HDAC5 Redistribution Assay

• Designed to assay compounds for their ability to modulate nuclear translocation of HDAC5
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimizedassay protocol
• Easy to use: Just plate cells, add compounds, and image

Class II histone deacetylases HDAC4,5,7, and 9 shuttle between the cytoplasm and the nucleus in a signal-dependent manner. In the nucleus, they bind to transcriptional regulators such as the MEF2 family and repress transcription from target promoters. In cardiomyocytes, HDAC5 and HDAC9 perform similar functions by restricting a MEF2-dependent fetal transcription program involved in cardiac hypertrophy, a leading cause of heart failure.

Hypertrophic agonists such as serotonin and phenylephrine stimulate PKD-dependent phosphorylation of HDAC5 and HDAC9 through a PKC-dependent pathway. Phosphorylation leads to nuclear exclusion of the HDAC, which is the dominant way of regulating HDAC5/9 activity in the cell. Compounds that induce nuclear retention of HDAC5/9 are therefore relevant drug candidates for the treatment of cardiac hypertrophy and other hypertrophic diseases.

Figure 1. Translocation of EGFP-HDAC5 in response to prazosin. Cells were treated with 50 μM phenylephrine (control, left panel) or 50 μM phenylephrine + 100 nM prazosin (right panel). Arrows indicate cytoplasm to nucleus translocation detected by the image analysis algorithm.

Figure 2. Prazosin concentration response curve in the HDAC5 Redistribution assay. Concentration response was measured in 9 point half log dilution series of prazosin. Cells were incubated with prazosin for 2 h. Cells were then fixed and the nucleus to cytoplasm translocation was measured using the Cellomics ArrayScan V^TI Reader and the RedistributionV3 BioApplication. % activity was calculated relative to the positive (100 nM prazosin) and negative control (0.25% DMSO). The EC₅₀ of prazosin is ~3.5 nM.