title: Fluorescence-based screening assays for the NAD⁺-dependent histone deacetylase smSirt2 from Schistosoma mansoni.
abstract: Sirtuins are NAD(+)-dependent histone deacetylases (HDACs) that cleave off acetyl but also other acyl groups from the ϵ-amino group of lysines in histones and other substrate proteins. Five sirtuin isoforms are encoded in the genome of the parasitic pathogen Schistosoma mansoni. During its life cycle, S. mansoni undergoes drastic changes in phenotype that are associated with epigenetic modifications. Previous work showed strong effects of hSirt2 inhibitors on both worm life span and reproduction. Thus, we postulate smSirt2 as a new antiparasite target. We report both the optimization of a homogeneous fluorescence-based assay and the development of a new heterogeneous fluorescence-based assay to determine smSirt2 activity. The homogeneous assay uses a coumarin-labeled acetyl lysine derivative, and the heterogeneous version is using a biotinylated and fluorescence-labeled oligopeptide. Magnetic streptavidin-coated beads allow higher substrate loading per well than streptavidin-coated microtiter plates and make it possible to screen for inhibitors of either smSirt2 or its human isoform (hSirt2) for selectivity studies. We also present hits from a pilot screen with inhibitors showing an IC50 lower than 50 µM. Binding of the hits to their targets is rationalized by docking studies using a homology model of smSirt2.
authors.affiliation: Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Schiedel
authors.affiliation: Département de Biologie Structurale Intégrative, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UDS), CNRS, INSERM, Illkirch, France.
authors.forename: Martin
authors.initials: M
authors.lastname: Marek
authors.affiliation: Center for Infection and Immunity of Lille (CIIL), INSERM U1019-CNRS UMR 8204, Université Lille Nord de France, Institut Pasteur de Lille, Lille, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Lancelot
authors.affiliation: Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany.
authors.forename: Berin
authors.initials: B
authors.lastname: Karaman
authors.affiliation: Kancera AB, Solna, Sweden.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Almlöf
authors.affiliation: Kancera AB, Solna, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Schultz
authors.affiliation: Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Sippl
authors.affiliation: Center for Infection and Immunity of Lille (CIIL), INSERM U1019-CNRS UMR 8204, Université Lille Nord de France, Institut Pasteur de Lille, Lille, France.
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Pierce
authors.affiliation: Département de Biologie Structurale Intégrative, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UDS), CNRS, INSERM, Illkirch, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Romier
authors.affiliation: Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany manfred.jung@pharmazie.uni-freiburg.de.
authors.forename: Manfred
authors.initials: M
authors.lastname: Jung
mesh_terms: D000818:Animals; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D044127:Epigenesis, Genetic / Q000187:drug effects; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D008958:Models, Molecular; D008968:Molecular Conformation; D009536:Niacinamide / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D012550:Schistosoma mansoni / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D056565:Sirtuin 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D020032:Tyrphostins / Q000737:chemistry / Q000494:pharmacology
chemical_list: D020032:Tyrphostins; D009536:Niacinamide; D056565:Sirtuin 2
keywords: Schistosoma mansoni; epigenetics; histone modifications; in vitro assay; sirtuin