TR-FRET assay for profiling HDAC10 inhibitors and PROTACs.

Area of Science:

• Biochemistry
• Drug Discovery
• Enzymology

Background:

• Quantitative biochemical characterization is crucial for drug discovery.
• Histone deacetylases (HDACs) are therapeutic targets, with HDAC10 uniquely using small molecule polyamines as substrates.
• Traditional assays for HDACs rely on peptide substrates, which are unsuitable for HDAC10.

Purpose of the Study:

• To adapt a TR-FRET ligand displacement assay for quantitative measurement of HDAC10 binding.
• To develop a method for characterizing HDAC10 inhibitors that does not require enzymatic activity.
• To enable the screening of investigational molecules targeting HDAC10.

Main Methods:

• Preparation of a small molecule dye conjugate (tracer).
• Production of a TwinStrep-GST-HDAC10 fusion protein.
• Utilizing Eu³⁺-labelled Strep-TactinXT® for detection.
• Performing TR-FRET ligand displacement assays for dose-response measurements.

Main Results:

• Successful adaptation of a TR-FRET assay for HDAC10.
• Demonstration of dose-response measurements for HDAC10-binding molecules.
• Generation of IC₅₀ values for investigational compounds.

Conclusions:

• The developed TR-FRET assay provides a robust method for quantifying HDAC10 binding.
• This assay circumvents the need for enzymatic turnover, offering a versatile tool for HDAC10 inhibitor screening.
• The methodology facilitates the biochemical characterization of potential therapeutic agents targeting HDAC10.