title: Quantification of histone H3 Lys27 trimethylation (H3K27me3) by high-throughput microscopy enables cellular large-scale screening for small-molecule EZH2 inhibitors.
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.
authors.affiliation: Lead Discovery Berlin-Screening, Global Drug Discovery, Bayer Pharma AG, Berlin, Germany.
authors.forename: Svenja
authors.initials: S
authors.lastname: Luense
authors.affiliation: Lead Discovery Berlin-Screening, Global Drug Discovery, Bayer Pharma AG, Berlin, Germany.
authors.forename: Philip
authors.initials: P
authors.lastname: Denner
authors.affiliation: Lead Discovery Berlin-Screening, Global Drug Discovery, Bayer Pharma AG, Berlin, Germany.
authors.forename: Amaury
authors.initials: A
authors.lastname: Fernández-Montalván
authors.affiliation: Department of Medicinal Chemistry Berlin, Medicinal Chemistry I, Microbiological Chemistry, Global Drug Discovery, Bayer Pharma AG, Berlin, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Hartung
authors.affiliation: Lead Discovery Berlin-Screening, Global Drug Discovery, Bayer Pharma AG, Berlin, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Husemann
authors.affiliation: Department of Oncology, Chromatin Modulation and Oncogenomics, Global Drug Discovery, Bayer Pharma AG, Berlin, Germany.
authors.forename: Carlo
authors.initials: C
authors.lastname: Stresemann
authors.affiliation: Lead Discovery Berlin-Screening, Global Drug Discovery, Bayer Pharma AG, Berlin, Germany stefan.prechtl@bayer.com.
authors.forename: Stefan
authors.initials: S
authors.lastname: Prechtl
mesh_terms: D057205:Automation, Laboratory; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery; D000071221:Enhancer of Zeste Homolog 2 Protein; D055785:Gene Knockdown Techniques; D057166:High-Throughput Screening Assays; D006657:Histones / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020128:Inhibitory Concentration 50; D008745:Methylation / Q000187:drug effects; D008853:Microscopy; D063151:Polycomb Repressive Complex 2 / Q000037:antagonists & inhibitors*; D034622:RNA Interference; D054852:Small Molecule Libraries; D013329:Structure-Activity Relationship
chemical_list: D006657:Histones; D054852:Small Molecule Libraries; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2
keywords: EZH2; KMT6; chromatin modulators; high-content analysis; histone methyltransferase