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

Dennis Wegener1, Christian Hildmann, Daniel Riester

  • 1Abteilung fuer Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Grisebachstr. 8, 37077 Goettingen, Germany.

Analytical Biochemistry
|September 27, 2003
PubMed
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Non-isotopic dual parameter competition assay suitable for high-throughput screening of histone deacetylases.

Bioorganic & medicinal chemistry letters·2009

A new assay enables sensitive, high-throughput screening of histone deacetylase (HDAC) inhibitors for cancer drug discovery. This fluorogenic method accurately measures HDAC activity, accelerating the search for novel therapeutics.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Drug Discovery

Background:

  • Histone deacetylases (HDACs) are crucial targets in cancer chemotherapy.
  • Existing screening methods for HDAC inhibitors can be limited in sensitivity and throughput.

Purpose of the Study:

  • To develop a highly sensitive, nonisotopic, homogeneous assay for high-throughput screening of HDAC inhibitors.
  • To validate the performance and suitability of the novel assay for drug discovery.

Main Methods:

  • A novel fluorogenic peptidic substrate for HDACs was designed, incorporating an epsilon-acetylated lysyl moiety and a 4-methylcoumarin-7-amide group.
  • Enzyme activity was measured by detecting fluorescence released after trypsin cleavage of deacetylated substrate.
  • The assay's performance was validated, assessing enzyme consumption, screening window, solvent tolerance, and enzyme compatibility.

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Main Results:

  • The assay demonstrated high sensitivity and a homogeneous format suitable for high-throughput screening.
  • An improved assay version showed significantly reduced enzyme consumption and an increased screening window coefficient.
  • The assay exhibited good tolerance to organic solvents and broad suitability for various HDAC-like enzymes.

Conclusions:

  • The novel fluorogenic assay provides a sensitive and efficient tool for screening HDAC inhibitors.
  • This assay will expedite research on HDAC-like enzymes and in vitro drug discovery efforts.
  • The method facilitates the identification of potential chemotherapeutic agents targeting malignant diseases.