title: Mechanistic analysis of a suicide inactivator of histone demethylase LSD1.
abstract: Lysine-specific demethylase 1 (LSD1) is a transcriptional repressor and a flavin-dependent amine oxidase that is responsible for the removal of methyl from lysine 4 of histone H3. In this study, we characterize the mechanism and scope of LSD1 inhibition by a propargylamine-derivatized histone H3 substrate (1). Unlike aziridinyl and cyclopropylamine-derivatized histone H3 peptide substrate analogues, compound 1 appears to covalently modify and irreversibly inactivate LSD1 with high potency. Accompanying this inactivation is a spectroscopic change, which shifts the absorbance maximum to 392 nm. Spectral changes associated with the 1-LSD1 complex and reactivity to decreased pH and sodium borohydride treatment were suggestive of a structure involving a flavin-linked inhibitor conjugate between N5 of the flavin and the terminal carbon of the inhibitor. Using a 13C-labeled inhibitor, NMR analysis of the 1-flavin conjugate was consistent with this structural assignment. Kinetic analysis of the spectroscopic shift induced by 1 showed that the flavin adduct formed in a reaction with kinetic constants similar to those of the LSD1 inactivation process. Taken together, these data support a mechanism of LSD1 inactivation by 1 involving amine oxidation followed by Michael addition to the propargylic imine. We further examined the potential for a biotinylated analogue of 1 (1-Btn) to be used as a tool in affinity pulldown experiments. Using 1-Btn, it was feasible to selectively pull down spiked and endogenous LSD1 from HeLa cell nuclear extracts, setting the stage for activity-based demethylase proteomics.
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Szewczuk
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Culhane
authors.forename: Maojun
authors.initials: M
authors.lastname: Yang
authors.forename: Ananya
authors.initials: A
authors.lastname: Majumdar
authors.forename: Hongtao
authors.initials: H
authors.lastname: Yu
authors.forename: Philip A
authors.initials: PA
authors.lastname: Cole
mesh_terms: D000595:Amino Acid Sequence; D019921:Biotinylation; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005415:Flavins / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006367:HeLa Cells; D056466:Histone Demethylases; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000302:isolation & purification; D013053:Spectrophotometry
chemical_list: D004791:Enzyme Inhibitors; D005415:Flavins; D056466:Histone Demethylases; C495195:KDM1A protein, human; D010089:Oxidoreductases, N-Demethylating; D005978:Glutathione