium releases photons that may be measured with scintillation counters or charge-coupled device (CCD) imagers. A variety of SPA bead formats allows utilization of various methyltransferase substrates, from biotinylated histone peptides to nucleosomes. The lack of a separation step and intrinsic sensitivity of the assay make SPA techniques amenable for HTS. However, the use of radioactivity is undesirable due to health risks as well as the cost of radiolabeled substrate and waste disposal. Thus the development and use of non-radioactive assays should be utilized whenever possible.

Simple substrate-to-product conversion is difficult to monitor in histone methyltransferase reactions where the spectral differences in SAM and SAH are insufficient for direct monitoring. Several coupled assays have been used to measure the SAH product of methyltransferases (Fig. 1C). The cysteine-free SAH hydrolase of Sulfolobus solfataricus generates a free thiol in homocysteine that may be measured with thiol-sensitive fluorophores [8Collazo E, Couture JF, Bulfer S, Trievel RC. A coupled fluorescent assay for histone methyltransferases Anal Biochem 2005; 342(1): 86-92.], including the malei-mide derivatives ThioGlo 1 [methyl-10-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-9-methoxy-3-oxo-3H-benzo[f]chromene-2-carboxylate] and CPM (7-diethylamino-3-(4’-maleimidyl-phenyl)-4-methylcoumarin) [8Collazo E, Couture JF, Bulfer S, Trievel RC. A coupled fluorescent assay for histone methyltransferases Anal Biochem 2005; 342(1): 86-92., 9Chung CC, Ohwaki K, Schneeweis JE, et al. A fluorescence-based thiol quantification assay for ultra-high-throughput screening for inhibitors of coenzyme A production Assay Drug Dev Technol 2008; 6(3): 361-74.]. The free thiol of homocysteine may also be measured by reaction with DTNB (5,5’-dithiobis-2-nitrobenzoic acid) to form the colored product TNB (5-thio-2-nitrobenzoic acid). Homocysteine may also be generated from SAH through the consecutive actions of SAH nucleosidase (SAHN) to generate adenine and S-ribosylhomocysteine and LuxS (S-ribosyl-homocysteinase) to cleave the latter intermediate to form Hcy [10Hendricks CL, Ross JR, Pichersky E, Noel JP, Zhou ZS. An enzyme-coupled colorimetric assay for S-adenosylmethionine-dependent methyltransferases Anal Biochem 2004; 326(1): 100-5.]. Alternatively, the adenine product of SAHN may be utilized by adenine deaminase to form hypoxanthine, where a decrease in UV-absorbance is monitored at 265 nm [11Dorgan KM, Wooderchak WL, Wynn DP, et al. An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases Anal Biochem 2006; 350(2): 249-55.]. The SAHH/ThioGlo assay described by Collazo et al. [8Collazo E, Couture JF, Bulfer S, Trievel RC. A coupled fluorescent assay for histone methyltransferases Anal Biochem 2005; 342(1): 86-92.] is likely the most straightforward and sensitive of these methods to measure SAH production, considering the single coupling enzyme and fluorescence detection. However, assay components must be carefully selected to minimize background fluorescence due to the reaction of free thiols with ThioGlo 1, and chemical blocking of free thiols of the substrate or enzyme may be considered. Coupled enzyme assays are inherently more difficult to optimize and susceptible to false positives when seeking enzyme inhibitors. Nonetheless, the removal of SAH in the coupling assays described here is advantageous for relief of SAH product inhibition, a characteristic common to histone methyltransferases.

Several biochemical assays have been developed that rely on antibody recognition of specific post-translational modifications. Enzyme-linked immunosorbent assays (ELISA) for histone methyltransferases involve immobilization of biotinylated histone peptide substrate on an avidin-coated microplate. Following enzymatic methylation of Lys or Arg residues on the immobilized peptide, a primary antibody specific to the PTM is captured on the plate. A secondary antibody conjugated with horseradish peroxidase (HRP) is used for chemiluminescence detection. The first protein arginine methyltransferase (PRMT) inhibitor AMI-1, a symmetrical sulfonated urea, was identified in an ELISA-based HTS assay of 9,000 diverse compounds using fungal Hmt1p (also known as Rmt1p) and the hnRNP protein Npl3p (Table 1) [12Cheng D, Yadav N, King RW, Swanson MS, Weinstein EJ, Bedford MT. Small molecule regulators of protein arginine methyltransferases J Biol Chem 2004; 279(23): 23892-9.]. AMI-1 is a non-specific arginine methyltransferase inhibitor, although it does not affect methylation of lysine.

A modern version of the ELISA is the dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) where the secondary antibody is labeled with a lanthanide chelate (typically europium) instead of HRP, and product formation is measured with time-resolved fluorescence (TRF). This technique was used to identify the first inhibitor of a histone lysine methyltransferase that was not competitive with the SAM cosubstrate. BIX-01294 was discovered in a screen of 125,000 compounds as a selective inhibitor of the G9a lysine methyltransferase (IC₅₀ 2.7 µM) and was competitive with the histone H3 peptide substrate (Table 1) [13Kubicek S, O'Sullivan RJ, August EM, et al. Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase Mol Cell 2007; 25(3): 473-81.]. BIX-01338 was also identified in this screen as a G9a inhibitor (IC₅₀ 4.7 µM), but it also inhibited other Lys and Arg methyltransferases and was SAM-competitive. Inhibitors of the arginine methyltransferase PRMT1 were also identified using a DELFIA assay to screen compounds selected by iterative virtual screening of the National Cancer Institute (NCI) diversity collection [14Spannhoff A, Heinke R, Bauer I, et al. Target-based approach to inhibitors of histone arginine methyltransferases J Med Chem 2007; 50(10): 2319-5.]. Two of the compounds active in the PRMT1 in vitro assay, allantodapsone and stilbamidine, were validated by demonstrating hypomethylation of H4R3 in HepG2 cells and inhibition of ERα-transcriptional activation in a cell-based reporter assay.

Antibody detection has also been used in a homogeneous assay utilizing AlphaScreen technology to measure histone G9a methyltransferase activity [15Quinn AM, Allali-Hassani A, Vedadi M, Simeonov A. A chemiluminescence-based method for identification of histone lysine methyltransferase inhibitors Mol Biosyst 2010; 6(5): 782-8.]. This dual bead-based technology utilizes laser excitation of donor beads, releasing a flow of singlet oxygen to generate chemiluminescent emission from acceptor beads in close proximity (Fig. 1D). Acceptor beads coated with secondary antibody are coupled to specific antibody detection of enzyme-modified substrates. AlphaScreen was used in the structural optimization of the BIX-01294 inhibitor of G9a, and IC₅₀ values obtained tracked closely with those measured in a SAHH/ThioGlo 1 coupled assay [16Liu F, Chen X, Allali-Hassani A, et al. Discovery of a 2,4-diamino-7-aminoalkoxyquinazoline as a potent and selective inhibitor of histone lysine methyltransferase G9a J Med Chem 2009; 52(24): 7950-3., 17Liu F, Chen X, Allali-Hassani A, et al. Protein lysine methyltransferase G9a inhibitors: design, synthesis., and structure activity relationships of 2.,4-diamino-7-aminoalkoxy-quinazolines J Med Chem 2010; 53(15): 5844-7.]. These studies led to the discovery of UNC0224 and UNC0321, the most potent G9a inhibitors identified to date (Table 1). The advantage of this assay compared to ELISA-based methods is the homogeneous nature of AlphaScreen where elimination of washing and substrate immobilization steps leads to an increased assay throughput. Importantly, the AlphaScreen assay was readily miniaturized to 1,536-well plate format allowing for its use in screening large compound collections [15Quinn AM, Allali-Hassani A, Vedadi M, Simeonov A. A chemiluminescence-based method for identification of histone lysine methyltransferase inhibitors Mol Biosyst 2010; 6(5): 782-8.].

AlphaScreen assays to measure histone methyltransferase activity may utilize direct coupling of the acceptor bead to the primary antibody in place of a secondary anti-immunoglobulin donor bead plus primary antibody. Some methyl mark antibodies directly conjugated to acceptor beads are commercially available (http://las.perkinelmer. com/). In vitro AlphaScreen assays most commonly use streptavidin-coated donor beads to bind biotinylated peptide substrates. However, donor beads may be conjugated with antibodies that recognize histone substrates at a site distinct from that modified by the methyltransferase or demethylase enzyme. This sandwich-based approach is suitable for enzymes with no or low activity towards peptide substrates as well as for cell-based quantitation of histone-modifying enzyme activity.

Time-resolved fluorescence resonance energy transfer (TR-FRET), like AlphaScreen, is a proximity based technique that may be used to measure enzymatic activity of histone-modifying enzymes. TR-FRET combines time-resolved fluorescence (TRF) and FRET in a homogeneous format where fluorescence emission of a donor dye overlaps with the excitation spectrum of an acceptor dye. Whereas AlphaScreen donor and acceptor molecules may be separated by up to 200 nm, the distance of interaction is typically limited to 1-10 nm for TR-FRET. To date, FRET has only been used to measure inhibition of HDAC activity in cells using a baculovirus-expressed GFP-p53 fusion protein [18Dudek JM, Horton RA. TR-FRET biochemical assays for detecting posttranslational modifications of p53 J Biomol Screen 2010; 15(5): 569-75.]. A terbium-labeled acetyl-p53 (Lys382) antibody was used to measure activity of the p300 histone acetyltransferase (HAT) using the non-histone GFP-p53 substrate where GFP is the FRET acceptor. We note that if an endpoint assay based on antibody detection is desired, the traditional method of stopping the enzymatic reaction by addition of acid will not be usable: instead, an addition of EDTA at a millimolar concentration can be used as an alternative.

The limitations of these techniques lie in the availability of specific antibodies, particularly in assays that require discrimination between mono- and dimethylated or di- and tri-methylated residues [19Kawamura A, Tumber A, Rose NR, et al. Development of homogeneous luminescence assays for histone demethylase catalysis and binding Anal Biochem 2010; 404(1): 86-93.]. Examination of more than 200 antibodies raised against 57 different histone modifications found that 25% of these failed specificity testing by Western blot and dot blot analysis, and 20% of the specific antibodies were deemed unsuitable for chromatin immunoprecipitation (ChIP) analysis [20Egelhofer TA, Minoda A, Klugman S, et al. An assessment of histone-modification antibody quality Nat Struct Mol Biol 2010; 18(1): 91-3.]. The high failure rate prompted the authors to create a website for posting these and new results of antibody specificity tests for the detection of epigenetic marks (http://compbio.med.harvard.edu/antibodies/). Where specific antibodies are not available, the use of tagged chromatin binding domains to measure changes in levels of specific post-translationally modified residues has been suggested (Wigle, T.J., personal communication), although it is unclear whether the micromolar affinities typical of reader domains for their cognate epigenetic mark would be sufficient for such applications. Additional complications in attempting to quantify PTMs with reader domains in a cellular environment may result from the observation by Fuchs et al. that neighboring PTMs can affect the affinity of chromatin-associated domains for a particular epigenetic mark [21Fuchs SM, Krajewski K, Baker RW, Miller VL, Strahl BD. Influence of combinatorial histone modifications on antibody and effector protein recognition Curr Biol 2011; 21(1): 53-8.]. Using a library of histone peptides with single or multiple PTMs immobilized in a peptide array, both enhanced and diminished binding of several reader domains were observed for combinations of PTMs. Using the same peptide array, the authors also found the specificity of some commercial antibodies raised against a specific epigenetic mark were affected by other PTMs present on the peptide bait [21Fuchs SM, Krajewski K, Baker RW, Miller VL, Strahl BD. Influence of combinatorial histone modifications on antibody and effector protein recognition Curr Biol 2011; 21(1): 53-8.].

The principles of capillary electrophoresis applied to a microfluidic chip (MCE) allow separation of nanoliter-sized samples based on the charge-to-mass ratio of the components of interest. The loss of lysine’s positive charge with acetylation results in facile electrophoretic separation of substrate and product peptides and offers a direct method for measuring HAT or HDAC activity. This type of electrophoretic mobility shift assay has been used to profile inhibitors of HDAC and HAT enzymes, including PCAF and GCN5L2 [22Fanslau C, Pedicord D, Nagulapalli S, et al. An electrophoretic mobility shift assay for the identification and kinetic analysis of acetyl transferase inhibitors Anal Biochem 2010; 402(1): 65-8., 23Blackwell L, Norris J, Suto CM, Janzen WP. The use of diversity profiling to characterize chemical modulators of the histone deacetylases Life Sci 2008; 82(21-22): 1050-8.]. A similar approach may be employed for changes in phosphorylation of histone peptides.

Methylation of lysine, however, does not change the positive charge of this amino acid, rendering a direct electrophoretic separation impossible. To address this limitation, methods originally developed for methyllysine mapping of histones by mass spectrometry (MS) have recently been applied to MCE. These techniques use the endoproteinase LysC to selectively cleave at the carboxyl side of unmodified lysine residues, leaving methylated lysine polypeptides intact (Fig. 1E). Such a histone mark-dependent cleavage allows for the production of fluorescent peptides of different charge-to-mass ratios. The utility of this method was demonstrated using LysC and fluorescent-labeled histone peptides for inhibitor screening and mechanism of action studies of the G9a methyltransferase and the LSD1 lysine demethylase [24Wigle TJ, Provencher LM, Norris JL, et al. Accessing protein methyltransferase and demethylase enzymology using microfluidic capillary electrophoresis Chem Biol 2010; 17(7): 695-704.]. The G9a MCE assay was used to determine the Morrison K_i (63 pM) of UNC0321, the 2,4-diamino-7-aminoalkoxy-quinazoline compound designed from the BIX-01294 chemical scaffold (Table 1) [17Liu F, Chen X, Allali-Hassani A, et al. Protein lysine methyltransferase G9a inhibitors: design, synthesis., and structure activity relationships of 2.,4-diamino-7-aminoalkoxy-quinazolines J Med Chem 2010; 53(15): 5844-7.]. The multiplicity of Lys residues in the N-terminal tails of histones, however, complicates generation of suitable peptides upon LysC cleavage and may require mutation or chemical modification of non-substrate Lys residues in substrate peptides. In addition to measuring lysine methyltransferase activity, protease-coupled MCE may be applicable to monitoring Arg methylation with the use of the ArgC endoproteinase [24Wigle TJ, Provencher LM, Norris JL, et al. Accessing protein methyltransferase and demethylase enzymology using microfluidic capillary electrophoresis Chem Biol 2010; 17(7): 695-704.].

Mass spectrometry (MS) has been an important tool in identifying novel PTMs as well as characterizing activities of histone-modifying enzymes [25Eberl HC, Mann M, Vermeulen M. Quantitative proteomics for epigenetics Chembiochem 2011; 12(2): 224-34.]. The majority of this work has been performed using a bottom-up approach where proteins are digested into small peptides prior to MS analysis. Tandem mass spectrometry (MS/MS) is used to map the site of modification and reconstruct protein identity using sequence information. The major disadvantage to this approach is that information on the combinatorial nature of histone modifications is lost. A top-down approach, on the other hand, refers to MS analysis of intact proteins to yield information on combinations of modifications and their relative abundance [26Garcia BA. What does the future hold for top down mass spectrometry? J Am Soc Mass Spectrom 2010; 21(2): 193-202.]. This is particularly useful in the analysis of heterogeneous mixtures of histones, for example in tissues and cell culture.

An intermediate approach to these techniques was constructed to mitigate the need for a high-resolution mass analyzer in top-down MS while retaining information on the interplay between PTMs on a single histone polypeptide. In middle-down MS, limited proteolysis of histones with endoproteinases such as GluC or AspN generates polypeptides of up to 50 residues [27Taverna SD, Ueberheide BM, Liu Y, et al. Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini Proc Natl Acad Sci USA 2007; 104(7): 2086-91.]. As the majority of PTMs are located on the N-terminal tails of these proteins, information on the complex spectrum of histone PTMs is gained with middle-down MS. This MS proteomics method has been used for global assessment of combinatorial PTMs and their tissue-specific abundance [28Garcia BA, Thomas CE, Kelleher NL, Mizzen CA. Tissue-specific expression and post-translational modification of histone H3 variants J Proteome Res 2008; 7(10): 4225-36.].

Quantitative MS has been used to measure enzymatic activity of histone-modifying enzymes. In vitro approaches generally target specific PTMs and use MS peak intensities of high resolution, well-defined peptides for relative quantitation of product formation (Fig. 2A). This label-free method has been used to measure activities of numerous histone-modifying enzymes [29Tsukada Y, Fang J, Erdjument-Bromage H, et al. Histone demethylation by a family of JmjC domain-containing proteins Nature 2006; 439(7078): 811-6.-31Osborne TC, Obianyo O, Zhang X, Cheng X, Thompson PR. Protein arginine methyltransferase 1: positively charged residues in substrate peptides distal to the site of methylation are important for substrate binding and catalysis Biochemistry 2007; 46(46): 13370-81.] and to determine inhibitor potency [32Chang Y, Zhang X, Horton JR, et al. Structural basis for G9a-like protein lysine methyltransferase inhibition by BIX-01294 Nat Struct Mol Biol 2009; 16(3): 312-7., 33Rose NR, Ng SS, Mecinovic J, et al. Inhibitor scaffolds for 2-oxoglutarate-dependent histone lysine demethylases J Med Chem 2008; 51(22): 7053-6.]. An internal standard for normalization that consists of a nonvarying, unmodified peptide may be used in conjunction with peptide intensity profiling to measure changes in histone modifications. Advances in the throughput of MS-based quantitation assays have increased the capacity for inhibitor screening in these sensitive and selective assays [34Roddy TP, Horvath CR, Stout SJ, et al. Mass spectrometric techniques for label-free high-throughput screening in drug discovery Anal Chem 2007; 79(21): 8207-13.]. For example, inhibition of the LSD1 histone demethylase has been measured using an eight-way parallel, staggered flow injection MS from samples in 384-well plate format [35Rye PT, Frick LE, LaMarr WA, Özbal CC. High-throughput Mass Spectrometric Detection of Histone 3 Demethylation Available from: http://www.biocius.com/pdfs/RapidFireHTMS_EpigeneticTargets_Poster.pdf [cited 2011 Jan];]. Other quantitative methods for measuring histone modifying enzymes utilize various labeling techniques, including stable isotope labeling with amino acids in cell culture (SILAC) [36Ong SE, Blagoev B, Kratchmarova I, et al. Stable isotope labeling by amino acids in cell culture, SILAC., as a simple and accurate approach to expression proteomics Mol Cell Proteomics 2002; 1(5): 376-86.], enzymatic incorporation of ¹⁸O [37Bantscheff M, Dumpelfeld B, Kuster B. Femtomol sensitivity post-digest (18)O labeling for relative quantification of differential protein complex composition Rapid Commun Mass Spectrom 2004; 18(8): 869-76.], iTRAQ [38Ross PL, Huang YN, Marchese JN, et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents Mol Cell Proteomics 2004; 3(12): 1154-69.], and chemical tagging of reactive side chains or groups with stable isotopes [39Smith CM, Gafken PR, Zhang Z, Gottschling DE, Smith JB, Smith DL. Mass spectrometric quantification of acetylation at specific lysines within the amino-terminal tail of histone H4 Anal Biochem 2003; 316(1): 23-33.]. Stable isotopes may also be incorporated into methylated histone residues using [¹³CD₃]-labeled SAM in place of the SAM methyl donor [40Ong SE, Mittler G, Mann M. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC Nat Methods 2004; 1(2): 119-26.].

Fig. (2) Histone demethylase assay formats. (A) Quantitative mass spectrometry measures demethylation of histone peptide substrates by identification and relative quantitation of peptides by MS/MS. Here, LSD1 demethylates dimethyl-H3K9 to consecutively generate mono-methylated and unmodified H3K9 peptides. (B) Activity of JmjC lysine demethylases may be measured by coupling formaldehyde production to NAD+ reduction using the coupling enzyme FDH and monitoring NADH formation. (C) Lysine demethylation catalyzed by LSD1 can be measured using the coupling enzymes HRP or FDH to quantitate hydrogen peroxide or formaldehyde products, respectively. FDH-coupled formaldehyde detection is also applicable to the testing of 2-OG–dependent JmjC-domain histone demethylases.

Given the low turnover of many histone methyltransferases, assays that measure displacement of SAM or a peptide substrate may be used to further characterize inhibitors or to select potential inhibitors. Fluorescence polarization (FP) assays measure changes in fluorescence anisotropy in the presence of compounds that displace fluorescently-labeled cognate peptides from the methyltransferase active site. Exploration of the SAR around the G9a inhibitor BIX-01294 was aided by measuring displacement of a fluorescein-labeled histone H3 peptide upon compound binding [16Liu F, Chen X, Allali-Hassani A, et al. Discovery of a 2,4-diamino-7-aminoalkoxyquinazoline as a potent and selective inhibitor of histone lysine methyltransferase G9a J Med Chem 2009; 52(24): 7950-3.]. A similar FP assay was developed to measure binding of histone H4 peptides to PRMT1 [41Feng Y, Xie N, Wu J, Yang C, Zheng YG. Inhibitory study of protein arginine methyltransferase 1 using a fluorescent approach Biochem Biophys Res Commun 2009; 379(2): 567-72.], and was used for the mechanistic analysis and comparison of binding affinities for published PRMT1 inhibitors (Table 1) [42Feng Y, Li M, Wang B, Zheng YG. Discovery and mechanistic study of a class of protein arginine methylation inhibitors J Med Chem 2010; 53(16): 6028-39.]. FP assays, however, require large amounts of enzyme and may not be appropriate for large-scale discovery of peptide-competitive inhibitors.

The options for cell-based assays to measure changes in posttranslational modifications of histone tails are limited and primarily semi-quantitative. The multiplicity of methyltransferases and a lack of characterization of their substrates make it difficult to assign activity to a particular enzyme. Transcriptional changes of genes known to be affected by histone methylation at a particular site are frequently used as surrogate markers of methyltransferase activity in cells [14Spannhoff A, Heinke R, Bauer I, et al. Target-based approach to inhibitors of histone arginine methyltransferases J Med Chem 2007; 50(10): 2319-5.]. ChIP at methyltransferase target genes may also be used to measure methylation changes at promoter-proximal sites. Measurements compared before and after knockdown of the methyltransferase by genetic or RNAi-based methods assist in validation of the inhibitor’s target.

Cell-based assays to measure changes in a particular epigenetic mark include fluorescence immunostaining with specific antibodies in live cells [43King ON, Li XS, Sakurai M, et al. Quantitative high-throughput screening identifies 8-hydroxyquinolines as cell-active histone demethylase inhibitors PLoS One 2010; 5(11): e15535.]. A similar technique is standard ELISA with fixed, permeabilized cells using a primary antibody directed against a specific PTM and a europium-labeled secondary antibody for TRF detection [14Spannhoff A, Heinke R, Bauer I, et al. Target-based approach to inhibitors of histone arginine methyltransferases J Med Chem 2007; 50(10): 2319-5.]. As discussed above, quantitation of methylation at a specific site is complicated by the availability of specific antibodies.