Sage Journals: Discover world-class research

Abstract

Histone posttranslational modifications are among the epigenetic mechanisms that modulate chromatin structure and gene transcription. Histone methylation and demethylation are dynamic processes controlled respectively by histone methyltransferases (HMTs) and demethylases (HDMs). Several HMTs and HDMs have been implicated in cancer, inflammation, and diabetes, making them attractive targets for drug therapy. Hence, the discovery of small-molecule modulators for these two enzyme classes has drawn significant attention from the pharmaceutical industry. Herein, the authors describe the development and optimization of homogeneous LANCE Ultra and AlphaLISA antibody-based assays for measuring the catalytic activity of two epigenetic enzymes acting on lysine 4 of histone H3: SET7/9 methyltransferase and LSD1 demethylase. Both the SET7/9 and LSD1 assays were designed as signal-increase assays using biotinylated peptides derived from the N-terminus of histone H3. In addition, the SET7/9 assay was demonstrated using full-length histone H3 protein as substrate in the AlphaLISA format. Optimized assays in 384-well plates are robust (Z′ factors ≥0.7) and sensitive, requiring only nanomolar concentrations of enzyme and substrate. All assays allowed profiling of known SET7/9 and LSD1 inhibitors. The results demonstrate that the optimized LANCE Ultra and AlphaLISA assay formats provide a relevant biochemical screening approach toward the identification of small-molecule inhibitors of HMTs and HDMs that could lead to novel epigenetic therapies.

Keywords

epigenetics SET7/9 LSD1 methyltransferase demethylase LANCE TR-FRET AlphaLISA

Introduction

Posttranslational modifications on histone proteins have been shown to play important roles in the regulation of chromatin structure and gene expression.¹ The reversible nature of histone covalent modifications or “marks,”² along with the deregulation of histone-modifying enzymes observed in diseased states such as cancer³ and neuropathologies,⁴ has promoted this enzyme group to an attractive class of drug targets.⁵

SET7/9 (KMT7), SMYD3, and members of the MLL (KMT2) protein family are histone methyltransferases that, together with LSD1 demethylase (KDM1), are known to affect the methylation level of histone H3 on the lysine 4 (H3K4) residue (reviewed by Ruthenburg et al.⁶). Although methylation of H3K4 by SET7/9 and MLL has been linked to active transcription,^7–9 demethylation elicited by LSD1 in association with the CoREST complex was suggested to repress gene expression.¹⁰ SET7/9 contains an evolutionary conserved SET-domain that uses S-adenosyl-L-methionine (SAM, or AdoMet) as a co-substrate to catalyze the methylation of lysine.^7,8 Unlike most other SET proteins that catalyze the addition of up to three methyl groups, SET7/9 catalyzes the transfer of a single methyl group to the unmodified H3K4 residue and is thus referred to as a histone lysine mono-methyltransferase.¹¹ On the other hand, LSD1 belongs to the flavin adenine dinucleotide (FAD)–dependent amine oxidase superfamily and specifically catalyzes the demethylation of mono- or dimethylated H3K4 residues with the concomitant formation of hydrogen peroxide and formaldehyde.¹⁰ In addition to being a repressor of transcription when associated with CoREST, LSD1 can also act as a transcriptional coactivator when complexed with the androgen receptor by demethylating mono- or dimethylated histone H3 lysine 9 residues.¹² Both SET7/9 and LSD1 have been associated with disease: SET7/9 with inflammatory disorders and diabetes¹³ and LSD1 with various types of cancer,¹⁴ making these two enzymes potential targets for therapeutic intervention.

Several methodological approaches have been used for quantifying the activity of histone methyltransferases (HMTs) and histone demethylases (HDMs), including radiometric assays,^8,15,16 enzyme-linked immunoassays,¹⁷ mass spectrometry,¹⁰ and enzyme-coupled detection of reaction by-products such as S-adenosyl-homocysteine (SAH, or AdoHcy),¹⁸ formaldehyde,¹⁰ or hydrogen peroxide.¹⁹ These methods suffer from drawbacks such as low throughput, lack of selectivity, high enzyme and peptide consumption, multiple wash steps, generation of hazardous waste, requirement for expensive equipment, or artifacts associated with the use of enzyme-coupled assays. The nonradioactive homogeneous LANCE Ultra and Alpha (AlphaScreen and AlphaLISA) platforms ( Fig. 1 ), based respectively on time-resolved fluorescence resonance energy transfer (TR-FRET)²⁰ and luminescent oxygen channeling assays,²¹ do not share these limitations and are now firmly established for the development and validation of high-throughput screening (HTS) campaigns.^22,23 In this regard, the AlphaScreen technology has been recently employed with success in the development of HTS assays for two epigenetic enzymes, G9a methyltransferase²⁴ and JMJD2E demethylase.²⁵

Figure 1.

Schematic representation of LANCE Ultra and AlphaLISA epigenetic assays. (A) Enzymatic assays using a biotinylated histone H3-derived peptide as substrate. In the first step, the enzyme modifies the substrate in the presence of the required cofactor to generate the reaction product; product detection can then be performed using LANCE Ultra or AlphaLISA antibody-based reagents directed against the mark of interest. In the LANCE Ultra setup (A, left), capture of the biotinylated reaction product by a europium-labeled antimark antibody (Eu-Ab, donor) and ULight-streptavidin (ULight-SA, acceptor) brings into close proximity donor and acceptor dye-bearing partners. Irradiation of complexed partners at 320 to 340 nm allows time-resolved fluorescence resonance energy transfer (TR-FRET) to occur between the europium-labeled antibody and the ULight-streptavidin, giving rise to light emission detected at 665 nm. In the AlphaLISA setup using a biotinylated peptide substrate (A, right), the reaction product is captured by streptavidin (SA) donor beads and anti-mark acceptor beads. Irradiation of complexed partners at 680 nm initiates in SA donor beads the production of singlet oxygen (¹O₂) molecules that reach the acceptor beads in proximity to generate a cascade of energy transfer reactions culminating in an amplified light emission detected at 615 nm. (B) Enzymatic assay using a full-length histone H3 substrate. A biotinylated antihistone H3 (C-ter) antibody is used for the capture of the full-length H3 protein by SA donor beads. For both LANCE Ultra and AlphaLISA assay setups, the intensity of emitted light is proportional to the level of substrate modification.

Using LANCE Ultra and AlphaLISA technologies, we undertook the development of sensitive and selective biochemical assays for measuring the activity of SET7/9 and LSD1 enzymes in a signal-increase mode. With both technologies, synthetic biotinylated peptides derived from the N-terminus of histone H3 (residues 1–21) were used as substrates ( Fig. 1A ). Furthermore, taking advantage of the AlphaLISA platform flexibility, we developed a SET7/9 assay using a full-length histone H3 protein substrate ( Fig. 1B ). In all cases, the methylation state of histone H3K4 residues was interrogated through the use of specific antibodies detecting either methylated or unmethylated H3K4 residues.

Materials and Methods

All biotinylated peptides, including substrate peptides histone H3 (1-21) (cat. 61702) and Lys4(me1)-histone H3 (1–21) (cat. 64355), were purchased from AnaSpec (Fremont, CA), whereas recombinant full-length histone H3 substrate (C110A) (cat. 31207) and modified positive controls were from Active Motif (Carlsbad, CA). S-(5′-adenosyl)-L-methionine chloride (cat. A7007), S-(5′-adenosyl)-L-homocysteine (cat. A9384), sinefungin (cat. S7559), trans-2-phenylcyclopropylamine (tranylcypromine; cat. P8511), and poly-L-lysine (cat. P1399) were purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human SET7/9 methyltransferase (cat. ALX-201-178-C100) and LSD1 demethylase (cat. 50100) were purchased from Enzo Life Sciences International, Inc. (Plymouth Meeting, PA) and BPS Bioscience, Inc. (San Diego, CA), respectively. All other reagents were from PerkinElmer (Waltham, MA) unless otherwise stated. For LANCE Ultra assays, these included europium (Eu)–labeled anti-H3K4me1-2 (cat. TRF0402) and anti-unmodified H3K4 (cat. TRF0404) antibodies, ULight-labeled Streptavidin (ULight-SA; cat. TRF0102), and 10× LANCE Detection Buffer (cat. CR97-100). For AlphaLISA assays, reagents included anti-H3K4me1-2 (cat. AL116) and anti-unmodified H3K4 (cat. AL119) acceptor beads, streptavidin (SA) donor beads (cat. 6760002), biotinylated anti-H3 (C-ter) antibody (cat. AL118), and 5× Epigenetics Buffer 1 Kit (cat. AL008). Assays for both technologies were performed in white opaque OptiPlate 384-well microplates (cat. 6007290) using TopSeal-A sealing film (cat. 6005185). All steps using streptavidin donor beads were performed under subdued laboratory lighting (<100 lux) using green filters (Roscolux filters #389) from Rosco (Stamford, CT) to avoid photo-depleting the beads. Plates were read on an EnVision Multilabel Plate Reader (PerkinElmer) in TR-FRET or Alpha mode according to the technology used.

LANCE Ultra enzymatic assays

LANCE Ultra SET7/9 and LSD1 assays were developed using a biotinylated histone H3-derived peptide (residues 1–21) as substrate. SET7/9 methyltransferase reactions were carried out in 50 mM Tris-HCl (pH 8.8) supplemented with 5 mM MgCl₂, 1 mM dithiothreitol (DTT), and 0.01% Tween-20 and were initiated by the addition of a mix of biotinylated histone H3 peptide and SAM cofactor. LSD1 demethylase reactions were carried out in 50 mM Tris-HCl (pH 9.0) supplemented with 50 mM NaCl, 1 mM DTT, and 0.01% Tween-20 and were initiated by the addition of biotinylated H3K4me1 peptide substrate. All enzymatic reactions were performed for a fixed period of time at room temperature in a 10-µL volume (see Table 1 for details on experimental conditions).

Table 1.

Optimized Conditions for SET7/9 Methyltransferase and LSD1 Demethylase Assays

Technology	Enzyme (Concentration)	Substrate (Concentration)	Cofactor (Concentration)	Reaction Time, min	Antimark Reagent
LANCE Ultra	SET7/9 (5 nM)	H3 peptide (200 nM)	SAM (300 nM)	60	Eu-anti-H3K4me1-2
	LSD1 (2 nM)	H3K4me1 peptide (200 nM)	—	60	Eu-anti-unmodified H3K4
AlphaLISA	SET7/9 (1 nM)	H3 peptide (50 nM)	SAM (100 nM)	30	Anti-H3K4me1-2 acceptor beads
	SET7/9 (0.3 nM)	Full-length histone H3 (300 nM)	SAM (10 µM)	10	Anti-H3K4me1-2 acceptor beads
	LSD1 (2 nM)	H3K4me1 peptide (80 nM)	—	60	Anti-unmodified H3K4 acceptor beads

SAM, S-adenosyl-L-methionine.

SET7/9 methyltransferase reactions were stopped by adding a mixture of Eu-labeled anti-H3K4me1-2 antibody and ULight-SA prepared in 1× LANCE Detection Buffer. LSD1 demethylase reactions were stopped by the addition of 300 µM tranylcypromine prepared in 1× LANCE Detection Buffer and incubated for 5 min before the addition of a mixture of Eu-labeled anti-unmodified H3K4 antibody and ULight-SA. For both assays, Eu-labeled antibody and ULight-SA were used at final concentrations of 2 nM and 50 nM, respectively (total assay volume of 20 µL). Capture of the reaction products by the Eu-labeled antibodies and ULight-SA was allowed to proceed for 60 min at room temperature before measuring TR-FRET signal (excitation at 320 nm, emission at 615 and 665 nm, delay time 50 µs, window time 100 µs).

AlphaLISA enzymatic assays

AlphaLISA SET7/9 assays were developed using two different substrates: a biotinylated histone H3-derived peptide (residues 1–21) and a recombinant full-length histone H3 protein substrate. The AlphaLISA LSD1 assay was developed using a biotinylated histone H3K4me1 (1–21) peptide substrate.

SET7/9 and LSD1 assays with biotinylated histone H3 peptide substrates

AlphaLISA SET7/9 methyltransferase and LSD1 demethylase reactions were run at room temperature in a 10-µL volume under conditions identical to those described for the LANCE Ultra assays. The enzymatic reactions were stopped by the addition of antibody-acceptor beads diluted in 1× Epigenetics Buffer 1. Anti-H3K4me1-2 acceptor beads were added to the SET7/9 reactions and anti-unmodified H3K4 acceptor beads to the LSD1 reactions. After 60 min at room temperature, SA donor beads prepared in 1× Epigenetics Buffer 1 were added, and the capture of the reaction product was allowed to proceed for an additional 30 min at room temperature before detecting the Alpha signal. For both assays, acceptor and SA donor beads were used at a final concentration of 20 µg/mL (total assay volume of 25 µL).

SET7/9 assay using a recombinant histone H3 protein substrate

SET7/9 reactions using a recombinant full-length histone H3 protein as substrate were conducted at room temperature in a 10-µL volume under the conditions described before for the biotinylated peptide substrate.

Methyltransferase reactions were stopped by the addition of 5 µL of high salt buffer consisting of 50 mM Tris-HCl (pH 7.4), 1 M NaCl, 0.1% Tween-20, and 0.3% poly-L-lysine. After 15 min at room temperature, capture of the reaction product was initiated by the addition of a mixture of anti-H3K4me1-2 acceptor beads and biotinylated antihistone H3 (C-ter) antibody. SA donor beads were added 60 min later, and reactions were incubated for an additional 30 min before detection of the Alpha signal. Alpha beads and biotinylated antihistone H3 (C-ter) antibody were used at a final concentration of 20 µg/mL and 1 nM, respectively (total assay volume of 25 µL). All detection reagents were prepared in a detection buffer containing 50 mM Tris-HCl (pH 7.4), 300 mM NaCl, 0.1% Tween-20, and 0.001% poly-L-lysine.

Data analysis

Data analysis was conducted using the GraphPad Prism 5 software package (GraphPad Software, San Diego, CA). Enzymatic progress curves were fitted using a one-site binding hyperbola equation. SAM and inhibitor concentration–response curves were generated by fitting the data using a four-parameter logistic equation and used for the determination of EC₅₀ (apparent K _m) and IC₅₀ values. Values are expressed as mean of triplicates ± standard deviation. Assay performance and robustness were evaluated using the Z′ factor equation.²⁶

Results

Assay development workflow

Developing LANCE Ultra and AlphaLISA reagents for the immunodetection of defined epigenetic marks requires the careful selection of appropriate antibodies.²⁷ These should allow the sensitive and specific detection of the mark(s) of interest while at the same time minimizing assay background due to cross-reactivity to unmodified substrate molecules. To this end, antibodies recognizing either H3K4me1-2 or unmodified H3K4 were selected and labeled with the LANCE europium chelate W1024 or covalently linked to AlphaLISA acceptor beads using standard protocols. The specificity of the resulting conjugates was characterized in titration experiments using histone H3-derived peptides and full-length histone H3 proteins bearing appropriate epigenetic marks (Suppl. Fig. S1). Interestingly, although the anti-unmodified lysine 4 histone H3 antibody was highly specific for the cognate sequence, the anti-methyl lysine 4 antibody was able to detect with similar sensitivity both the mono- and dimethylated forms of histone H3 lysine 4 (H3K4me1-2).

The goal of our enzymatic assay development process was to develop and optimize robust and sensitive assays that can be used for the identification of compounds modulating enzyme activity. The first step of assay development consisted of selecting a substrate concentration between the EC₆₀ and EC₉₀ values taken from substrate titration curves. This allowed maximizing enzyme velocity while eliminating the risk of “hooking” on the substrate. A “hook” effect is usually observed in three-component assays when substrate concentrations exceed the binding capacity of the system.²⁸ Following determination of optimal substrate concentration, enzyme progress curves were generated at increasing enzyme concentrations in the presence of saturating cofactor (when required). An enzyme concentration and reaction time providing a linear signal increase over time were selected and used for all subsequent experiments. For methyltransferase assays, SAM titration curves were performed next, at the selected enzyme, substrate, and incubation time conditions. SAM concentrations near the apparent K _m values were further selected in order for the assays to be sensitive to SAM-competitive inhibitors. Assay linearity was then reconfirmed prior to assessing DMSO tolerance and performing inhibitor concentration–response curves, as well as Z′ factor determination.

SET7/9 methyltransferase assay using a histone H3 peptide substrate

Using a biotinylated histone H3 (1–21) peptide substrate, LANCE Ultra and AlphaLISA SET7/9 methyltransferase assays were optimized following the steps described above. Based on peptide titration data (not shown), 200 nM and 50 nM peptide substrate were selected for the LANCE Ultra and AlphaLISA assays, respectively. Enzyme progress curves were generated ( Fig. 2A , B ), and a 60-min reaction time using 5 nM SET7/9 was selected for LANCE Ultra, whereas a 30-min reaction time using 1 nM enzyme was chosen for AlphaLISA experiments. The effect of SAM concentration on the methyltransferase activity was then investigated, where both LANCE Ultra and AlphaLISA assays showed equivalent apparent K _m concentrations for SAM, being 75 and 79 nM, respectively ( Fig. 2C , D ). These values are significantly lower than what has been reported in the literature for SET7/9 assays conducted with H3-derived peptides (17 µM; see Ibañez et al.²⁹). However, it should be noted that in the Ibañez et al.²⁹ assay protocol, much higher SET7/9 enzyme and peptide substrate concentrations (200- to 1000-fold) were used, a difference that might account for this discrepancy.

Figure 2.

LANCE Ultra and AlphaLISA SET7/9 methyltransferase assay optimization using a biotinylated histone H3 peptide substrate. (A, B) Enzymatic progress curves. Enzymatic progress curves were run at increasing SET7/9 concentrations and stopped at the indicated time points. Peptide substrate and S-adenosyl-L-methionine (SAM) concentrations were (A) 200 nM and 300 µM, respectively, for the LANCE Ultra assay and (B) 50 nM and 100 µM for the AlphaLISA assay. (C, D) Determination of the apparent K_m (K_m app) value for SAM. Enzymatic reactions were carried out at increasing SAM concentrations using (C) 5 nM enzyme, 200 nM peptide substrate, and a 60-min incubation time in the LANCE Ultra setup and (D) 1 nM enzyme, 50 nM peptide substrate, and a 30-min incubation time in the AlphaLISA setup. (E, F) Inhibition of SET7/9 methyltransferase activity. Increasing concentrations of inhibitor were preincubated 10 min with the enzyme prior to initiating the methyltransferase reaction with a mixture of peptide substrate and SAM cofactor. Enzymatic reactions were carried out in the presence of 1% DMSO using (E) 5 nM enzyme, 200 nM peptide substrate, 300 nM SAM, and a 60-min incubation time in the LANCE Ultra setup and (F) 1 nM enzyme, 50 nM peptide substrate, 100 nM SAM, and a 30-min incubation time in the AlphaLISA setup. SAH, S-adenosyl-L-homocysteine.

A concentration of 100 nM SAM (i.e., near the apparent K_m) was selected for the AlphaLISA assay, whereas 300 nM (~4× apparent K_m) was chosen for LANCE Ultra to obtain a larger assay window. Under the selected conditions (summarized in Table 1 ), the LANCE Ultra assay was linear for up to 60 min (r² = 0.925) with a signal to background (S/B) ratio of 7.3 at 60 min, whereas the AlphaLISA assay was linear for up to 30 min (r² = 0.980) with an S/B ratio of 60 at 30 min (data not shown).

To assess the sensitivity of the LANCE Ultra and AlphaLISA assays toward small-molecule modulators, two nonselective SAM-competitive HMT inhibitors were tested: sinefungin, a structural analog of SAM isolated from Streptomyces, and SAH, a by-product of all SAM-promoted methyltransferase reactions. Both compounds inhibited SET7/9 activity in a concentration-dependent manner with IC₅₀ values of 769 and 359 nM for sinefungin and 93 and 16 µM for SAH in the LANCE Ultra and AlphaLISA assays, respectively ( Fig. 2E , F ). The lower IC₅₀ values obtained with AlphaLISA are consistent with the threefold lower SAM concentration used in the enzymatic reaction. Our data indicate that, under these assay conditions, sinefungin behaves as a more potent inhibitor of SET7/9 than SAH.

Robustness of the optimized LANCE Ultra and AlphaLISA assays was determined by evaluating their respective Z′ factor values. Inhibited signal was measured using sinefungin at 300 µM in LANCE Ultra and 100 µM for the AlphaLISA assay. Calculated Z′ factor values were above 0.80 with both technologies ( Table 2 ), with S/B ratios of 9.4 in LANCE Ultra and 36 in AlphaLISA. Assay stability was demonstrated for both platforms, with Z′ factor values higher than 0.70 following overnight incubation. Taken together, these results demonstrate the robustness of the optimized SET7/9 methyltransferase peptide assays on both detection platforms and their suitability for HTS.

Table 2.

Statistical Evaluation of Assay Quality

	LANCE Ultra		AlphaLISA
Enzyme	Z′	S/B	Z′	S/B
Biotinylated histone H3 peptide substrate
SET7/9	0.80 (0.86)	9.4 (11)	0.81 (0.71)	36 (20)
LSD1	0.88 (0.82)	20 (14)	0.80 (0.83)	643 (262)
Full-length histone H3 substrate
SET7/9	—	—	0.76 (0.70)	36 (50)

Z′ factor values and signal-to-background (S/B) ratios were determined by analyzing 48 assay wells for both total and inhibited signals. The signal was read following a standard 60-min (LANCE Ultra) or 30-min (AlphaLISA) incubation with detection reagents. Values in parentheses were obtained following an overnight incubation. See text for details.

SET7/9 methyltransferase assay using a full-length histone H3 substrate

In contrast to TR-FRET, the AlphaLISA technology theoretically allows measuring interactions between binding partners over distances up to 250 nm.²¹ This characteristic makes the Alpha technology a highly suitable tool for developing assays aimed at measuring protein–peptide^30,31 and protein–protein³² interactions, as well as enabling the detection of full-length proteins bearing specific posttranslational modifications.^23,32 The AlphaLISA platform was thus selected for developing a SET7/9 methyltransferase assay using recombinant full-length histone H3 as substrate. The use of detection buffers optimized for short histone H3-derived peptide substrates led to high background levels and a concomitant loss of antibody specificity. Further buffer optimization experiments (not shown) proved that high salt concentration was essential to minimize assay background, whereas the addition of a higher concentration of poly-L-lysine was required to stop the enzymatic reaction. In this regard, the exact mechanism of action of poly-L-lysine as an inhibitor of SET7/9 is not known and was not further studied. However, it could be hypothesized that the addition of a large amount of nonmodified lysine residues will successfully outcompete the ones present on a given substrate, hence bringing its enzymatic modification to a halt. A buffer solution combining both 1 M NaCl and 0.3% poly-L-lysine was thus used to stop the enzymatic reactions prior to the addition of acceptor beads and biotinylated antihistone H3 (C-ter) antibody. With these protocol adjustments in place, development and optimization of the SET7/9 protein assay were resumed through experiments similar to those performed for the SET7/9 assay using biotinylated peptide substrate.

To determine the optimal substrate concentration to be used in the assay, full-length histone H3 titration experiments were performed and a concentration of 300 nM histone H3 protein was selected for further assay optimization (data not shown). Enzyme progress curves were then performed at different SET7/9 concentrations ( Fig. 3A ). These curves showed a decline of SET7/9 activity over time, with almost no residual activity after 15 min. On the basis of these results, we selected a reaction time of 10 min, using an enzyme concentration of 0.3 nM.

Figure 3.

AlphaLISA SET7/9 methyltransferase assay optimization using full-length histone H3 protein substrate. (A) Enzymatic progress curves. Enzymatic progress curves were run at increasing SET7/9 concentrations and stopped at the indicated time points. Protein substrate and S-adenosyl-L-methionine (SAM) concentrations were 300 nM and 100 µM, respectively. (B) Determination of the apparent (app) K_m value for SAM. Enzymatic reactions were carried out at increasing SAM concentrations using 0.3 nM enzyme, 300 nM protein substrate, and a 10-min incubation time. (C) Inhibition of SET7/9 methyltransferase activity. Increasing concentrations of inhibitor were preincubated 10 min with the enzyme prior to initiating the methyltransferase reaction with a mixture of protein substrate and SAM cofactor. Enzymatic reactions were carried out in the presence of 1% DMSO, using 0.3 nM enzyme, 300 nM protein substrate, 10 µM SAM, and a 10-min incubation time. SAH, S-adenosyl-L-homocysteine.

The apparent K_m value for SAM was then evaluated at the selected enzyme concentration and incubation time ( Fig. 3B ). A value of 12 µM was obtained, which is in agreement with literature data (6 µM, Trievel et al.¹⁵; 3.5 µM, Dirk et al.³³). A concentration of 10 µM SAM was selected for the optimized protein assay. Interestingly, this apparent K_m value is considerably higher than the one obtained for the AlphaLISA peptide assay (79 nM), a fact that could be attributable to the different nature of the substrates. Linearity of the optimized enzymatic reaction as a function of time was then confirmed for up to 10 min (r² = 0.941) with an S/B ratio of 29 (data not shown). Optimized assay conditions are summarized in Table 1 .

Concentration–response curves for sinefungin and SAH were performed, and both compounds inhibited SET7/9 activity with IC₅₀ values of 47 µM for sinefungin and 175 µM for SAH ( Fig. 3C ). As expected, IC₅₀ values were higher than those obtained in the SET7/9 methyltransferase peptide assay because a higher SAM concentration was used for the protein assay. However, the same rank order of potency was obtained for the two compounds regardless of the substrate used ( Figs. 2F and 3C ). A Z′ factor value of 0.76 with an S/B ratio of 36 were obtained using 10 mM sinefungin for inhibited samples and, importantly, performance was maintained after an overnight incubation ( Table 2 ). These results confirm the AlphaLISA SET7/9 methyltransferase protein assay robustness as well as its suitability for HTS.

LSD1 demethylase assay using a H3K4me1 peptide substrate

The availability of LANCE Ultra and AlphaLISA reagents specifically detecting unmethylated lysine 4 allowed developing LSD1 demethylase signal increase assays measuring the appearance of the demethylated product. Because LSD1 has been reported to demethylate monomethyl-lysine 4 on histone H3-derived peptides at least 20 residues long,¹⁹ a biotinylated H3K4me1 peptide (residues 1–21) was selected as substrate for these assays. Following titration of the H3K4me1 peptide, a concentration of 200 nM was chosen for LANCE Ultra and 80 nM for AlphaLISA assays (data not shown). Enzyme progress curves showed that signal increased linearly with time for up to 2 h when 2 nM LSD1 was used ( Fig. 4A , B ). On the basis of these results, we selected a 60-min reaction time and a 2-nM LSD1 enzyme concentration for the subsequent experiments with both technologies ( Table 1 ). Under these conditions, S/B ratios were equal to 18 and 292 for LANCE Ultra and AlphaLISA assays, respectively. The redox cofactor FAD is essential for LSD1 activity and is strongly embedded in the amine oxidase domain. As such, FAD addition to the enzyme reaction buffer is not necessary for optimal enzymatic activity as it is already present in recombinant LSD1 enzyme preparations.¹⁰

Figure 4.

LANCE Ultra and AlphaLISA LSD1 demethylase assay optimization using biotinylated H3K4me1 peptide substrate. (A, B) Enzymatic progress curves. Enzymatic progress curves were run at increasing LSD1 concentrations and stopped at the indicated time points. Peptide substrate concentrations were (A) 200 nM for the LANCE Ultra assay and (B) 80 nM for the AlphaLISA assay. (C, D) Inhibition of LSD1 demethylase activity. Increasing concentrations of inhibitor were preincubated 10 min with the enzyme prior to initiating the demethylase reaction with the peptide substrate. Enzymatic reactions were carried out in the presence of 1% DMSO using (C) 2 nM enzyme, 200 nM peptide substrate, and a 60-min incubation time in the LANCE Ultra setup and (D) 2 nM enzyme, 80 nM peptide substrate, and a 60-min incubation time in the AlphaLISA setup.

The antidepressant drug tranylcypromine is a monoamine oxidase inhibitor reported to inhibit the activity of LSD1 demethylase.³⁴ Data in Figure 4C , D indicate that tranylcypromine inhibits LSD1 enzyme activity in a concentration-dependent manner with similar IC₅₀ values of 74 and 32 µM for the LANCE Ultra and AlphaLISA assays, respectively. Given that tranylcypromine is an irreversible covalent inhibitor of enzyme activity, comparison of IC₅₀ values reported herein with those published in the literature has not been performed due to significant differences in assay conditions. Finally, assay robustness was assessed by Z′ factor determination, where inhibited signal was measured using 3 mM tranylcypromine. Values of 0.88 and 0.80 were obtained, with S/B ratios of 20 and 643 for LANCE Ultra and AlphaLISA assays, respectively, demonstrating their suitability for HTS ( Table 2 ).

Discussion

In this study, we presented the development and optimization of LANCE Ultra and AlphaLISA assays for two epigenetic enzymes controlling the methylation state of histone H3 lysine 4 residues. Optimized assays were highly sensitive, requiring low enzyme and substrate concentrations, which confers a significant advantage in terms of cost over less sensitive methods as liquid chromatography/mass spectrometry or enzyme-coupled assays. Importantly, the use of low nanomolar enzyme concentrations can enable structure–activity relationship studies where the use of very low concentrations of small-molecule compounds is required. Our assays also allowed inhibitor profiling and were highly robust, as highlighted by Z′ factor values equal or above 0.7. Notably, assay quality was maintained following an overnight incubation, allowing both online and off-line plate reading.

Although the presented data focused on the development of SET7/9 and LSD1 assays, the same antimark specific detection reagents could also be used for studying other enzymes involved in the modification of the H3K4 residue. For instance, a signal increase SIRT1 assay measuring the deacetylation of H3K4 residues has been optimized using reagents detecting unmodified H3K4 (unpublished data). Similarly, demethylation of an H3K4 trimethylated substrate could also be monitored using either the H3K4me1-2 or unmodified H3K4 detection reagents. In brief, the assay development approach described here is broadly applicable to other epigenetic marks and in fact has been used for developing LANCE Ultra and AlphaLISA reagents detecting H3K9me2, H3K9ac, H3K27ac, H3K27me1-2, H3K27me3, and H3K36me2 (unpublished data). This could aid in the identification of novel small-molecule compounds of pharmacological value targeting important epigenetic enzymes such as EZH2, the Jumonji domain–containing family of demethylases, acetyltransferases such as p300 and pCAF, and deacetylases from the HDAC and sirtuin classes.

Footnotes

All authors are employees of PerkinElmer. None of the authors or PerkinElmer received at any time payment or services from a third party for any aspect of the submitted work. There are no other relationships/conditions/circumstances that present a potential conflict of interest.

Supplementary material for this article is available on the Journal of Biomolecular Screening Web site at .

References

Bannister

A. J.

Kouzarides

Regulation of Chromatin by Histone Modifications. Cell Res. 2011, 21, 381–395.

Shi

Whetstine

J. R.

Dynamic Regulation of Histone Lysine Methylation by Demethylases. Mol. Cell 2007, 25, 1–14.

Chi

Allis

C. D.

Wang

G. G.

Covalent Histone Modifications—Miswritten, Misinterpreted and Mis-erased in Human Cancers. Nat. Rev. Cancer 2010, 10, 457–469.

Urdinguio

R. G.

Sanchez-Mut

J. V.

Esteller

Epigenetic Mechanisms in Neurological Diseases: Genes, Syndromes, and Therapies. Lancet Neurol. 2009, 8, 1056–1072.

Kelly

T. K.

De Carvalho

D. D.

Jones

P. A.

Epigenetic Modifications as Therapeutic Targets. Nat. Biotechnol. 2010, 28, 1069–1078.

Ruthenburg

A. J.

Allis

C. D.

Wysocka

Methylation of Lysine 4 on Histone H3: Intricacy of Writing and Reading a Single Epigenetic Mark. Mol. Cell 2007, 25, 15–30.

Wang

Cao

Xia

Erdjument-Bromage

Borchers

Tempst

Zhang

Purification and Functional Characterization of a Histone H3-Lysine 4-Specific Methyltransferase. Mol. Cell 2001, 8, 1207–1217.

Nishioka

Chuikov

Sarma

Erdjument-Bromage

Allis

C. D.

Tempst

Reinberg

Set9, a Novel Histone H3 Methyltransferase That Facilitates Transcription by Precluding Histone Tail Modifications Required for Heterochromatin Formation. Genes Dev. 2002, 16, 479–489.

Milne

T. A.

Briggs

S. D.

Brock

H. W.

Martin

M. E.

Gibbs

Allis

C. D.

Hess

J. L.

MLL Targets SET Domain Methyltransferase Activity to Hox Gene Promoters. Mol. Cell 2002, 10, 1107–1117.

10.

Shi

Lan

Matson

Mulligan

Whetstine

J. R.

Cole

P. A.

Casero

R. A.

Shi

Histone Demethylation Mediated by the Nuclear Amine Oxidase Homolog LSD1. Cell 2004, 119, 941–953.

11.

Xiao

Jing

Wilson

J. R.

Walker

P. A.

Vasisht

Kelly

Howell

Taylor

I. A.

Blackburn

G. M.

Gamblin

S. J.

Structure and Catalytic Mechanism of the Human Histone Methyltransferase SET7/9. Nature 2003, 421, 652–656.

12.

Metzger

Wissmann

Yin

Muller

J. M.

Schneider

Peters

A. H.

Günther

Buettner

Schüle

LSD1 Demethylates Repressive Histone Marks to Promote Androgen-Receptor-Dependent Transcription. Nature 2005, 437, 436–439.

13.

Reddy

M. A.

Miao

Shanmugam

Yee

J. K.

Hawkins

Ren

Natarajan

Role of the Histone H3 Lysine 4 Methyltransferase, SET7/9, in the Regulation of NF-kappaB-Dependent Inflammatory Genes: Relevance to Diabetes and Inflammation. J. Biol. Chem. 2008, 283, 26771–26781.

14.

Hayami

Kelly

J. D.

Cho

H. S.

Yoshimatsu

Unoki

Tsunoda

Field

H. I.

Neal

D. E.

Yamaue

Ponder

B. A.

Overexpression of LSD1 Contributes to Human Carcinogenesis through Chromatin Regulation in Various Cancers. Int. J. Cancer 2011, 128, 574–586.

15.

Trievel

R. C.

Beach

B. M.

Dirk

L. M.

Houtz

R. L.

Hurley

J. H.

Structure and Catalytic Mechanism of a SET Domain Protein Methyltransferase. Cell 2002, 111, 91–103.

16.

Dhayalan

Dimitrova

Rathert

Jeltsch

A Continuous Protein Methyltransferase (G9a) Assay for Enzyme Activity Measurement and Inhibitor Screening. J. Biomol. Screen. 2009, 14, 1129–1133.

17.

Kubicek

O’Sullivan

R. J.

August

E. M.

Hickey

E. R.

Zhang

Teodoro

M. L.

Rea

Mechtler

Kowalski

J. A.

Homon

C. A.

. Reversal of H3K9me2 by a Small-Molecule Inhibitor for the G9a Histone Methyltransferase. Mol. Cell 2007, 25, 473–481.

18.

Collazo

Couture

J. F.

Bulfer

Trievel

R. C.

A Coupled Fluorescent Assay for Histone Methyltransferases. Anal. Biochem. 2005, 342, 86–92.

19.

Forneris

Binda

Vanoni

M. A.

Battaglioli

Mattevi

Human Histone Demethylase LSD1 Reads the Histone Code. J. Biol. Chem. 2005, 280, 41360–41365.

20.

Hemmilä

LANCE™: Homogeneous Assay Platform for HTS. J. Biomol. Screen. 1999, 4, 303–308.

21.

Ullman

E. F.

Kirakossian

Switchenko

A. C.

Ishkanian

Ericson

Wartchow

C. A.

Pirio

Pease

Irvin

B. R.

Singh

. Luminescent Oxygen Channeling Assay (LOCI): Sensitive, Broadly Applicable Homogeneous Immunoassay Method. Clin. Chem. 1996, 42, 1518–1526.

22.

Eglen

R. M.

Reisine

Roby

Rouleau

Illy

Bosse

Bielefeld

The Use of AlphaScreen Technology in HTS: Current Status. Curr. Chem. Genomics 2008, 1, 2–10.

23.

Pedro

Padros

Beaudet

Schubert

H. D.

Gillardon

Dahan

Development of a High-Throughput AlphaScreen Assay Measuring Full-Length LRRK2(G2019S) Kinase Activity Using Moesin Protein Substrate. Anal. Biochem. 2010, 404, 45–51.

24.

Quinn

A. M.

Allali-Hassani

Vedadi

Simeonov

A Chemiluminescence-Based Method for Identification of Histone Lysine Methyltransferase Inhibitors. Mol. Biosyst. 2010, 6, 782–788.

25.

Kawamura

Tumber

Rose

N. R.

King

O. N.

Daniel

Oppermann

Heightman

T. D.

Schofield

Development of Homogeneous Luminescence Assays for Histone Demethylase Catalysis and Binding. Anal. Biochem. 2010, 404, 86–93.

26.

Zhang

J. H.

Chung

T. D.

Oldenburg

K. R.

A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J. Biomol. Screen. 1999, 4, 67–73.

27.

Fuchs

S. M.

Krajewski

Baker

R. W.

Miller

V. L.

Strahl

B. D.

Influence of Combinatorial Histone Modifications on Antibody and Effector Protein Recognition. Curr. Biol. 2011, 21, 53–58.

28.

Fernando

S. A.

Wilson

G. S.

Studies of the ‘Hook’ Effect in the One-Step Sandwich Immunoassay. J. Immunol. Methods 1992, 151, 47–66.

29.

Ibañez

Mcbean

J. L.

Astudillo

Y. M.

Luo

An Enzyme-Coupled Ultrasensitive Luminescence Assay for Protein Methyltransferases. Anal. Biochem. 2010, 401, 203–210.

30.

Quinn

A. M.

Bedford

M. T.

Espejo

Spannhoff

Austin

C. P.

Oppermann

Simeonov

A Homogeneous Method for Investigation of Methylation-Dependent Protein-Protein Interactions in Epigenetics. Nucleic Acids Res. 2010, 38, e11.

31.

Wigle

T. J.

Herold

J. M.

Senisterra

G. A.

Vedadi

Kireev

D. B.

Arrowsmith

C. H.

Frye

S. V.

Janzen

W. P.

Screening for Inhibitors of Low-Affinity Epigenetic Peptide-Protein Interactions: An AlphaScreen-Based Assay for Antagonists of Methyl-lysine Binding Proteins. J. Biomol. Screen. 2010, 15, 62–71.

32.

Arcand

Roby

Bosse

Lipari

Padros

Beaudet

Marcil

Dahan

Single-Well Monitoring of Protein-Protein Interaction and Phosphorylation-Dephosphorylation Events. Biochemistry 2010, 49, 3213–3215.

33.

Dirk

L. M.

Flynn

E. M.

Dietzel

Couture

J. F.

Trievel

R. C.

Houtz

R. L.

Kinetic Manifestation of Processivity during Multiple Methylations Catalyzed by SET Domain Protein Methyltransferases. Biochemistry 2007, 46, 3905–3915.

34.

Schmidt

D. M.

McCafferty

D. G.

Trans-2-Phenylcyclopropylamine is a Mechanism-Based Inactivator of the Histone Demethylase LSD1. Biochemistry 2007, 46, 4408–4416.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.20 MB

Development of Homogeneous Nonradioactive Methyltransferase and Demethylase Assays Targeting Histone H3 Lysine 4

Abstract

Keywords

Introduction

Materials and Methods

LANCE Ultra enzymatic assays

AlphaLISA enzymatic assays

SET7/9 and LSD1 assays with biotinylated histone H3 peptide substrates

SET7/9 assay using a recombinant histone H3 protein substrate

Data analysis

Results

Assay development workflow

SET7/9 methyltransferase assay using a histone H3 peptide substrate

SET7/9 methyltransferase assay using a full-length histone H3 substrate

LSD1 demethylase assay using a H3K4me1 peptide substrate

Discussion

Footnotes

References

Supplementary Material