title: Kinetic method for the large-scale analysis of the binding mechanism of histone deacetylase inhibitors.
abstract: Performing kinetic studies on protein ligand interactions provides important information on complex formation and dissociation. Beside kinetic parameters such as association rates and residence times, kinetic experiments also reveal insights into reaction mechanisms. Exploiting intrinsic tryptophan fluorescence a parallelized high-throughput Förster resonance energy transfer (FRET)-based reporter displacement assay with very low protein consumption was developed to enable the large-scale kinetic characterization of the binding of ligands to recombinant human histone deacetylases (HDACs) and a bacterial histone deacetylase-like amidohydrolase (HDAH) from Bordetella/Alcaligenes. For the binding of trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), and two other SAHA derivatives to HDAH, two different modes of action, simple one-step binding and a two-step mechanism comprising initial binding and induced fit, were verified. In contrast to HDAH, all compounds bound to human HDAC1, HDAC6, and HDAC8 through a two-step mechanism. A quantitative view on the inhibitor-HDAC systems revealed two types of interaction, fast binding and slow dissociation. We provide arguments for the thesis that the relationship between quantitative kinetic and mechanistic information and chemical structures of compounds will serve as a valuable tool for drug optimization.
authors.affiliation: University of Applied Sciences Darmstadt, Department of Chemical Engineering and Biotechnology, Schnittspahnstrasse 12, 64287 Darmstadt, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Meyners
authors.affiliation: Imperial College London, Department of Chemistry, South Kensington Campus, London SW7 2AZ, UK; University of Cambridge, Department of Chemistry, Lensfield Rd, CB2 1EW, UK.
authors.forename: Matthias G J
authors.initials: MG
authors.lastname: Baud
authors.affiliation: Imperial College London, Department of Chemistry, South Kensington Campus, London SW7 2AZ, UK.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Fuchter
authors.affiliation: University of Applied Sciences Darmstadt, Department of Chemical Engineering and Biotechnology, Schnittspahnstrasse 12, 64287 Darmstadt, Germany. Electronic address: franz-josef.meyer-almes@h-da.de.
authors.forename: Franz-Josef
authors.initials: FJ
authors.lastname: Meyer-Almes
mesh_terms: D000421:Alcaligenes / Q000201:enzymology; D001884:Bordetella / Q000201:enzymology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors / Q000378:metabolism*; D006655:Histone Deacetylases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding
chemical_list: D005456:Fluorescent Dyes; D056572:Histone Deacetylase Inhibitors; D008024:Ligands; D006655:Histone Deacetylases
keywords: Binding assay; Binding mechanism; Histone deacetylases; Inhibitors; Kinetics