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Published on: August 10, 2017
First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Selective Inhibitors with Cellular
Daniel Herp1, Johannes Ridinger2,3,4, Dina Robaa5
1Institute of Pharmaceutical Sciences, University of Freiburg, Albertstraße 25, 79104, Freiburg, Germany.
Abstract:
Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, especially cancer. Five HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc-dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement, e. g., in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of most other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like acetyl-putrescine or -spermidine. Hence, it is also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin-labelled acetyl-spermidine derivative to measure its PDAC activity, which is suitable for high-throughput screening. Using this assay, we identified potent inhibitors of HDAC10-mediated spermidine deacetylation in vitro. Based on the oligoamine preference of HDAC10, we also designed inhibitors with a basic moiety in appropriate distance to the zinc binding hydroxamate that showed potent inhibition of HDAC10 with high selectivity, and we solved a HDAC10-inhibitor structure using X-ray crystallography. We could demonstrate selective cellular target engagement for HDAC10 but a lysosomal phenotype in neuroblastoma cells that was previously associated with HDAC10 inhibition was not observed. Thus, we have developed new chemical probes for HDAC10 that allow further clarification of the biological role of this enzyme.
Insights
Researchers developed a new assay to study HDAC10, a polyamine deacetylase (PDAC). This led to the discovery of potent and selective HDAC10 inhibitors, aiding further research into this enzyme's biological roles.
Area of Science:
- Biochemistry
- Epigenetics
- Drug Discovery
Background:
- Histone deacetylases (HDACs) are crucial epigenetic regulators implicated in various diseases, including cancer.
- While several HDAC inhibitors are approved for cancer therapy, HDAC10 remains understudied due to a lack of effective enzymatic assays.
- HDAC10 exhibits unique polyamine deacetylase (PDAC) activity, distinct from the lysine deacetylation of other HDACs.
Purpose of the Study:
- To develop a robust enzymatic assay for measuring HDAC10 polyamine deacetylase activity.
- To identify potent and selective inhibitors of HDAC10.
- To investigate the biological role of HDAC10 in diseases like neuroblastoma.
Main Methods:
- Development of a novel fluorescent enzymatic conversion assay using an aminocoumarin-labeled acetyl-spermidine derivative for HDAC10 activity measurement.
- High-throughput screening to identify inhibitors of HDAC10-mediated spermidine deacetylation.
- Structure-based inhibitor design targeting HDAC10's oligoamine preference and zinc-binding site.
- X-ray crystallography to determine the structure of HDAC10 in complex with an inhibitor.
- Cellular target engagement studies and phenotypic analysis in neuroblastoma cells.
Main Results:
- The first fluorescent assay for HDAC10 PDAC activity, suitable for high-throughput screening, was successfully established.
- Potent inhibitors of HDAC10-mediated spermidine deacetylation were identified in vitro.
- Novel HDAC10 inhibitors with high selectivity were designed based on enzyme substrate preference and validated through structural analysis.
- Selective cellular engagement of HDAC10 was confirmed, but a previously associated lysosomal phenotype in neuroblastoma cells was not observed.
Conclusions:
- A novel, high-throughput compatible fluorescent assay for HDAC10 activity has been developed.
- Potent and selective chemical probes for HDAC10 have been identified and characterized.
- These new tools will facilitate further investigation into the specific biological functions and therapeutic potential of HDAC10.
