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Updated: Aug 25, 2025
Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS
Published on: August 10, 2017
Fluorescence Polarization-Based Competition Assays to Evaluate Histone Deacetylase 6 Inhibitors
Qirat F Ashraf1,2, Erica J Quilates1,2, Olasunkanmi O Olaoye1,2
1Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, ON, Canada.
Abstract:
Histone deacetylase 6 (HDAC6) is an emerging clinical target for the treatment of several hematological cancers and central nervous system disorders. HDAC6 catalyzes the deacetylation of lysine residues on substrates such as tubulin, with profound implications in key cellular processes, including cellular motility and migration. This critical deacetylation activity occurs at the catalytic domain 2 (CD2) of HDAC6, and small molecule inhibitors of HDAC6 are designed to target CD2. We briefly highlight previously reported strategies for recombinant bacterial expression and purification of the HDAC6 CD2. We aim to discuss competition assays that have been used to evaluate the potency of potential HDAC6 inhibitors against CD2 via displacement of pre-bound fluorescent HDAC-probes. Moreover, we elaborate on previous protocols that have been employed in inhibitor screening and present an HDAC6-selective probe that also enables rapid and reliable high-throughput screening of new chemical entities designed to target the HDAC6 CD2.
Insights
Histone deacetylase 6 (HDAC6) is a key target for cancer and neurological disease treatments. A new HDAC6-selective probe facilitates high-throughput screening of novel inhibitors targeting its catalytic domain 2 (CD2).
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Histone deacetylase 6 (HDAC6) is a significant therapeutic target for hematological cancers and CNS disorders.
- HDAC6 regulates cellular processes like motility and migration through tubulin deacetylation at its catalytic domain 2 (CD2).
- Small molecule inhibitors are developed to target the HDAC6 CD2 for therapeutic intervention.
Purpose of the Study:
- To review methods for recombinant HDAC6 CD2 expression and purification.
- To discuss competition assays for evaluating HDAC6 inhibitor potency against CD2.
- To present a novel HDAC6-selective probe for high-throughput screening (HTS) of new chemical entities targeting CD2.
Main Methods:
- Recombinant bacterial expression and purification of HDAC6 CD2.
- Development and application of competition assays using fluorescent probes.
- High-throughput screening using a newly developed HDAC6-selective probe.
Main Results:
- Established protocols for HDAC6 CD2 production.
- Demonstrated utility of competition assays for inhibitor potency evaluation.
- Introduced a selective probe enabling efficient HTS of HDAC6 CD2 inhibitors.
Conclusions:
- HDAC6 CD2 is a validated target for therapeutic development.
- Competition assays and the novel probe are effective tools for inhibitor screening.
- The developed probe supports rapid and reliable HTS for new HDAC6-targeting drugs.
