Improved fluorogenic histone deacetylase assay for high-throughput-screening applications

Histone deacetylases (HDACs) are key targets for chemotherapeutic intervention in malignant diseases. In this paper, a highly sensitive, nonisotopic, homogenous assay for high-throughput screening of HDAC inhibitors is presented. The assay is based on a new fluorogenic peptidic substrate of HDACs co...

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Bibliographic Details
Published in:Analytical biochemistry 2003-10, Vol.321 (2), p.202-208
Main Authors: Wegener, Dennis, Hildmann, Christian, Riester, Daniel, Schwienhorst, Andreas
Format: Article
Language:English
Subjects:
Acetylation
Acetylpolyamine amidohydrolase
Amidohydrolases - pharmacology
Animals
Coumarins - chemistry
Drug Evaluation, Preclinical
Enzyme Inhibitors - pharmacology
Fluorescent Dyes - chemistry
Fluorogenic substrates
Fluorometry - methods
High-throughput screening assay
Histone deacetylase
Histone Deacetylases - metabolism
Hydroxamic Acids - pharmacology
Kinetics
Liver - enzymology
Rats
Trypsin - metabolism
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Summary:Histone deacetylases (HDACs) are key targets for chemotherapeutic intervention in malignant diseases. In this paper, a highly sensitive, nonisotopic, homogenous assay for high-throughput screening of HDAC inhibitors is presented. The assay is based on a new fluorogenic peptidic substrate of HDACs comprising an ε-acetylated lysyl moiety and an adjacent 4-methylcoumarin-7-amide moiety at the C terminus of the peptide chain. Upon deacetylation of the acetylated lysyl moiety, molecules are recognized as substrates by trypsin, which releases highly fluorescent 7-amino-4-methylcoumarin molecules in a subsequent step of the assay. The fluorescence increase is directly proportional to the amount of deacetylated substrate molecules, i.e., HDAC activity. Validation of an improved version of the assay revealed (i) a significantly lower enzyme consumption, (ii) an increased screening window coefficient, (iii) a good tolerance toward organic solvents, and (iv) a good suitability for a whole range of different HDAC-like enzymes. The novel assay thus will expedite studies of HDAC-like enzymes and in vitro screening for drug discovery.
ISSN:0003-2697
1096-0309
DOI:10.1016/S0003-2697(03)00426-3