Development and implementation of a high-throughput AlphaLISA assay for identifying inhibitors of EZH2 methyltransferase

Assay Drug Dev Technol. 2013 Apr;11(3):152-62. doi: 10.1089/adt.2012.481. Epub 2013 Feb 14.


The methylation state of lysine residues within histone H3 is a major determinant of active and inactive regions of the genome. Enhancer of Zeste homolog 2 (EZH2) is a histone lysine methyltransferase that is part of the polycomb repressive complex 2 (PRC2). Elevated EZH2 expression levels have been linked to hypertrimethylation of histone H3 lysine 27 (H3K27), repression of tumor repressor genes, and the onset of several types of cancers. We used the AlphaLISA technology to develop a high-throughput assay for identifying small molecule inhibitors of EZH2. AlphaLISA Acceptor Beads coated with antibodies directed against methylated H3K27 provided a sensitive method of detecting EZH2 activity through measurement of K27 methylation of a biotinylated H3-based peptide substrate. Optimized assay conditions resulted in a robust assay (Z'>0.7) which was successfully implemented in a high-throughput screening campaign. Small molecule inhibitors identified by this method may serve as powerful tools to further elucidate the potential importance of EZH2 in the development and treatment of cancer.

MeSH terms

  • Algorithms
  • Antibodies / chemistry
  • Buffers
  • Data Interpretation, Statistical
  • Enhancer of Zeste Homolog 2 Protein
  • Enzyme Inhibitors / pharmacology*
  • Fluorescence Polarization Immunoassay
  • High-Throughput Screening Assays / methods*
  • Humans
  • Indicators and Reagents
  • Methylation
  • Peptides / chemistry
  • Polycomb Repressive Complex 2 / antagonists & inhibitors*
  • Recombinant Proteins / chemistry
  • Reference Standards
  • Reproducibility of Results
  • Streptavidin


  • Antibodies
  • Buffers
  • Enzyme Inhibitors
  • Indicators and Reagents
  • Peptides
  • Recombinant Proteins
  • Streptavidin
  • EZH2 protein, human
  • Enhancer of Zeste Homolog 2 Protein
  • Polycomb Repressive Complex 2