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Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS
Published on: August 10, 2017
Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS
Claudia A Simões-Pires1, Vincent Zwick1, Sylvian Cretton1
1Section des Sciences Pharmaceutiques, Universités des Geneva-Lausanne (EPGL).
Abstract:
The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography - mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.
Insights
This study presents a novel cell-based method to measure histone deacetylase (HDAC) 1 and HDAC6 activity. The assay uses ultra-high-performance liquid chromatography-mass spectrometry for accurate, endogenous HDAC inhibition profiling in drug discovery.
Area of Science:
- Biochemistry
- Pharmacology
- Cell Biology
Background:
- Histone deacetylase (HDAC) inhibitors are crucial in cancer therapy.
- Isoform-selective HDAC inhibitors, particularly for HDAC1 and HDAC6, are of significant interest for targeted drug discovery.
- Existing methods often rely on cell-free assays, which may not accurately reflect endogenous enzyme activity.
Purpose of the Study:
- To develop and validate a novel cell-based method for determining the inhibitory activity of compounds on HDAC1 and HDAC6.
- To provide a more physiologically relevant assay compared to cell-free biochemical methods.
- To facilitate the discovery of isoform-selective HDAC inhibitors.
Main Methods:
- Utilized ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) for quantitative analysis.
- Employed specific synthetic substrates incubated with treated and untreated HeLa cells to measure HDAC isoform activity.
- Validated the method using known HDAC inhibitors: trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific).
Main Results:
- The UHPLC-MS method accurately quantified endogenous HDAC1 and HDAC6 activity in a cellular context.
- The assay demonstrated sensitivity to known HDAC inhibitors, confirming its reliability.
- The method proved effective in identifying HDAC6-selective compounds in prior neuroblast studies.
Conclusions:
- This cell-based UHPLC-MS assay offers a robust and physiologically relevant approach for profiling HDAC1 and HDAC6 inhibitory activity.
- The method is adaptable to various cell lines and automation, supporting high-throughput screening in drug discovery.
- It advances the development of targeted therapies by enabling precise assessment of HDAC isoform selectivity.
