title: Histone deacetylase inhibitor assay based on fluorescence resonance energy transfer.
abstract: Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Furthermore, in recent years HDACs occupied a major position as key targets for chemotherapeutic intervention in malignant diseases. However, progress in the development of these new chemotherapeutics is largely dependent on the existence of bioassays well-suited to inhibitor screening. Herein, we present the first nonisotopic competition binding assay for HDACs. The assay principle has been demonstrated using the well-established HDAC homolog FB188 histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes species FB188. The assay is based on a new fluorescent HDAC inhibitor that shows fluorescence resonance energy transfer with tryptophans upon binding to the enzyme. In a competition situation with other HDAC inhibitors the displacement of the fluorescent inhibitor is accompanied by a decrease of fluorescence resonance energy transfer. The assay is well suited to kinetic studies of inhibitor binding and to HDAC inhibitor identification, e.g., in the context of high-throughput inhibitor screening in drug discovery.
authors.affiliation: Department of Molekular Genetics and Praeparative Molekular Biology, Institute for Microbiology and Genetics, Grisebachstr. 8, 37077 Goettingen, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Riester
authors.forename: Christian
authors.initials: C
authors.lastname: Hildmann
authors.forename: Andreas
authors.initials: A
authors.lastname: Schwienhorst
authors.forename: Franz-Josef
authors.initials: FJ
authors.lastname: Meyer-Almes
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D001667:Binding, Competitive; D001884:Bordetella / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D056572:Histone Deacetylase Inhibitors; D000581:Amidohydrolases; D006655:Histone Deacetylases