Title:A High Throughput Scintillation Proximity Imaging Assay for Protein Methyltransferases
Volume: 15
Issue: 5
Author(s): Glorymar Ibanez, David Shum, Gil Blum, Bhavneet Bhinder, Constantin Radu, Christophe Antczak, Minkui Luo and Hakim Djaballah
Affiliation:
Keywords:
Drug discovery, EuHMTase1, inhibitor, protein methyltransferases, red shifted imaging beads, SET7/9, SET8,
SETD2, SPA technology
Abstract: Protein methyltransferases (PMTs) orchestrate epigenetic modifications through post-translational methylation
of various protein substrates including histones. Since dysregulation of this process is widely implicated in many cancers,
it is of pertinent interest to screen inhibitors of PMTs, as they offer novel target-based opportunities to discover small
molecules with potential chemotherapeutic use. We have thus developed an enzymatic screening strategy, which can be
adapted to scintillation proximity imaging assay (SPIA) format, to identify these inhibitors. We took advantage of
S-adenosyl-L-[3H-methyl]-methionine availability and monitored the enzymatically catalyzed [3H]-methyl addition on
lysine residues of biotinylated peptide substrates. The radiolabeled peptides were subsequently captured by streptavidin
coated SPA imaging PS beads. We applied this strategy to four PMTs: SET7/9, SET8, SETD2, and EuHMTase1, and
optimized assay conditions to achieve Z' values ranging from 0.48 to 0.91. The robust performance of this SPIA for the
four PMTs was validated in a pilot screen of approximately 7,000 compounds. We identified 80 cumulative hits across the
four targets. NF279, a suramin analogue, was found to specifically inhibit SET7/9 and SETD2 with IC50 values of 1.9 and
1.1 μM, respectively. Another identified compound, Merbromin, a topical antiseptic, was classified as a pan-active
inhibitor of the four PMTs. These findings demonstrate that our proposed SPIA strategy is generic for multiple PMTs and
can be successfully implemented to identify novel and specific inhibitors of PMTs. The specific PMT inhibitors may
constitute a new class of anti-proliferative agents for potential therapeutic use.
