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Improved Fluorogenic Histone Deacetylase Assay for High-Throughput-Screening Applications

Improved Fluorogenic Histone Deacetylase Assay for High-Throughput-Screening Applications

Dennis Wegener, Christian Hildmann, Daniel Riester, Andreas Schwienhorst

Anal Biochem. 2003 Oct 15;321(2):202-8.

PMID: 14511685

Abstract:

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

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42413455	HDAC Substrate 2, fluorogenic			Price

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