PerkinElmer, 1744 William St., Montreal, Quebec, Canada H3J 1R4
Sponsored by PerkinElmer
In this work, we describe the development of enzymatic assays to perform screenings for modulators of H3K9 acetyltransferases, and H3K4 and H3K9 methyltransferases, using homogeneous proximity assays based on two non-radioactive technologies: LANCE® Ultra timeresolved fluorescence energy transfer and AlphaLISA® bead-based chemiluminescent assays. To this end, we used as substrate a biotinylated peptide derived from the N-terminus of Histone H3 (residues 1-21) to assay acetyltransferase p300 and methyltransferases G9a and SET7/9. In all cases, we found conditions highly suitable for screening: enzyme concentrations between 0.05 to 5 nM and substrate concentrations between 50 and 500 nM. Acetyl-CoA or Sadenosylmethionine (SAM) concentrations were in the 0.1-20 M range, enabling sensitive screenings for compounds competing with these cofactors. Entire assays could be carried out in less than 4 hours, with order-of-potency for known inhibitors (anacardic acid, sinefungin, SAH) in good correlation with published literature.
The results presented herein demonstrate how to optimally assess p300, G9a, and SET7/9 activities in vitro using a biotinylated H3-derived peptide in non-radioactive, homogeneous assay formats. This could enable simple and fast screenings of compound libraries, facilitating the discovery of novel modulators of histone modifying enzymes.