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Quantification of histone H3 Lys27 trimethylation (H3K27me3) by high-throughput microscopy enables cellular large-scale screening for small-molecule EZH2 inhibitors
- Source :
- Journal of Biomolecular Screening
- Publication Year :
- 2014
-
Abstract
- EZH2 inhibition can decrease global histone H3 lysine 27 trimethylation (H3K27me3) and thereby reactivates silenced tumor suppressor genes. Inhibition of EZH2 is regarded as an option for therapeutic cancer intervention. To identify novel small-molecule (SMOL) inhibitors of EZH2 in drug discovery, trustworthy cellular assays amenable for phenotypic high-throughput screening (HTS) are crucial. We describe a reliable approach that quantifies changes in global levels of histone modification marks using high-content analysis (HCA). The approach was validated in different cell lines by using small interfering RNA and SMOL inhibitors. By automation and miniaturization from a 384-well to 1536-well plate, we demonstrated its utility in conducting phenotypic HTS campaigns and assessing structure-activity relationships (SAR). This assay enables screening of SMOL EZH2 inhibitors and can advance the mechanistic understanding of H3K27me3 suppression, which is crucial with regard to epigenetic therapy. We observed that a decrease in global H3K27me3, induced by EZH2 inhibition, comprises two distinct mechanisms: (1) inhibition of de novo DNA methylation and (II) inhibition of dynamic, replication-independent H3K27me3 turnover. This report describes an HCA assay for primary HTS to identify, profile, and optimize cellular active SMOL inhibitors targeting histone methyltransferases, which could benefit epigenetic drug discovery.
- Subjects :
- Computational biology
macromolecular substances
Biochemistry
Methylation
Analytical Chemistry
Histones
Small Molecule Libraries
Histone H3
Inhibitory Concentration 50
Structure-Activity Relationship
Cell Line, Tumor
Drug Discovery
Humans
Enhancer of Zeste Homolog 2 Protein
Epigenetics
EZH2
Original Research
Automation, Laboratory
Microscopy
biology
Dose-Response Relationship, Drug
Drug discovery
histone methyltransferase
Cell Cycle
Polycomb Repressive Complex 2
high-content analysis
Molecular biology
chromatin modulators
High-Throughput Screening Assays
Histone
Histone methyltransferase
High-content screening
Gene Knockdown Techniques
biology.protein
Molecular Medicine
KMT6
RNA Interference
Epigenetic therapy
Biotechnology
Subjects
Details
- ISSN :
- 1552454X
- Volume :
- 20
- Issue :
- 2
- Database :
- OpenAIRE
- Journal :
- Journal of biomolecular screening
- Accession number :
- edsair.doi.dedup.....7eea132c47a4c6efbe74bbc4b72075ea