title: Development and validation of a generic fluorescent methyltransferase activity assay based on the transcreener AMP/GMP assay.
abstract: 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 a high-throughput screening (HTS) environment or are applicable to a limited number of enzymes. The authors developed a generic methyltransferase assay method using fluorescent immunodetection of adenosine monophosphate (AMP), which is formed from the MT reaction product S-adenosylhomocysteine in a dual-enzyme coupling step. The detection range of the assay; its suitability for HTS, including stability of reagents following dispensing and after addition to reactions; and the potential for interference from drug-like molecules was investigated. In addition, the use of the assay for measuring inhibitor potencies with peptide or intact protein substrates was examined through pilot screening with selected reference enzymes including HMT G9a. By combining a novel enzymatic coupling step with the well-characterized Transcreener AMP/GMP assay, the authors have developed a robust HTS assay for HMTs that should be broadly applicable to other types of methyltransferases as well.
authors.affiliation: 1BellBrook Labs, Madison, Wisconsin.
authors.forename: Tony A
authors.initials: TA
authors.lastname: Klink
authors.forename: Matt
authors.initials: M
authors.lastname: Staeben
authors.forename: Kim
authors.initials: K
authors.lastname: Twesten
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Kopp
authors.forename: Meera
authors.initials: M
authors.lastname: Kumar
authors.forename: Rebecca Schall
authors.initials: RS
authors.lastname: Dunn
authors.forename: Cori A
authors.initials: CA
authors.lastname: Pinchard
authors.forename: Karen M
authors.initials: KM
authors.lastname: Kleman-Leyer
authors.forename: Martin
authors.initials: M
authors.lastname: Klumpp
authors.forename: Robert G
authors.initials: RG
authors.lastname: Lowery
mesh_terms: D000249:Adenosine Monophosphate / Q000276:immunology / Q000378:metabolism*; D000918:Antibody Specificity; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D006157:Guanosine Monophosphate / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D057230:Limit of Detection; D012435:S-Adenosylhomocysteine / Q000737:chemistry* / Q000378:metabolism; D054852:Small Molecule Libraries
chemical_list: D005456:Fluorescent Dyes; D006657:Histones; D054852:Small Molecule Libraries; D000249:Adenosine Monophosphate; D006157:Guanosine Monophosphate; D012435:S-Adenosylhomocysteine; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; C540312:SETD7 protein, human