TY - JOUR
T1 - Accessing protein methyltransferase and demethylase enzymology using microfluidic capillary electrophoresis
AU - Wigle, Tim J.
AU - Provencher, Laurel M.
AU - Norris, Jacqueline L.
AU - Jin, Jian
AU - Brown, Peter J.
AU - Frye, Stephen V.
AU - Janzen, William P.
N1 - Funding Information:
We thank Feng Liu and Xin Chen for synthesizing the analogs of UNC0224, Amy Van Deusen for building compound plates, and Irene Chau for providing the PRMT3 enzyme. We also acknowledge the efforts of Seth Cohen in facilitating this research. The project described was supported by Award Number RC1GM090732 from the National Institute Of General Medical Sciences, the Carolina Partnership, Caliper Life Sciences, the Ontario Research Fund and the Structural Genomics Consortium, a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research, and the Wellcome Trust.
PY - 2010
Y1 - 2010
N2 - 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.
AB - 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.
KW - CHEMBIO
KW - DNA
UR - http://www.scopus.com/inward/record.url?scp=77955399089&partnerID=8YFLogxK
U2 - 10.1016/j.chembiol.2010.04.014
DO - 10.1016/j.chembiol.2010.04.014
M3 - Article
C2 - 20659682
AN - SCOPUS:77955399089
SN - 1074-5521
VL - 17
SP - 695
EP - 704
JO - Chemistry and Biology
JF - Chemistry and Biology
IS - 7
ER -