Combinatorial Chemistry & High Throughput Screening

Rathnam Chaguturu 
iDDPartners, 3 Edith Court
Princeton Junction
NJ 08550
USA

Back

A High Throughput Scintillation Proximity Imaging Assay for Protein Methyltransferases

Author(s): Glorymar Ibanez, David Shum, Gil Blum, Bhavneet Bhinder, Constantin Radu, Christophe Antczak, Minkui Luo and Hakim Djaballah

Affiliation: HTS Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

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.

Order Reprints Order Eprints Rights & PermissionsPrintExport

Article Details

VOLUME: 15
ISSUE: 5
Page: [359 - 371]
Pages: 13
DOI: 10.2174/138620712800194468