title: Accessing protein methyltransferase and demethylase enzymology using microfluidic capillary electrophoresis.
abstract: The discovery of small molecules targeting the >80 enzymes that add (methyltransferases) or remove (demethylases) methyl marks from lysine and arginine residues, most notably present in histone tails, may yield unprecedented chemotherapeutic agents and facilitate regenerative medicine. To better enable chemical exploration of these proteins, we have developed a highly quantitative microfluidic capillary electrophoresis assay to enable full mechanistic studies of these enzymes and the kinetics of their inhibition. This technology separates small biomolecules, i.e., peptides, based on their charge-to-mass ratio. Methylation, however, does not alter the charge of peptide substrates. To overcome this limitation, we have employed a methylation-sensitive endoproteinase strategy to separate methylated from unmethylated peptides. The assay was validated on a lysine methyltransferase (G9a) and a lysine demethylase (LSD1) and was employed to investigate the inhibition of G9a by small molecules.
authors.affiliation: Center for Integrative Chemical Biology and Drug Discovery Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, The University of North Carolina, CB #7363, 120 Mason Farm Road, Chapel Hill, NC 27599-7363, USA.
authors.forename: Tim J
authors.initials: TJ
authors.lastname: Wigle
authors.forename: Laurel M
authors.initials: LM
authors.lastname: Provencher
authors.forename: Jacqueline L
authors.initials: JL
authors.lastname: Norris
authors.forename: Jian
authors.initials: J
authors.lastname: Jin
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Brown
authors.forename: Stephen V
authors.initials: SV
authors.lastname: Frye
authors.forename: William P
authors.initials: WP
authors.lastname: Janzen
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D004353:Drug Evaluation, Preclinical; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods*; D056466:Histone Demethylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011495:Histone-Lysine N-Methyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008745:Methylation; D046210:Microfluidic Analytical Techniques; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D054852:Small Molecule Libraries / Q000378:metabolism / Q000494:pharmacology
chemical_list: D010455:Peptides; D054852:Small Molecule Libraries; D012436:S-Adenosylmethionine; D056466:Histone Demethylases; D011495:Histone-Lysine N-Methyltransferase