title: A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening.
abstract: Most cellular assays that quantify the efficacy of histone deacetylase (HDAC) inhibitors measure hyperacetylation of core histone proteins H3 and H4. Here we describe a new approach, directly measuring cellular HDAC enzymatic activity using the substrate Boc-K(Ac)-7-amino-4-methylcoumarin (AMC). After penetration into HeLa cervical carcinoma or K562 chronic myeloid leukemia cells, the deacetylated product Boc-K-AMC is formed which, after cell lysis, is cleaved by trypsin, finally releasing the fluorophor AMC. The cellular potency of suberoylanilide hydroxamic acid, LBH589, trichostatin A, and MS275 as well-known HDAC inhibitors was determined using this assay. IC(50) values derived from concentration-effect curves correlated well with EC(50) values derived from a cellomics array scan histone H3 hyperacetylation assay. The cellular HDAC activity assay was adapted to a homogeneous format, fully compatible with robotic screening. Concentration-effect curves generated on a Tecan Genesis Freedom workstation were highly reproducible with a signal-to-noise ratio of 5.7 and a Z' factor of 0.88, indicating a very robust assay. Finally, a HDAC-inhibitor focused library was profiled in a medium-throughput screening campaign. Inhibition of cellular HDAC activity correlated well with cytotoxicity and histone H3 hyperacetylation in HeLa cells and with inhibition of human recombinant HDAC1 in a biochemical assay. Thus, by using Boc-K(Ac)-AMC as a cell-permeable HDAC substrate, the activity of various protein lysine-specific deacetylases including HDAC1-containing complexes is measurable in intact cells in a simple and homogeneous manner.
authors.affiliation: Therapeutic Area Oncology, ALTANA Pharma-a member of the Nycomed Group, Byk-Gulden Str. 2, 78467 Konstanz, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Ciossek
authors.forename: Heiko
authors.initials: H
authors.lastname: Julius
authors.forename: Heike
authors.initials: H
authors.lastname: Wieland
authors.forename: Thomas
authors.initials: T
authors.lastname: Maier
authors.forename: Thomas
authors.initials: T
authors.lastname: Beckers
mesh_terms: D000107:Acetylation; D001331:Automation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D012371:Robotics; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases