EPIgeneous Methyltransferase assay: a new HTRF Universal SAH detection assay to assess methyltransferase activity TM T. Roux1, N. Douayry1, S. Junique1, L. Sergeant1, G. Donsimoni1, E. Bourrier1, C.P. Walsh3, R.F. Smith3, K.T. Howitz3, R. LaRose2 , F. Degorce1 and E. Trinquet1 1 Cisbio Bioassays, Codolet, France | 2Cisbio US, Bedford, MA, USA | 3 Reaction Biology Corp., Malvern, PA, USA Introduction Epigenetics is one of the fastest growing Enzymes and related substrates validated fields in research because of their potential to Methyltransferases Substrates identify new drugs for therapeutic targets and G9a, EZH2 complex, SET7/9 H3 (1-21) or (1-50) peptide especially in oncology. A key focus of epigenetics research is on methyltransferases because PRMT1, SET8 H4 (1-25) peptide of their abundance and ability to methylate DOT1L, SETD2, MLL1 complex, Oligonucleosome NSD1, NSD2 histones and various other substrates. Cisbio SET7/9 p53 has developped a universal methyltransferase mix and read assay using HTRF technology DNMT1 DNA (poly(dI-dC)) that provides the sensitivity and flexibility of hN7, WNV, NSP14, NSP10-16 RNA substrates. This assay, which directly quantifies COMT Dopamine S-5’-adenosyl-L-homocysteine (SAH), has been successfully validated on a variety of enzymes G9a Recombinant full H3 and substrates (see table). This study presents the principle, protocol and specifications of the assay as well as assay performances and optimization on an enzyme of interest: DOT1L with nucleosome as the substrate. Assay Principle and reagents SAM SAH 337 nm FRET 665 nm 620 nm Me NO FRET SAH-d2 Anti SAH cryptate SAH enzyme The assay consists of an enzymatic and Enzymatic step (10 µl) detection step. The methyltransferase activity is + 42 µlµl enzyme compounds assessed by measuring the conversion of SAM (or enzymatic buffer) + 2 µl SAM (S-5’adenosyl-L-methionine) to SAH. In order + 2 µl substrate to directly measure SAH release, an anti SAH antibody labeled with terbium cryptate and a SAH-d2 tracer are used. The SAH released by the enzymatic reaction competes with the SAH-d2 labeled leading to a decrease of the HTRF signal. Oligonucleosome and all methyltransferase Enzymatic step enzymes, but those for RNA and dopamine substrates, were provided by Reaction Biology Corp. Detection step (20µl) + 2 µl Detection buffer 1 + (10min @ RT) + 4 µl SAH-d2 + 4 µl anti-SAH Cryptate read Incubate RT or 30°C Incubate 1h at RT Detection step DOT1L / Oligonucleosome assay 1. DOT1L titration 2. Oligonucleosome and SAM titrations Detection of DOT1L specific activity and identification of optimal enzyme concentration. 20,000 16,000 Human recombinant DOT1L was serially diluted to the indicated concentrations and the assay carried out with 10ng/μl (= 77 nM) oligonucleosome as substrate and 2 μM SAM for 2 h at 30°C. The negative controls (no SAM or no nucleosome) show the measurement of the enzymatic specific activity. A DOT1L concentration of 4.5 nM (EC80) was selected for further experiments. This concentration lead to 10% conversion of SAM into SAH EC50= 0.4 nM 12,000 SAM + Nucleosome SAM alone Nucleosome alone 8,000 4,000 0 -14 -12 -10 -8 -6 Determination of optimal substrate and SAM concentrations. 20,000 HTRF Ratio (665/620) HTRF Ratio (665/620) 24,000 12,000 SAM 4 µM SAM 2 µM SAM 1 µM SAM 0.5 µM 8,000 4,000 10 -11 10 -10 3. Inhibitor titration EPIgeneous TM methyltransferase assay validated by measuring activity of SGC0946 inhibitor. This assay was performed using 0.5 μM SAM (EC40), 77 nM oligonucleosome (EC80) and 4.5 nM DOT1L (EC80). The enzymatic reaction was stopped with the detection reagents after a 2 h incubation at 30°C. 12,000 •IC50 of SGC0946 is in good agreement with the literature (2). IC50= 0.01nM 10 -7 10 -6 •As expected, BIX01294 which is a G9a selective inhibitor does not inhibit DOT1L. 4,000 -12 -10 -8 -6 -4 [Inhibitor] (logM) Validated Methyltransferases 100 80 60 40 20 0 -20 -11 -10 -9 -8 [Enzyme] (LogM) 10,000 This assay was performed using 0.5 μM SAM (EC40), 77 nM oligonucleosome (EC80) and 4.5 nM DOT1L (EC80). Z' = 0.57 S/B = 2.6 8,000 Assay robustness demonstrated through Z’ factor determination. The Z’ factor was obtained with biological balanced conditions and underlines the robustness of the assay and its suitability for HTS in biological relevant conditions. 6,000 4,000 No inhibitor 2,000 0 5 10 15 20 25 30 35 40 nb of well Assay sensitivity 120 -12 12,000 • Controls of inhibitors without enzyme show that they do not affect the detection reagents. -2 SGC0946 @ 10nM -7 -6 -5 9 MLL1 complex / Nucleosome DOT1L / Nucleosome SETD2 / Nucleosome G9a / H3 peptide EZH2 complex / H3 peptide PRMT1 / H4 peptide SET7-9 / H3 peptide SET7-9 / p53 DNMT1 / DNA COMT / Dopamine hN7 / RNA WestNile / RNA NSP10-16 / RNA NSP14 / RNA The HTRF EPIgeneousTM Methyltransferase assay has been validated with a large set of enzymes and different substrates, as listed in the introduction table. The graph above summarizes the activities detected for a selection of enzyme/substrate combinations. Enzymatic reactions were carried out at RT or 30°C for 1 to 2 h with 0.5 to 10 µM of SAM and related substrates according to enzymes tested. Reactions were stopped with detection reagents addition in 384 low volume plate (20 µL) and then read on Pherastar FS (flashlamp) after 1 h incubation. conclusion We have developped a universal methyltransferase mix and read assay using HTRF technology that provides: • The flexibility of substrates. The assay is validated with a large set of methyltransferase sub families so far: PKMT and PRMT on histone peptides, nucleosomes or other proteins (p53) ; DNA MT ; RNA MT and COMT with dopamine. 8 S/B (= Assay window) 8,000 Enzyme activity (%) 10 -8 14,000 HTRF Ratio (665/620) BIX01294 BIX01294 w/o enzyme 16,000 HTRF Ratio (665/620) 10 -9 4. Z’ factor SGC0946 SGC0946 w/o enzyme -14 0.5 µM of SAM, a concentration below reported Km of 0.67 µM (1), is selected for subsequent experiments. For oligonucleosome, 77 nM (EC80) is selected for further tests. [Oligoucleosome] logM log [Enzyme] M -16 Oligonucleosome was titrated with several concentrations of SAM. DOT1L is used at 4.5 nM and incubated with SAM and substrate 2 h at 30°C. 16,000 7 SAM 200 µM SAM 40 µM SAM 20 µM SAM 10 µM SAM 2 µM SAM 0.4 µM 6 5 4 3 2 1 0 1% 4% 11% 33% 100% SAM / SAH conversion (%) The graph represents the assay windows obtained with a range of SAM concentrations and at different conversion percentages of SAM into SAH. The assay has been optimized to be suitable for a large range of SAM concentrations in the enzymatic step (0.4 – 200 µM). With these concentrations of SAM, the assay is able to assess the enzymatic activity with 4 to 100% turn over of the enzyme (hence 4 to 100% conversion of SAM into SAH). • Enough sensitivity to work in biological relevant conditions (avoiding enzyme saturation) as shown with DOT1L / nucleosome assay example. This study demonstrates that all enzymes and substrates developed by Reaction Biology Corp. are fully compatible with this new Universal SAH assay, and enable the optimization of robust assay formats for the study and the screening of methyltransferase targets. • Non radioactive assay with high sensitivity by measuring the release of SAH. Avoid false positives and counter screening due to coupling enzymes and indirect measurement format. References • Flexible enzymatic assay conditions: large SAM concentration range compatibility (0.4 – 200 µM) (2) Yu et al. Nature commun., 2012 (1) Richon et al. Chem Biol Drug Des, 2011 &Yao et al. J Am Chem Soc, 2011 Europe and other countries +33(0)466 796 705 U.S. and Canada 1-888-963-4567 China +86 10 8080 9288 www.cisbio.com