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

Gauthier, Nancy; Caron, M.; Pedro, Liliana; Arcand, Mathieu; Blouin, Julie; Labonté, Anne; Normand, Claire; Paquet, Valérie E.; Rodenbrock, Anja; Roy, Marjolaine; Rouleau, Nathalie; Beaudet, Lucille; Padrós, Jaime; Rodrı́guez-Suárez, Roberto

doi:10.1177/1087057111416659

Cited by 39 publications

(53 citation statements)

References 34 publications

(56 reference statements)

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“…IC 50 values were also fairly consistent with literature values of 359 nM for sinefungin and 16 µM for SAH. 3 Both compounds showed slightly higher potency in our assay, which could be attributed to transfer of the compound via the Echo liquid handler, eliminating potential loss of compounds, particularly those with hydrophobic characteristics, from sticking to tips. [22][23][24][25] Z′ factors for the miniaturized assays were excellent-0.82 and above in the miniaturized assay volumes, in both 384-and 1536-well formats, compared to 0.73 in the manually pipetted 25 µL 384-well assay.…”

Section: Alphalisa Methyltransferase Assaymentioning

confidence: 99%

“…The plates were sealed, centrifuged for 1 s at 1000 rpm, and then incubated for 30 min at RT. Conditions were adapted from Gauthier et al 3 Anti-methyl-Histone H3 Lysine 4 (H3K4me1-21) acceptor beads were then transferred to the assay plates with the Echo 525 liquid handler to a final concentration of 20 µg/ mL. Plates were resealed, briefly centrifuged at 1000 rpm, shaken for 15 s on a Teleshake 384 (Thermo Scientific, Waltham, MA) magnetic shaker, and then incubated for 60 min at RT in the dark.…”

Section: Alphalisa Methyltransferase Assay Set7/9 Inhibitor Titrationmentioning

confidence: 99%

“…1,2 Epigenetic changes are thought to play a role in many diseases, including cancer, 2 cardiovascular disease, neuropathologies, inflammation, and diabetes. 1,3 A class of enzymes known as methyltransferases play a role in histone modification, which affects the regulation of gene transcription. 1 These enzymes are therefore intriguing targets for drug discovery.…”

Section: Introductionmentioning

confidence: 99%

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Miniaturization of High-Throughput Epigenetic Methyltransferase Assays with Acoustic Liquid Handling

Edwards¹,

Lesnick²,

Wang³

et al. 2016

SLAS Technology

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Epigenetics continues to emerge as an important target class for drug discovery and cancer research. As programs scale to evaluate many new targets related to epigenetic expression, new tools and techniques are required to enable efficient and reproducible high-throughput epigenetic screening. Assay miniaturization increases screening throughput and reduces operating costs. Echo liquid handlers can transfer compounds, samples, reagents, and beads in submicroliter volumes to high-density assay formats using only acoustic energy-no contact or tips required. This eliminates tip costs and reduces the risk of reagent carryover. In this study, we demonstrate the miniaturization of a methyltransferase assay using Echo liquid handlers and two different assay technologies: AlphaLISA from PerkinElmer and EPIgeneous HTRF from Cisbio.

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Section: Alphalisa Methyltransferase Assaymentioning

confidence: 99%

Section: Alphalisa Methyltransferase Assay Set7/9 Inhibitor Titrationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Miniaturization of High-Throughput Epigenetic Methyltransferase Assays with Acoustic Liquid Handling

Edwards¹,

Lesnick²,

Wang³

et al. 2016

SLAS Technology

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“…10 Recently, homogeneous nonradioactive antibody-based assays (LANCE Ultra and AlphaLISA) have been described. 11 During assay development, we investigated the use of a similar peptide-based AlphaScreen method. However, the screen described here, based on a nonwash radiometric FlashPlate assay previously described for HAT activity, 12 provided a greater signal window.…”

Section: Optimization Of Pmt Activity Assaymentioning

confidence: 99%

Small-Molecule Inhibitors of the Protein Methyltransferase SET7/9 Identified in a High-Throughput Screen

et al. 2012

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Aberrant expression of chromatin-modifying enzymes (CMEs) is associated with a range of human diseases, including cancer. CMEs are now an important target area in drug discovery. Although the role that histone and protein (lysine) methyltransferases (PMTs) play in the regulation of transcription and cell growth is increasingly recognized, few smallmolecule inhibitors of this class of enzyme have been reported. Here we describe an assay suitable for primary compound screening for the identification of PMT inhibitors. The assay followed the methylation of histones in the presence of the PMT SET7/9 and the radioactive cofactor S-adenosyl-methionine using scintillating microplates (FlashPlate) and was used to screen approximately 65 000 compounds (% coefficient of variation = 10%; Z′ = 0.6). The hits identified from a library of more than 63 000 diverse small molecules included a series of rhodanine compounds with micromolar activity. A screen of the National CancerInstitute Diversity Set (2000 compounds) identified an orsein derivative that inhibited SET7/9 (~20 µM) and showed modest growth inhibition associated with the expected cellular phenotype of reduced histone methylation in a human tumor cell line. The assay represents a useful tool for the identification of inhibitors of PMT activity.

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“…Initially, the compounds were screened in a FRET-based LANCE ultra G9a histone H3-lysine N-methyltransferase assay 26 that measures the dimethylation of a biotinylated histone H3 (1−21) peptide at lysine 9. Previous studies have suggested that EHMT1 and EHMT2 both can mono-, di-, and trimethylate histone H3 at lysine 9.…”

mentioning

confidence: 99%

Analogues of the Natural Product Sinefungin as Inhibitors of EHMT1 and EHMT2

Devkota

Lohse

Liu

et al. 2014

ACS Med. Chem. Lett.

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A series of analogues of the natural product sinefungin lacking the amino acid moiety was synthesized and probed for their ability to inhibit EHMT1 and EHMT2. This study led to inhibitors 3b and 4d of methyltransferase activity of EHMT1 and EHMT2 and it demonstrates that such analogues constitute an interesting scaffold to develop selective methyltransferase inhibitors. Surprisingly, the inhibition was not competitive toward AdoMet. KEYWORDS: Sinefungin, methyltransferase inhibitors, EHMT1, EHMT2 H istone modifications have a central role in regulating gene expression 1 and it has been suggested that they constitute an epigenetic code.2 The enzymes capable of transferring acetyl, methyl, and other groups are referred to as writers, whereas enzymes responsible for removing the modifications are referred to as erasers. Furthermore, a range of domains capable of recognizing the modified substrates are termed readers. Protein lysine methyltransferases (PKMTs) are a group of epigenetic writers, responsible for the transfer of one to three methyl groups from S-adenosyl-L-methionine(s) (AdoMet, 1, Figure 1) to the ε-amino group of the target lysine residues in histone tails 3 and some nonhistone targets. 4 To date, more than 50 PKMTs have been identified and EHMT1 (GLP, G9a like proteins) and EHMT2 (G9a) are among the most well-studied PKMTs. They are capable of mono-and dimethylating lysine 9 residue of histone 3 (H3K9) in euchromatin 5 but have also been found to methylate nonhistone substrates such as tumor suppressor p53, 6 Wiz, and reptin. 7 EHMT1/2-mediated methylation has been implicated to be pivotal for processes such as embryo development, 8 germ cell development and meiosis, 9 and lymphocyte functioning, 10,11 as well as cognition and adaptive behaviors.12 Genetic variations of EHMT1/2 have been associated with human diseases such as cancer, inflammatory diseases, and neurogenerative disorders.12−17 Removal of EHMT1 causes subtelomeric deletion syndrome with severe mental retardation, 13,14 along with reduced locomotor activity and exploration.12 Increased expression of EHMT2 has been associated with lung, prostate, and hepatocellular carcinoma. 15,16 Interestingly, studies also suggest that EHMT2 deficiency impairs immune response 6 while knock down of EHMT1/2 causes severe growth defects inducing embryonic lethality.8 There has been a growing interest to identify potent inhibitors of these enzymes. Since the discovery of the first PKMT inhibitor, chaetocin, 17 in 2005,

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

Cited by 39 publications

References 34 publications

Miniaturization of High-Throughput Epigenetic Methyltransferase Assays with Acoustic Liquid Handling

Miniaturization of High-Throughput Epigenetic Methyltransferase Assays with Acoustic Liquid Handling

Small-Molecule Inhibitors of the Protein Methyltransferase SET7/9 Identified in a High-Throughput Screen

Analogues of the Natural Product Sinefungin as Inhibitors of EHMT1 and EHMT2

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