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Updated: Jul 17, 2026
Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
Published on: August 5, 2022
Histone deacetylase inhibitor assay based on fluorescence resonance energy transfer
Daniel Riester1, Christian Hildmann, Andreas Schwienhorst
1Department of Molekular Genetics and Praeparative Molekular Biology, Institute for Microbiology and Genetics, Grisebachstr. 8, 37077 Goettingen, Germany.
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.
Insights
A novel nonisotopic competition binding assay for histone deacetylases (HDACs) has been developed. This assay facilitates high-throughput screening for new HDAC inhibitors crucial for cancer drug discovery.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Histone deacetylases (HDACs) are critical enzymes regulating gene expression in eukaryotes.
- HDACs are increasingly targeted for cancer chemotherapy.
- Developing effective HDAC inhibitors requires robust bioassays for screening.
Purpose of the Study:
- To introduce the first nonisotopic competition binding assay for HDACs.
- To enable efficient screening of potential HDAC inhibitors.
- To support drug discovery efforts for novel cancer therapeutics.
Main Methods:
- Demonstrated assay principle using FB188 histone deacetylase-like amidohydrolase.
- Utilized a novel fluorescent HDAC inhibitor.
- Employed fluorescence resonance energy transfer (FRET) between the inhibitor and enzyme tryptophans.
Main Results:
- The assay measures displacement of the fluorescent inhibitor by competing HDAC inhibitors.
- A decrease in FRET indicates displacement and inhibitor binding.
- The assay is suitable for kinetic studies and inhibitor identification.
Conclusions:
- The developed assay is a valuable tool for HDAC inhibitor screening.
- This assay can accelerate the identification of new chemotherapeutics targeting HDACs.
- Facilitates high-throughput screening in drug discovery programs.
