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Development and Validation of a Generic Fluorescence Methyltransferase Activity Assay Using the Transcreener® AMP2/GMP2 Assay Tony A. Klink, Matt Staeben, Rebecca Josvai, Meera Kumar, Karen Kleman-Leyer, and Robert G. Lowery BellBrook Labs, LLC, Madison, WI, USA 150 100 1 µM 5 µM 10 µM 50 µM 50 0 0 5 10 15 The Transcreener® EPIGEN Methyltransferase Assay Principle. SAH produced by the target methyltransferase is converted to AMP by coupling enzymes which allows homogenous fluorescent detection using the Transcreener ® AMP2/GMP2 Assay. B) 100 125 ∆ mP ∆ mP 100 150 ∆ mP 125 50 100 0 0 5 10 15 20 25 0 % Conversion 15 minutes 30 minutes 1 hour 2 hours 5 10 15 20 25 % Conversion 5 hours 8 hours 48 hours 0 Hour 2.5 Hours 4.5 Hours 6.5 Hours SAM/SAH standard curves (10 µM initial SAM) were used to examine A) equilibration and stability of signal after addition to coupling enzyme reactions and B) stability of pre-combined detection reagents prior to addition to the plate. Both studies were performed at room temperature. 0.35 0.70 0.80 0.78 0.86 0.85 0.85 0.85 0.87 0.87 0.89 0.84 0.88 0.87 0.89 0.93 0.93 0.91 0.91 75 50 50 Set7/Set9 25 25 G9a SUV39H1 0 8.5 Hours 14 Hours 19 Hours Figure 3. Signal and Reagent Stability for >16 hours. 50 µM 0.06 0.53 0.72 0.71 0.83 0.84 0.87 0.86 0.88 0.89 0.91 75 25 0 30 10 µM -0.49 0.04 0.43 0.59 0.73 0.72 0.82 0.82 0.86 0.83 0.85 A) Standard curves mimic MT reactions (formation of SAH from SAM) show large assay window (>100 mP signal) at less than 20% SAM conversion over the full range of initial SAM concentrations tested. B) Table with Z’ values at percent SAM conversion showing the lower limit of detection (yellow) and initial values that are ≥0.5 (green). 150 50 25 5 µM -1.7 -1.3 -0.22 0.11 0.49 0.50 0.70 0.72 0.79 0.76 0.80 Figure 2. Initial Velocity Detection from 1 µM to 50 µM SAM. 175 75 20 1 µM % SAM Conversion Figure 1. 125 100 % SAM Conversion 0.5 1.0 2.0 3.0 5.0 7.5 10 15 20 25 30 50 100 200 200 0 0 1000 2000 3000 4000 0 50 100 150 200 250 300 350 [G9a] ng/mL 5000 [Enzyme] ng/mL Figure 4. Universal HMT Detection. Enzyme titrations demonstrate dose responses for three HMTs, Set7/Set9, G9a, and SUV39H1 using Histone H3 peptide (1-25) at the SAM Km (6 µM, 2 µM, and 12 µM, respectively). HMT reactions were run for 60 minutes followed by addition of quench and detection reagents including the SAM-SAH ® 2 coupling enzyme mixture and the Transcreener AMP /GMP2 monoclonal antibody and tracer. without acceptor substrate 1 µM Histone H3 (full length) 10 µM Histone H3 Peptide 1-25 100 µM Histone H3 Peptide 1-25 Figure 5. Accommodation of Different Acceptor Substrates. G9a histone methyltransferase reactions were performed with native protein (1 µM) or peptide substrates (10 µM or 100 µM) at 1 µM SAM. Reactions were processed as described in Figure 4. Conclusions 6 3 SAH, pmol 4 2 • Excellent Z’ values and >100 mP signal are achieved using [SAM] from 1 µM to 50 µM under initial velocity conditions. 3 • Greater than 16 hour deck and signal stability ensures compatibility with automated HTS infrastructure. 2 sinefungin chaetocin 1 1 0 • The Transcreener® EPIGEN Methyltransferase Assay combines coupling enzymes with the well characterized Transcreener® AMP2/GMP2 FP reagents to enable universal homogenous methyltransferase enzyme detection. 4 234 ng/mL 117 ng/mL 59 ng/mL 29 ng/mL 14.6 ng/mL 5 SAH, pmol ∆ mP A) B) A) ∆ mP Overview Methylation is a ubiquitous covalent modification used to control the function of diverse biomolecules including hormones, neurotransmitters, xenobiotics, proteins, nucleic acids and lipids. Histone methyltransferases (HMTs) are currently of high interest as drug targets because of their role in epigenetic regulation, however most HMT assay methods are either not amenable to an HTS environment or are applicable to a limited number of enzymes. We developed a generic methyltransferase assay method using fluorescent immunodetection of AMP, which is formed from the MT reaction product S-adenosylhomocysteine in a dual enzyme coupling step. The assay format shows >100 mP signal with Z’>0.5 at initial rate conditions for 1 µM to 50 µM SAM. The suitability for HTS is demonstrated using 384-well plates with >16 hour deck (prior to plate addition) and signal (stability after plate addition) stability at room temperature. The activity of three HMTs (G9a, Set7/Set9, SUV39H1) were followed with using histone H3 peptides while G9a activity was assessed using both peptide and full length histone H3. In addition, inhibitor potencies were determined with G9a HMT. By combining a novel enzymatic coupling step with the well characterized Transcreener® AMP2/GMP2 Assay, we have developed a robust HTS assay for HMTs which should be broadly applicable to other types of methyltransferases as well. • The universal nature of the assay is demonstrated using three HMTs with either peptide or full-length protein substrates in a single assay. • Inhibitor potency measurements were demonstrated using G9a HMT and two known HMT inhibitors, sinefungin and chaetocin. 0 10 20 30 40 Time (minutes) 50 Figure 6. G9a Histone Methyltransferase Initial Rates. 60 Initial velocity conditions were determined using saturating histone H3 peptide (1-25) of 50 µM with [SAM] at the Km = 2 µM. Quantity of SAH was determined using a 2 µM SAM/SAH standard curve as shown in Figure 2. 0 0.1 1 10 100 1000 [Inhibitor] µM Figure 7. Pharmacology. Dose response curves were generated for G9a methyltransferase sensitivity with known inhibitors (chaetocin and sinefungin). The IC50 values for sinefungin and chaetocin were 4.7 and 770 µM, respectively. The G9a enzyme reactions progressed to 25% SAM conversion with a Z’ >0.5. Acknowledgements Funding for this work was provided by the National Institute of General Medical Sciences grant #R44GM073290 © 2011 BellBrook Labs. All Rights Reserved. BellBrook Labs, 5500 Nobel Drive, Suite 250, Madison, WI, 53711 866.313.7881 or 608.443.2400