TR-FRET assay for profiling HDAC10 inhibitors and PROTACs

Quantitative biochemical characterization of the binding/inhibitory properties of investigative substances against their protein targets and anti-targets is a necessary step in modern drug discovery campaigns. The histone deacetylase family of proteins comprises eleven Zn²⁺ dependent enzymes, members of which are regularly investigated as therapeutic drug targets. The binding of histone deacetylases by small molecule inhibitors or PROTACs is typically measured in enzymatic assays that use acylated lysine-containing peptides as substrates. Histone deacetylase 10, however, is unique within the family in that it recognizes acetylated small molecule polyamines, as opposed to peptides, as substrates. We have therefore adapted a TR-FRET ligand displacement assay for histone deacetylase 10, which does not rely on enzymatic turnover of a substrate. In this chapter, we describe the preparation of the three different assay components: a small molecule dye conjugate "tracer", a TwinStrep-GST-HDAC10 fusion protein, and Eu³⁺-labelled Strep-TactinXT®. Lastly, we describe how to combine these reagents and perform dose-response measurements of investigational HDAC10-binding molecules to produce IC₅₀ values.