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A fluorogenic histone deacetylase assay well suited for high-throughput activity screening
Dennis Wegener1, Frank Wirsching, Daniel Riester
1Department of Molecular Genetics and Preparative Molecular Biology, Institute for Microbiology and Genetics, Grisebachstrasse 8, 37077 Goettingen, Germany.
Abstract:
Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Recent findings suggest that HDACs could be key targets for chemotherapeutic intervention in malignant diseases. A convenient and sensitive fluorogenic assay for HDAC activity would therefore expedite studies of HDAC in transcriptional regulation and in vitro screening for drug discovery. In this study, novel fluorogenic substrates of HDACs were synthesized with an epsilon-acetylated lysyl moiety and an adjacent MCA moiety at the C terminus of the peptide chain. Upon deacetylation of the acetylated lysyl moiety, molecules became substrates for trypsin, which released highly fluorescent AMC molecules in a subsequent step of the assay. The fluorescence increased in direct proportion to the amount of deacetylated substrate molecules, i.e., HDAC activity. The nonisotopic, homogeneous assay is well suited for high-throughput HDAC inhibitor screening.
Insights
Researchers developed a new fluorogenic assay to measure histone deacetylase (HDAC) activity. This assay enables sensitive detection and facilitates high-throughput screening for potential cancer drugs targeting HDACs.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Histone deacetylases (HDACs) are crucial enzymes regulating gene expression in eukaryotes.
- HDACs are increasingly recognized as potential therapeutic targets for cancer treatment.
- A sensitive assay for HDAC activity is needed for research and drug discovery.
Purpose of the Study:
- To develop a novel, sensitive, and homogeneous fluorogenic assay for measuring HDAC activity.
- To create a tool for expediting studies on HDACs in transcriptional regulation.
- To facilitate high-throughput screening of potential HDAC inhibitors for drug discovery.
Main Methods:
- Synthesis of novel fluorogenic substrates containing an epsilon-acetylated lysyl moiety and an MCA moiety.
- Assay principle relies on sequential deacetylation by HDACs and subsequent trypsin cleavage.
- Release of highly fluorescent AMC molecules upon enzymatic activity, measured by fluorescence intensity.
Main Results:
- The assay demonstrated a direct correlation between fluorescence intensity and HDAC activity.
- The developed fluorogenic substrates are effective for detecting HDAC activity.
- The assay is suitable for nonisotopic, homogeneous, and high-throughput screening.
Conclusions:
- A novel fluorogenic assay for HDAC activity has been successfully developed.
- This assay provides a sensitive and convenient method for studying HDACs.
- The assay is well-suited for high-throughput screening of HDAC inhibitors in drug discovery efforts.