title: Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate.
abstract: Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry.
authors.affiliation: Institute of Pharmaceutical Sciences, Albert-Ludwigs-University of Freiburg, Freiburg, Germany.
authors.forename: Martin L
authors.initials: ML
authors.lastname: Schmitt
authors.affiliation: Institute of Pharmaceutical Sciences, Albert-Ludwigs-University of Freiburg, Freiburg, Germany.
authors.forename: Kathrin I
authors.initials: KI
authors.lastname: Ladwein
authors.affiliation: Institute of Pharmacy, Martin-Luther-University of Halle-Wittenberg, Halle, Germany.
authors.forename: Luca
authors.initials: L
authors.lastname: Carlino
authors.affiliation: Institute of Pharmaceutical Sciences, Albert-Ludwigs-University of Freiburg, Freiburg, Germany German Cancer Consortium (DKTK), Heidelberg, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Schulz-Fincke
authors.affiliation: Department of Urology/Women's Hospital and Center for Clinical Research, University of Freiburg Medical Center, Freiburg, Germany.
authors.forename: Dominica
authors.initials: D
authors.lastname: Willmann
authors.affiliation: Department of Urology/Women's Hospital and Center for Clinical Research, University of Freiburg Medical Center, Freiburg, Germany.
authors.forename: Eric
authors.initials: E
authors.lastname: Metzger
authors.affiliation: Adolf-Butenandt Institute and Munich Center of Integrated protein science (CIPS), Ludwig Maximilians University of Munich, Munich, Germany.
authors.forename: Pierre
authors.initials: P
authors.lastname: Schilcher
authors.affiliation: Adolf-Butenandt Institute and Munich Center of Integrated protein science (CIPS), Ludwig Maximilians University of Munich, Munich, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Imhof
authors.affiliation: Department of Urology/Women's Hospital and Center for Clinical Research, University of Freiburg Medical Center, Freiburg, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Schüle
authors.affiliation: Institute of Pharmacy, Martin-Luther-University of Halle-Wittenberg, Halle, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Sippl
authors.affiliation: Institute of Pharmaceutical Sciences, Albert-Ludwigs-University of Freiburg, Freiburg, Germany manfred.jung@pharmazie.uni-freiburg.de.
authors.forename: Manfred
authors.initials: M
authors.lastname: Jung
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D019921:Biotinylation; D055808:Drug Discovery; D057890:Epigenomics; D005453:Fluorescence; D056466:Histone Demethylases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006657:Histones / Q000737:chemistry*; D006801:Humans; D007118:Immunoassay; D007313:Insecta; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
chemical_list: D000906:Antibodies; D006657:Histones; D010455:Peptides; D056466:Histone Demethylases; C495195:KDM1A protein, human; D008239:Lysine
keywords: LSD1; chemoinformatics; enzyme assays or enzyme kinetics; epigenetics; medicinal chemistry