title: Development of a universal radioactive DNA methyltransferase inhibition test for high-throughput screening and mechanistic studies.
abstract: DNA methylation is an important epigenetic mark in eukaryotes, and aberrant pattern of this modification is involved in numerous diseases such as cancers. Interestingly, DNA methylation is reversible and thus is considered a promising therapeutic target. Therefore, there is a need for identifying new small inhibitors of C5 DNA methyltransferases (DNMTs). Despite the development of numerous in vitro DNMT assays, there is a lack of reliable tests suitable for high-throughput screening, which can also give insights into inhibitor mechanisms of action. We developed a new test based on scintillation proximity assay meeting these requirements. After optimizing our assay on human DNMT1 and calibrating it with two known inhibitors, we carried out S-Adenosyl-l-Methionine and DNA competition studies on three inhibitors and were able to determine each mechanism of action. Finally, we showed that our test was applicable to 3 other methyltransferases sources: human DNMT3A, bacterial M.SssI and cellular extracts as well.
authors: CNRS-Pierre Fabre USR n° 3388 ETaC, CRDPF, 3 Avenue H. Curien, 31035 Toulouse Cedex 01, France.
authors: Christina
authors: C
authors: Gros
authors: Laura
authors: L
authors: Chauvigné
authors: Anaïs
authors: A
authors: Poulet
authors: Yoann
authors: Y
authors: Menon
authors: Frédéric
authors: F
authors: Ausseil
authors: Isabelle
authors: I
authors: Dufau
authors: Paola B
authors: PB
authors: Arimondo
mesh_terms: D002457:Cell Extracts; D002460:Cell Line; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors*; D019175:DNA Methylation / Q000187:drug effects; D004121:Dimethyl Sulfoxide; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D012997:Solvents; D014316:Tritium
chemical_list: D002457:Cell Extracts; D004791:Enzyme Inhibitors; D012997:Solvents; D014316:Tritium; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; D004121:Dimethyl Sulfoxide
references: Semin Cell Dev Biol. 2010 Apr;21(2):238-46
references: 19892028
references: JAMA. 2008 Mar 19;299(11):1345-50
references: 18349095
references: Curr Med Chem. 2010;17(33):4052-71
references: 20939822
references: Cell. 2007 Feb 23;128(4):683-92
references: 17320506
references: N Engl J Med. 2003 Nov 20;349(21):2042-54
references: 14627790
references: Anal Biochem. 2003 Sep 1;320(1):1-12
references: 12895464
references: Epigenetics. 2011 Nov;6(11):1373-7
references: 22048250
references: Science. 2011 Feb 25;331(6020):1036-40
references: 21163962
references: J Nutr Biochem. 2011 Mar;22(3):242-51
references: 20573497
references: Biochem Pharmacol. 2011 Feb 1;81(3):364-78
references: 21044612
references: Nat Genet. 1998 Jul;19(3):219-20
references: 9662389
references: Anal Biochem. 2010 Jun 15;401(2):203-10
references: 20227379
references: J Biol Chem. 1977 Oct 25;252(20):7265-72
references: 332688
references: J Biomol Screen. 1999;4(2):67-73
references: 10838414
references: Biochemistry. 1998 Oct 27;37(43):15162-9
references: 9790680
references: J Mol Biol. 2001 Jun 22;309(5):1201-8
references: 11399089
references: Anal Biochem. 2012 Apr 1;423(1):171-7
references: 22342622
references: Chembiochem. 2012 Jan 2;13(1):157-65
references: 22170584
references: Cancer Cell. 2012 Mar 20;21(3):430-46
references: 22439938
references: Chembiochem. 2011 Jun 14;12(9):1337-45
references: 21633996
references: J Mol Biol. 1997 Jan 10;265(1):56-67
references: 8995524
references: PLoS Genet. 2012 Jun;8(6):e1002750
references: 22761581
references: Nature. 2007 Sep 13;449(7159):248-51
references: 17713477
references: Biochemistry. 2010 Apr 13;49(14):2999-3008
references: 20210320
references: Biol Reprod. 2009 Jul;81(1):155-64
references: 19228594
references: Anal Chem. 2011 Jun 15;83(12):4996-5004
references: 21545095
references: PLoS One. 2010 Aug 24;5(8):e12388
references: 20808780
references: Curr Med Chem. 2008;15(1):92-106
references: 18220765
references: Biochemistry. 2005 Jul 12;44(27):9472-85
references: 15996102
references: Methods Biochem Anal. 2005;46:1-265
references: 16350889
references: Nat Genet. 2007 Mar;39(3):391-6
references: 17322882
references: J Biol Chem. 1999 Nov 12;274(46):33002-10
references: 10551868
references: Chem Biol. 2012 May 25;19(5):572-8
references: 22633409
references: Nucleic Acids Res. 1997 Nov 15;25(22):4666-73
references: 9358180
references: Curr Neurol Neurosci Rep. 2011 Oct;11(5):464-73
references: 21671162
references: Chembiochem. 2011 Jan 24;12(2):206-22
references: 21243710
references: Carcinogenesis. 2010 Jan;31(1):27-36
references: 19752007
references: Science. 2003 Apr 18;300(5618):489-92
references: 12702876
references: J Biol Chem. 2005 Dec 9;280(49):40749-56
references: 16230360
references: Nat Chem Biol. 2011 Jul 10;7(8):566-74
references: 21743462
references: Science. 2003 Apr 18;300(5618):455
references: 12702868
references: EMBO J. 2002 Aug 1;21(15):4183-95
references: 12145218
references: Anal Biochem. 2008 Feb 15;373(2):296-306
references: 18028865
references: Nat Rev Genet. 2010 Apr;11(4):285-96
references: 20300089
references: Science. 2012 Feb 10;335(6069):709-12
references: 22323818
references: J Biol Chem. 1999 Nov 12;274(46):33011-9
references: 10551869
references: Biochimie. 2012 Nov;94(11):2280-96
references: 22967704
references: Hum Mol Genet. 2000 Oct;9(16):2395-402
references: 11005794
references: Proc Natl Acad Sci U S A. 1999 May 25;96(11):6107-12
references: 10339549
references: J Mol Biol. 1998 Feb 6;275(5):747-58
references: 9480766
references: Eur J Biochem. 2002 Oct;269(20):4981-4
references: 12383256
references: Bioorg Chem. 2004 Aug;32(4):234-43
references: 15210338
references: Mol Cell Biol. 2002 Jan;22(2):480-91
references: 11756544
references: Mol Cell Biol. 2003 Aug;23(16):5594-605
references: 12897133
references: Mol Cell. 2010 Jan 15;37(1):46-56
references: 20129054
references: ACS Chem Biol. 2013 Mar 15;8(3):543-8
references: 23294304
references: J Biol Chem. 2005 May 6;280(18):17986-91
references: 15757890