A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Current assays for histone deacetylase (HDAC) inhibitors often rely on indirect measurements like histone hyperacetylation.
• A direct, homogeneous assay for cellular HDAC activity is needed for efficient drug screening.

Purpose of the Study:

• To develop and validate a novel assay for directly measuring intracellular HDAC enzymatic activity.
• To assess the utility of this assay for screening HDAC inhibitors in various cell lines and in a medium-throughput format.

Main Methods:

• Utilized a cell-permeable substrate, Boc-K(Ac)-7-amino-4-methylcoumarin (AMC), to measure intracellular HDAC activity.
• Adapted the assay to a homogeneous format compatible with robotic screening platforms.
• Validated the assay by comparing results with a histone H3 hyperacetylation assay and profiling a library of HDAC inhibitors.

Main Results:

• The assay successfully measured cellular HDAC activity in HeLa and K562 cells.
• Potency values (IC50) correlated well with those from a histone H3 hyperacetylation assay.
• The homogeneous assay demonstrated high reproducibility (Z' = 0.88) and was suitable for medium-throughput screening.
• Inhibition of cellular HDAC activity correlated with cytotoxicity and histone H3 hyperacetylation.

Conclusions:

• A novel, homogeneous assay directly measures cellular HDAC activity using a cell-permeable substrate.
• This assay is robust, reproducible, and suitable for screening HDAC inhibitors in intact cells.
• The assay provides a valuable tool for drug discovery targeting HDACs.